Compositions and methods for detecting biological contaminant

Abstract

To provide compositions and methods useful to the determination of whether a microbial contaminant is present in a biological therapeutic production process.SOLUTION: Provided are an artificial positive amplification control plasmid and unique quantitative PCR detection probe are provided, which enables the rapid and real-time detection of a false positive result. Also provided is a composition comprising an artificial nucleotide sequence, a fluorophore, and a quencher. Here, the artificial nucleotide sequence comprises at its three-prime end no more than five consecutive nucleic acids that are identical to any one sequence in a group of sequences.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 139,321, filed on Mar. 27, 2015, which is hereby specifically incorporated herein by reference in its entirety.

[0002] Field of the Invention The present invention generally relates to processes for manufacturing biological molecules by cell culture. More specifically, but not exclusively, the present invention relates to compositions and methods for detecting biological contaminants in cell cultures.

Background Art

[0003] Background of the Invention Biopharmaceuticals, particularly therapeutic antibodies, are produced by mammalian cell cultures. Chinese hamster ovary (CHO) cells are the most commonly used host cells. These production systems are prone to exogenous and endogenous viral infections, which pose potential safety concerns regarding biopharmaceuticals. Therefore, virus clearance procedures and virus quantification are used to improve the safety of drugs. Steps employed to reduce the virus load include nanofiltration, virus inactivation by heat or pH retention, and chromatography. The virus load and virus removal efficacy can be monitored by time - consuming infectivity assays or rapid quantitative assays such as real - time PCR or quantitative polymerase chain reaction (Q - PCR).

[0004] Q-PCR requires that appropriate negative and positive controls be reliable. For the control of proper nucleic acid extraction, a nucleic acid extraction control is added to the test sample. If the nucleic acid extraction control is negative or outside the expected recovery range during Q-PCR, the sample is rejected. Conversely, if the nucleic acid extraction control is positive or within the expected recovery range during Q-PCR, the nucleic acid extraction from the test sample is considered reliable. Negative controls such as sample-free buffers are usually included in Q-PCR assays. The presence of a positive signal in the negative control may mean contamination of the Q-PCR or nucleic acid extraction reagents by viral substances.

[0005] Positive amplification controls may also be included in Q-PCR virus quantity assays. Such positive controls may contain preserved viral nucleic acid sequences that are amplified using primers that amplify authentic viral contaminants. The inability of Q-PCR to detect a positive control may indicate that the amplification procedure would not have detected viral contaminants if they were present in the test sample.

[0006] The use of positive amplification controls that mimic the target contaminants poses problems of its own. If the test sample is contaminated with the positive control, even in very small amounts, the test sample may show false positives given the excellent sensitivity of Q-PCR. False positives can be reduced to some extent by using low levels of positive amplification controls, using a separate room for positive control operations, using a UNG / dUTP system that selectively degrades PCR products containing dUTP, using single-use containers and displacement pipettes, and thoroughly decontaminating the work area and equipment. Despite the careful use of those reduction measures, false positive results still occur during PCR testing.

[0007] In biopharmaceutical manufacturing, the risk of false positives for biological contaminants cannot be ignored and can result in costly corrective actions. There is a great need for systems and methods to determine whether a given positive PCR result is a true positive or a false positive caused by cross-contamination of the positive control. The Applicants have developed and disclose herein a positive control composition, system, and method that enable real-time determination of false positive Q-PCR signals.

Summary of the Invention

[0008] The Applicants have solved the problem of identifying in real time whether a positive Q-PCR signal for a target contaminant in a test sample is a true positive or a false positive due to cross-contamination. The Applicants have created a positive amplification control (PAC) plasmid that contains a biological contaminant target sequence (i.e., positive control sequence) and a unique artificial plasmid-specific sequence. This unique artificial plasmid-specific sequence enables an assay technician to specifically identify the plasmid if it is present in the sample. Thus, if a positive contaminant signal is detected and it is determined that the unique artificial plasmid-specific sequence is absent, the technician can be confident that the result is a true positive result. Conversely, in the case of a false positive event, the presence of the unique artificial plasmid sequence enables the technician to quickly rule out the apparent positive result as a false positive.

[0009] In some embodiments, the unique artificial plasmid-specific sequence of the positive control (the "unique sequence"; also known as PACP or positive growth control polynucleotide) is detected using a fluorescently labeled artificial oligonucleotide detection probe (the "unique detection probe" or "UDP") included in the Q-PCR reaction mixture. The unique detection probe includes a nucleic acid polymer covalently attached to a fluorophore and a quencher. The "unique" nucleic acid polymer is designed to specifically anneal to the unique sequence and be incorporated into the amplicon copies of the unique sequence during PCR. The "unique" nucleic acid polymer is designed such that under the hybridization conditions employed in the performance of the assay of interest, it does not recognize or anneal to any naturally occurring parvovirus. In one embodiment, the "unique" nucleic acid polymer comprises 17 to 20 nucleotides, where no more than 7 to 10 consecutive internal nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence. In one embodiment, the "unique" nucleotide polymer comprises 17 to 20 nucleotides, where no more than 7 to 10 consecutive nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence as set forth in SEQ ID NOs: 9 and 12 - 37. If the nucleic acid polymer of the unique detection probe is not incorporated into the amplicon copy, the quencher remains in close proximity to the fluorophore. If the fluorophore is excited, the quencher absorbs the emitted light and prevents its detection (by FRET or contact quenching). When the nucleic acid polymer is incorporated into the amplicon copy (i.e., when the unique sequence is present in the sample), the fluorophore and the quencher are released from the unique detection probe and are thus spatially separated. In this case, when the fluorophore is excited, the quencher is far enough away that it cannot efficiently quench the emitted light. Thus, the emitted light can be detected. Therefore, when the unique sequence is present in the sample, the detectable emission wavelength increases in intensity as PCR progresses. When the unique sequence is absent, the quencher functions and no emission wavelength is detected even as PCR progresses.In one embodiment, the fluorophore is attached at or near the 5'-end of the nucleic acid polymer and the quencher is attached at or near the 3'-end. In an alternative embodiment, the fluorophore is attached at or near the 3'-end of the nucleic acid polymer and the quencher is attached at or near the 5'-end.

[0010] Any fluorophore-quencher pair known currently or discovered later can be used in the practice of the present invention. (See, e.g., S.A. Marras, 「Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes」, Methods Mol. Biol. 2006; 335:3-16). In some embodiments, the fluorophores have excitation wavelengths of 495 nm, 538 nm, or 646 nm, and emission wavelengths of 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye having an absorption peak in the range of 430 nm to 672 nm. In some embodiments, the quencher is selected from the group consisting of DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, and DHQ-3. In one embodiment, the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm (e.g., FAM), and the quencher is BHQ-1. In one embodiment, the nucleic acid polymer comprises the nucleic acid sequence of TIFF2025105635000001.tif4128.

[0011] One aspect of the present invention is the unique detection probe itself as described above, containing a nucleic acid polymer and a linked fluorophore and quencher. Another aspect of the present invention is the positive amplification control (PAC) plasmid itself containing a biological contaminant target sequence and a unique artificial plasmid-specific sequence, and its use as a positive control for assessing the presence of a target biological contaminant in a cell culture. This PAC plasmid is used as a control for the success of a PCR amplification reaction designed to amplify a target biological contaminant sequence. For example, a separate and parallel PCR reaction is performed that contains the same components and is conducted under the same parameters as the test sample, but contains the positive control plasmid instead of the test sample. If the positive control plasmid-containing sample generates a positive "contaminant" signal while the test sample does not, the test sample can be concluded to lack the target biological contaminant. In some embodiments, the test sample is obtained from a mammalian cell culture, such as a bioreactor culture containing CHO cells modified to produce a therapeutic protein of interest.

[0012] In one embodiment, the PAC plasmid contains (a) a target amplification polynucleotide (TAP) sequence, such as, for example, a parvovirus nucleic acid sequence or the sequence of another target contaminant, and (b) a plasmid amplification control polynucleotide (PACP) sequence. The PACP sequence (sense or click strand) is complementary to the nucleic acid polymer (antisense or Watson strand) of the unique sequence probe.

[0013] In one aspect, the PAC plasmid is employed in a separate Q-PCR reaction in parallel with the Q-PCR reaction containing the test sample. The TAP sequence is designed to represent the target biological contaminant. For example, if the Q-PCR reaction of the test sample cannot produce the TAP amplicon and the Q-PCR of the positive control (i.e., the sample containing the PAC plasmid) cannot produce the TAP amplicon, it can be inferred that the Q-PCR reaction has failed. In one aspect, the target biological contaminant is a rodent parvovirus, and the TAP sequence includes a rodent parvovirus sequence. In one aspect, the TAP sequence includes all or a portion of the parvovirus NS1 sequence that is conserved across several rodent parvovirus strains. See Cotmore, et al., "Replication Initiator Protein NS1 of the parvovirus Minute Virus of Mice Binds to Modular Divergent Sites Distributed throughout Duplex Viral DNA", J. Virol. 2007 Dec; 81(23):13015-13027. In some aspects, several rodent parvovirus strains include the mouse minute virus prototype strain (MVMp), the mouse minute virus immunosuppressive strain (MVMi), the mouse minute virus Cutter strain (MVMc), mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MVP-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a (RPV-1a), rat minute virus (RMV), and the University of Massachusetts strain of rat virus L (RV-Umass). See O.-W. Merten, "Virus Contaminations of Cell Cultures - A Biotechnological View", Cytotechnology. 2002 July; 39(2):91-116.In one aspect, the TAP array includes the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, and the complementary sequence of SEQ ID NO:4.

[0014] In other aspects, the invention is directed to a PCR cocktail composition and a method of using the PCR cocktail to detect a target contaminant in a test sample and to eliminate false positives resulting from contamination of the test sample by the PAC.

[0015] In one aspect, the PCR cocktail contains, among other things, a target contaminant-specific forward oligonucleotide primer, a target contaminant-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the unique detection probe (UDP) described above, and a target contaminant-specific reverse oligonucleotide primer. In aspects where the target contaminant is a rodent parvovirus, the PCR cocktail contains, among other things, a rodent parvovirus-specific forward oligonucleotide primer, a rodent parvovirus-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the unique detection probe (UDP) described above, and a rodent parvovirus-specific reverse oligonucleotide primer. Each detection probe (i.e., a target contaminant-specific oligonucleotide detection probe such as a rodent parvovirus-specific oligonucleotide detection probe and an artificial oligonucleotide detection probe) contains a nucleic acid sequence linked to a fluorophore at one end (either 5' or 3') and a quencher at the other end (either 3' or 5' respectively).

[0016] In the above case, the nucleic acid sequences of the rodent parvovirus-specific forward oligonucleotide primer and the rodent parvovirus-specific oligonucleotide detection probe hybridize to the antisense strand of the parvovirus. The rodent parvovirus-specific reverse oligonucleotide primer hybridizes to the sense strand of the parvovirus. In one embodiment, the parvovirus sequence to which the primer and the parvovirus-specific oligonucleotide probe hybridize is a conserved rodent parvovirus sequence. In some cases, the conserved parvovirus sequence is a parvovirus NS1 sequence, such as the nucleic acid sequence set forth in SEQ ID NO:9, for example. By using the conserved sequence, it is believed that a single probe is effective for detecting multiple strains of rodent parvovirus.

[0017] In one aspect, the artificial oligonucleotide detection probe does not hybridize to either parvovirus nucleic acid or any biological contaminant sequence. The nucleic acid of the artificial oligonucleotide detection probe is synthetic and is considered not to hybridize to any biological contaminant nucleic acid sequence under any stringency. In one aspect, the nucleic acid of the artificial oligonucleotide detection probe (also known as the "unique" nucleic acid polymer or unique sequence) is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions employed in the subject assay. In one aspect, the "unique" nucleic acid polymer contains 17 to 20 nucleotides, where no more than 7 to 10 consecutive internal nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence. In one aspect, the "unique" nucleotide polymer contains 17 to 20 nucleotides, where no more than 7 to 10 consecutive nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence as set forth in SEQ ID NO: 9 and 12 - 37. However, the nucleic acid of the artificial oligonucleotide detection probe (i.e., the unique sequence) hybridizes to the PACP sequence of the PAC plasmid. Thus, the artificial oligonucleotide detection probe detects the PAC plasmid but does not detect parvovirus or other biological contaminant sequences.

[0018] In one aspect, a PCR cocktail can be used to determine whether a PAC plasmid giving a false positive result is present in a test biological sample. In this case, if the test sample exhibits a Q-PCR signal positive for the parvovirus-specific oligonucleotide probe and a Q-PCR signal negative for the artificial oligonucleotide detection probe, the test sample is presumed to be free of PAC contamination (i.e., a true positive).

[0019] In one aspect, the target contaminant-specific forward oligonucleotide primer comprises the sequence of SEQ ID NO:1; the target contaminant-specific oligonucleotide detection probe nucleic acid comprises the sequence of SEQ ID NO:2; the artificial oligonucleotide detection probe nucleic acid (i.e., UDP) comprises the sequence of SEQ ID NO:3; and the target contaminant-specific reverse oligonucleotide primer comprises the sequence of SEQ ID NO:4.

[0020] In other aspects, the present invention provides systems and methods for detecting biological contaminants in a test sample. In this case, the test sample is a cell culture, such as an industrial-scale mammalian cell culture for the production of a therapeutic protein. Mammalian cells useful in the practice of the present invention include, but are not limited to, CHO cells, CHO-K1 cells, and EESYR cells (see U.S. Patent No. 7,771,997). The systems and methods include the use of a nucleic acid extraction control (NEC) in addition to the primers, probes, cocktails, and PAC plasmids used as described above. In one aspect, the NEC is M13K07 phage, which is included in the test sample prior to nucleic acid extraction. When nucleic acid extraction is appropriate for the purpose of detecting contaminant DNA or RNA, the NEC nucleic acid (e.g., M13K07 nucleic acid) is detected by Q-PCR in the "spiked" test sample. In certain aspects, the Q-PCR reaction mixture contains a target contaminant-specific forward oligonucleotide primer, a target contaminant-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the unique detection probe (UDP) described above, a target contaminant-specific reverse oligonucleotide primer, an NEC-specific forward oligonucleotide primer, an NEC-specific oligonucleotide detection probe, and an NEC-specific reverse oligonucleotide primer. In one aspect, the test sample is taken from a therapeutic protein-producing cell culture, which has been spiked with an NEC (e.g., M13K07 phage) prior to nucleic acid extraction and subsequent Q-PCR analysis.

[0021] In a specific embodiment, the target contaminant is a rodent parvovirus. In that case, (1) the target contaminant-specific forward oligonucleotide primer is a rodent parvovirus-specific forward oligonucleotide primer, more specifically comprising the sequence of SEQ ID NO:1; (2) the target contaminant-specific oligonucleotide detection probe is a rodent parvovirus-specific oligonucleotide detection probe, more specifically comprising the nucleic acid sequence of SEQ ID NO:2; (3) the artificial oligonucleotide detection probe is a unique detection probe (UDP), more specifically comprising the nucleic acid sequence of SEQ ID NO:3; (4) the target contaminant-specific reverse oligonucleotide primer is a rodent parvovirus-specific reverse oligonucleotide primer, more specifically comprising the nucleic acid sequence of SEQ ID NO:4; (5) the NEC-specific forward oligonucleotide primer is an M13 forward oligonucleotide primer, more specifically comprising the nucleic acid sequence of SEQ ID NO:5; (6) the NEC-specific oligonucleotide detection probe is an M13 detection probe, more specifically comprising the nucleic acid sequence of SEQ ID NO:6; and (7) the NEC-specific reverse oligonucleotide primer is an M13 reverse oligonucleotide primer, more specifically comprising the nucleic acid of SEQ ID NO:8. In one aspect of the method, if an NEC Q-PCR signal is detected (i.e., the signal is within the expected recovery range), it can be inferred that the nucleic acid extraction of the test sample was successful. On the other hand, if no NEC signal is detected (i.e., the signal is outside the expected recovery range), it can be inferred that the nucleic acid extraction of the test sample failed, and any negative Q-PCR target contaminant signal is considered invalid (i.e., false negative). [Invention 1001] A composition comprising an artificial nucleotide sequence, a fluorophore, and a quencher, wherein the artificial nucleotide sequence comprises, at its 3'-end, 5 or fewer consecutive nucleic acids identical to any one of the sequences of SEQ ID NO:9 and 12 - 37. [Invention 1002] The composition of the present invention 1001, wherein the fluorophore is selected from the group of fluorophores having an excitation wavelength in the range of 495 nm or more and 680 nm or less, and an emission wavelength in the range of 515 nm or more and 710 nm or less. [The present invention 1003] The composition of the present invention 1002, wherein the quencher is selected from the group of dyes having a peak of absorption in the range of 430 nm or more and 672 nm or less. [The present invention 1004] The composition of the present invention 1001, wherein the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm. [The present invention 1005] The composition of the present invention 1004, wherein the fluorophore is FAM. [The present invention 1006] The composition of the present invention 1005, wherein the quencher is BHQ-1. [The present invention 1007] The composition of the present invention 1001, wherein the fluorophore is bound to the 5'-end of the artificial nucleotide sequence, and the quencher is bound to the 3'-end of the artificial nucleotide sequence. [The present invention 1008] The artificial nucleotide sequence is The composition of the present invention 1001, which contains the nucleic acid sequence of TIFF2025105635000002.tif4128. [The present invention 1009] a. Rodent parvovirus-specific forward oligonucleotide primer; b. Rodent parvovirus-specific oligonucleotide detection probe; c. Artificial oligonucleotide detection probe; d. Rodent parvovirus-specific reverse oligonucleotide primer; e. M13-specific forward oligonucleotide primer; f. M13-specific oligonucleotide detection probe; and g. M13-specific reverse oligonucleotide primer A composition comprising. [The present invention 1010] The composition of the present invention 1009, wherein the oligonucleotide sequences of a, b, and d each contain the NS-1 sequence of a rodent parvovirus selected from the group consisting of the murine minute virus prototype strain (MVMp), the murine minute virus immunosuppressive strain (MVMi), the murine minute virus Cutter strain (MVMc), murine parvovirus 1b (MPV-1b), murine parvovirus 1a (MPV-1a), murine parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat minute virus, and the Umass strain of rat virus L (RV-Umass). [The present invention 1011] The composition of the present invention 1009, wherein the artificial oligonucleotide detection probe contains a nucleotide sequence having, at its 3'-end, a continuous nucleic acid of 5 or less nucleotides identical to any one of the sequences of SEQ ID NO: 9 and 12 to 37. [The present invention 1012] The composition of the present invention 1010, wherein the oligonucleotide sequences of a, b, and d hybridize to SEQ ID NO: 37. [The present invention 1013] The composition of the present invention 1009, wherein each detection probe of b, c, and f contains a fluorophore and a quencher such that the fluorophores for each detection probe emit light at different wavelengths. [The present invention 1014] The composition of the present invention 1013, wherein the fluorophore of each probe is selected from the group of fluorophores having an excitation wavelength in the range of 495 nm or more and 680 nm or less, and an emission wavelength in the range of 515 nm or more and 710 nm or less. [The present invention 1015] The composition of the present invention 1014, wherein the quencher is selected from the group of dyes having a peak of absorption in the range of 430 nm or more and 672 nm or less. [The present invention 1016] The artificial oligonucleotide detection probe (c) contains the fluorophore FAM and the quencher BHQ-1; the rodent parvovirus-specific oligonucleotide detection probe (b) contains the fluorophore VIC and the minor groove-binding non-fluorescent quencher (MGBNFQ); and the M13-specific oligonucleotide detection probe contains the fluorophore Cy5 and the quencher BHQ2, the composition of the present invention 1013. [The present invention 1017] The rodent parvovirus-specific forward oligonucleotide primer (a) contains the nucleic acid sequence of SEQ ID NO:1; the rodent parvovirus-specific reverse oligonucleotide primer (d) contains the nucleic acid sequence of SEQ ID NO:4; and the rodent parvovirus-specific oligonucleotide detection probe (b) contains the nucleic acid sequence of SEQ ID NO:2, the composition of the present invention 1012. [The present invention 1018] The artificial oligonucleotide detection probe (c) contains the nucleic acid sequence of SEQ ID NO:3, the composition of the present invention 1011. [The present invention 1019] The M13-specific forward oligonucleotide primer (e) contains the nucleic acid sequence of SEQ ID NO:5; the M13-specific oligonucleotide detection probe (f) contains the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer (g) contains the nucleic acid sequence of SEQ ID NO:7, the composition of the present invention 1009. [The present invention 1020] a. A step of mixing a plurality of components to prepare a reaction mixture, wherein the components include (i) a nucleic acid sample derived from a test sample, (ii) an oligonucleotide, and (iii) a DNA polymerase, the said step; b. A step of subjecting the reaction mixture to a polymerase chain reaction (PCR); c. During PCR, a step of monitoring the production of (i) a target amplification polynucleotide (TAP), (ii) a nucleic acid extraction control amplification polynucleotide (NACP), and (iii) a plasmid amplification control polynucleotide (PACP); and d. A step of comparing the production of TAP with the production of NACP and PACP Including the presence of TAP produced during PCR, the presence of NACP, and the absence of PACP, a method for detecting biological contaminants in a test sample, which indicates that the test sample contains biological contaminants and does not contain a positive amplification control plasmid. [Inventive concept 1021] The method of Inventive concept 1020, wherein the oligonucleotide comprises (a) a rodent parvovirus-specific forward oligonucleotide primer, (b) a rodent parvovirus-specific oligonucleotide detection probe, (c) an artificial oligonucleotide detection probe, (d) a rodent parvovirus-specific reverse oligonucleotide primer, (e) an M13-specific forward oligonucleotide primer, (f) an M13-specific oligonucleotide detection probe, and (g) an M13-specific reverse oligonucleotide primer. [Inventive concept 1022] The method of Inventive concept 1020, wherein the test sample is obtained from a mammalian cell culture or a purified fraction thereof. [Inventive concept 1023] The method of Inventive concept 1020, wherein M13K07 phage is added to the test sample. [Inventive concept 1024] The method of Inventive concept 1020, wherein the nucleic acid sample is prepared by subjecting approximately 1 mL of the test sample to lysis, proteolysis, and heat denaturation, followed by mixing the sample with an extraction control sample, and then extracting nucleic acid from the sample. [Inventive concept 1025] The method of Inventive concept 1024, wherein the extraction control sample is M13K07 phage. [Inventive concept 1026] (a) The rodent parvovirus-specific forward oligonucleotide primer contains the nucleic acid sequence of SEQ ID NO:1; (b) The rodent parvovirus-specific oligonucleotide detection probe contains a VIC fluorophore, a minor groove-binding non-fluorescent quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2; (c) The artificial oligonucleotide detection probe contains a FAM fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3; (d) The rodent parvovirus-specific reverse oligonucleotide primer contains the nucleic acid sequence of SEQ ID NO:4; (e) The M13-specific forward oligonucleotide primer contains the nucleic acid sequence of SEQ ID NO:5; (f) The M13-specific oligonucleotide detection probe contains a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer contains the nucleic acid sequence of SEQ ID NO:7, the method of the present invention 1025. [The present invention 1027] The method of the present invention 1020, wherein the component contains uracil-N-glycosylase (UNG). [The present invention 1028] The method of the present invention 1020, which includes a step of incubating the reaction mixture at 50 °C for at least 2 minutes. [The present invention 1029] The PCR step (b) is (i) A step of incubating the reaction mixture at 95 °C for 2 minutes; followed by (ii) (1) 8 cycles of denaturation at 95 °C for 10 seconds, followed by (2) annealing for 30 seconds, wherein the annealing temperature of the first cycle of the 8 cycles is 70 °C, the annealing temperature decreases by 1 °C per cycle, and the annealing temperature of the last cycle of the 8 cycles is 62 °C, the said 8 cycles; followed by (iii)(1)A DNA amplification of at least 40 cycles including a denaturation step at 95°C for 10 seconds, followed by (2) an annealing step at 62°C for 30 seconds, wherein the rate of temperature change from the denaturation temperature to the annealing temperature is about 4.4°C per second, and from the annealing temperature to the denaturation temperature is about 2.2°C per second, said DNA amplification The method of the present invention 1028, comprising [The present invention 1030] (a) Monitoring the production of TAP by measuring fluorescence at 533 - 580 nm in each amplification cycle; (b) Monitoring the production of NACP by measuring fluorescence at 618 - 660 nm in each amplification cycle; and (c) Monitoring the production of PACP by measuring fluorescence at 465 - 510 nm in each amplification cycle, the method of the present invention 1026. [The present invention 1031] The method of the present invention 1020, comprising comparing the results obtained from the method performed on a test sample with an external positive control and an external negative control, and if the negative control or the positive control fails, the results obtained from the method performed on the test sample are rejected. [The present invention 1032] The external positive control is a. A step of mixing a plurality of positive control components to prepare a positive control mixture, wherein the positive control components include (i) a positive amplification control (PAC) plasmid, and (ii) a positive control oligonucleotide cocktail, and (iii) do not include a test sample, said step; b. Subjecting the positive control mixture to a polymerase chain reaction (PCR) of the positive control; and c. During the PCR, a step of monitoring the production of (i) a target amplification polynucleotide (TAP), (ii) a nucleic acid extraction control amplification polynucleotide (NACP), and (iii) a plasmid amplification control polynucleotide (PACP) The method of the present invention 1031, which includes the presence of TAP, NACP, and PACP produced during PCR, indicates that PCR is functioning properly, and the absence of any one or more of TAP, NACP, or PACP indicates that the positive control has failed. [The present invention 1033] The method of the present invention 1032, wherein the PAC plasmid contains (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid. [The present invention 1034] (i) The parvovirus nucleic acid sequence contains the sequence of SEQ ID NO:37; (ii) the M13K07 nucleic acid sequence contains the sequence of SEQ ID NO:8; and (iii) the artificial nucleic acid sequence unique to the plasmid contains the sequence of SEQ ID NO:10. The method of the present invention 1033. [The present invention 1035] The method of the present invention 1034, wherein the PAC plasmid contains the nucleic acid sequence of SEQ ID NO:11. [The present invention 1036] The positive control oligonucleotide cocktail A rodent parvovirus-specific forward oligonucleotide primer containing the nucleic acid sequence of SEQ ID NO:1; A rodent parvovirus-specific oligonucleotide detection probe containing a VIC fluorophore, an MGBNFQ quencher, and the nucleic acid sequence of SEQ ID NO:2; An artificial oligonucleotide detection probe containing a FAM fluorophore, a BHQ quencher, and the nucleic acid sequence of SEQ ID NO:3; A rodent parvovirus-specific reverse oligonucleotide primer containing the nucleic acid sequence of SEQ ID NO:4; An M13-specific forward oligonucleotide primer containing the nucleic acid sequence of SEQ ID NO:5; An M13-specific oligonucleotide detection probe containing a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and An M13-specific reverse oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:7 The method of the present invention 1033 comprising the same [The present invention 1037] Positive control PCR comprises a. incubating the positive control mixture at 95°C for 2 minutes; followed by b. eight cycles of (i) denaturation at 95°C for 10 seconds, followed by (ii) annealing for 30 seconds, where the first annealing temperature of the eight cycles is 70°C, the annealing temperature decreases by 1°C per cycle, and the last annealing temperature of the eight cycles is 62°C; and followed by c. DNA amplification for 40 cycles comprising (i) a denaturation step at 95°C for 10 seconds, followed by (ii) an annealing step at 62°C for 30 seconds The method of the present invention 1036 comprising the same [The present invention 1038] The production of TAP in positive control PCR is monitored by measuring fluorescence at 533 - 580 nm in each amplification cycle, the production of NACP in positive control PCR is monitored by measuring fluorescence at 618 - 660 nm in each amplification cycle; and the production of PACP in positive control PCR is monitored by measuring fluorescence at 465 - 510 nm in each amplification cycle, the method of the present invention 1036 [The present invention 1039] a. Positive amplification control (PAC) plasmid b. Rodent parvovirus-specific forward oligonucleotide primer c. Rodent parvovirus-specific oligonucleotide detection probe d. Artificial oligonucleotide detection probe e. Rodent parvovirus-specific reverse oligonucleotide primer f. M13-specific forward oligonucleotide primer g. M13-specific oligonucleotide detection probe h. M13-specific reverse oligonucleotide primer; and i. Buffer A composition comprising [Inventive concept 1040] The composition of Inventive concept 1039, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid. [Inventive concept 1041] The composition of Inventive concept 1040, wherein the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO:37, the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO:8, and the unique nucleic acid sequence comprises the sequence of SEQ ID NO:10. [Inventive concept 1042] The composition of Inventive concept 1041, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:11. [Inventive concept 1043] The rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1; The rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2; The artificial oligonucleotide detection probe comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3; The rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4; The M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; The M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and The M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7, The composition of Inventive concept 1042.

Modes for Carrying Out the Invention

[0022] Detailed Description of the Invention Before describing the present invention, it should be understood that the present invention is not necessarily limited to the specific methods and experimental conditions described, because such methods and conditions can vary. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the technical terms used herein are for the purpose of merely describing specific embodiments and are not intended to be limiting.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in relation to a particular recited numerical value means that the value can vary by up to 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, as well as all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0024] Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but the preferred methods and materials are described hereinafter. All publications mentioned herein are incorporated by reference in their entirety. Other aspects will become apparent from the following detailed description.

[0025] The following detailed description is presented to enable a person skilled in the art to make and use the invention as described herein.

[0026] The present invention relates to improved materials and methods for detecting any biological contaminants in any cell culture producing a therapeutic protein. Specifically, the present invention relates to materials and methods for quantitative polymerase chain reaction (Q-PCR) that employ elements or steps of a positive amplification control that are readily detectable to exclude false positives.

[0027] PCR and Quantitative PCR As used herein, the term "polymerase chain reaction" ("PCR") refers to a method for making copies of nucleic acids (e.g., DNA) by employing multiple cycles of denaturation (separation of template DNA strands), annealing (hybridization of single-stranded oligonucleotides to single-stranded template DNA strands), and DNA synthesis (catalyzed by a DNA polymerase to synthesize a new DNA strand primed from the 3' end of the hybridized oligonucleotide using the template DNA strand as a template). To effect amplification, at least two different oligonucleotide primers (commonly known simply as "primers") are used in the PCR reaction. One primer, commonly called the forward primer, hybridizes to the antisense strand of the template DNA and forms the 5' end of the newly synthesized sense strand. The other primer, commonly called the reverse primer, hybridizes to the sense strand of the template DNA and forms the 5' end of the newly synthesized antisense strand. In each cycle, each template strand is copied to form a new double-stranded DNA molecule, also known as an "amplicon". Thus, an unlimited amount of oligonucleotide primers, DNA polymerase (i.e., Taq polymerase or other thermostable DNA polymerases; see Innis et al., DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA, 85(24) Proc Natl Acad Sci U S A. 9436-40 (1988)), and nucleotide triphosphates are used to double the number of DNA molecules (templates and amplicons) in each cycle. PCR is described in U.S. Patent No. 4,683,202 (issued July 28, 1987). See also PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

[0028] As used herein, the term "cycle" means one round of (1) melting of a DNA strand called "denaturation", followed by (2) hybridization of an oligonucleotide primer to the resulting single-stranded DNA according to the law of base pairing, a process called "annealing", and (3) polymerization of a new DNA strand starting at the 3' end of the oligonucleotide primer and proceeding in the 5' to 3' direction, a process called "amplification" or "extension". Generally, polymerization uses a DNA polymerase enzyme such as Taq polymerase to catalyze the formation of phosphodiester bonds between adjacent deoxynucleotide triphosphates ("dNTPs") along the exposed single-stranded template DNA according to the law of base pairing. Denaturation, annealing, and amplification are carried out at specific temperatures, based in part on the GC content of the DNA template and oligonucleotide primer, as well as the length of the DNA strand to be copied. The temperatures of denaturation and annealing, as well as the ionic strength of the reaction buffer, control the stringency of hybridization and the fidelity of DNA copying.

[0029] "Quantitative PCR" or "qPCR" or "Q-PCR" (also known as "real-time PCR") is a type of PCR that enables the monitoring of amplicon formation during the PCR cycle process. Using Q-PCR, the amount of a specific template DNA in a sample can be quantified. Q-PCR incorporates at least one oligonucleotide detection probe into the reaction mixture in addition to a forward oligonucleotide primer and a reverse oligonucleotide primer. A detection probe is a single-stranded oligonucleotide that hybridizes to the sense or antisense strand of the target template DNA somewhere between the forward primer binding site and the reverse primer binding site. During the annealing stage, the oligonucleotide detection probe anneals to the single-stranded template. As polymerization occurs, the probe is cleaved and degraded by the 5' nuclease activity of DNA polymerase. Thus, as amplification of a specific template sequence occurs, the detection probe is degraded at an exponential rate.

[0030] Q-PCR oligonucleotide detection probes are generally constructed with a conjugated fluorophore (also known as a reporter fluorescent dye or simply "reporter") and a conjugated quencher. In most cases, the fluorophore is conjugated to or near the 5'-end of the oligonucleotide, and the quencher is conjugated to or near the 3'-end of the oligonucleotide. However, any executable configuration can be used in the practice of the present invention. When the oligonucleotide detection probe is intact, the fluorophore and the quencher are in proximity such that the quencher absorbs the light emitted by the excited fluorophore, thereby significantly reducing detectable fluorophore emission. When the oligonucleotide detection probe is cleaved or degraded, the fluorophore and the quencher are released and consequently spatially separated. The quencher is no longer close enough to quench the fluorophore emission. As specific amplicons are formed, more oligonucleotide detection probes are cleaved, more fluorophores and quenchers are released, and thus more fluorophore / quencher pairs are separated, increasing the amplitude of the fluorescence emission. In other words, the increasing fluorophore emission signal correlates with the amount of specific target DNA in the sample. For an overview of Q-PCR, see Ian M. Mackay et al., Survey and Summary: Real-Time PCR in Virology, 30(6) Nucleic Acids Research 1292-1305 (2002).

[0031] Fluorescence quenching can occur by direct contact between a reporter and a quencher (also known as static quenching), or by fluorescence resonance energy transfer (FRET) between the reporter and the quencher if both are within each other's Förster radius. A specific fluorophore can be excited by light of one or more specific wavelengths, or a range of wavelengths with a maximum value, which is called the excitation wavelength. After the fluorophore is excited, it returns to the ground state and emits light at a wavelength longer than the excitation wavelength, which is called the emission wavelength. During FRET, a second fluorophore, dye, lanthanide series molecule, etc., having an absorption spectrum that matches or overlaps the emission spectrum of the fluorophore, absorbs the light emitted by the excited fluorophore within the Förster radius, thereby quenching or reducing the wavelength of the fluorophore emission. Contact or static quenching occurs when the reporter and the quencher form a triplet complex in the ground state of the fluorophore. This triplet complex is non-fluorescent, i.e., essentially non-excitable, and thus does not emit light at the expected emission wavelength.

[0032] For an overview of static quenching and FRET, see Salvatore A. E. Marras et al., Efficiencies of Fluorescence Resonance Energy Transfer and Contact-Mediated Quenching in Oligonucleotide Probes, 30(21) Nucleic Acids Research e122, pp.1-8 (2002). Marras et al. also consider the selection of reporter / quencher pairs for use in the application of Q-PCR.

[0033] Nucleic acid The terms "polynucleotide", "oligonucleotide", "probe", "primer" or "nucleotide primer" or "oligonucleotide primer", "template" or "template nucleic acid" or "template DNA" are used herein according to their ordinary meanings to those skilled in the art of molecular biology. For a detailed explanation of each, see, for example, PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

[0034] As used herein, "amplicon" refers to a DNA product generated by amplification of a template nucleic acid sequence by PCR. As PCR proceeds and the template is amplified, the newly formed DNA amplicons serve as templates for subsequent rounds of DNA synthesis.

[0035] Cell culture The present invention is directed to an improved Q-PCR method for detecting biological contaminants in cell cultures. Cell cultures are often used to produce complex biological molecules for therapeutic use, such as antibodies, capture molecules, and Fc fusion proteins. These cultures must remain free of biological contaminants. Detection of contaminants is important to determine whether a particular batch should be discarded or subjected to remediation.

[0036] Cell cultures include a culture medium and cells typically derived from a single cell line. In this case, the cell line includes cells that can produce a biotherapeutic protein. Examples of cell lines routinely used for producing proteinaceous biopharmaceuticals include, among others, primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. One or more of several specific CHO cell variants, such as the Chinese hamster ovary (CHO) cell line or the CHO-K1 cell line, are optimized for large-scale protein production. The EESYR® cell line is a specialized CHO cell line optimized for enhanced production of a protein of interest. For a detailed description of the EESYR® cells, see U.S. Patent No. 7,771,997 (issued August 10, 2010).

[0037] "Cell culture" or "culture" means the growth and propagation of cells outside of a multi-cellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. For example, Animal cell culture: A Practical Approach(See D. Rickwood, ed., 1992). Mammalian cells can be cultured either in suspension or attached to a solid substrate. Regardless of the presence or absence of microcarriers, and in batch, fed-batch, continuous, semi-continuous, or perfusion modes, fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors are available for mammalian cell culture. A cell culture medium or a concentrated feed medium can be added continuously or at intervals to the culture during cultivation (i.e., batch-fed). For example, the culture can be fed once a day, every other day, every two days, or when the concentration of a specific medium component being monitored directly or indirectly deviates from the desired range.

[0038] Animal cells such as CHO cells or EESYR® cells can be cultured in small-scale cultures, such as in a 125 ml container with about 25 ml of medium, a 250 ml container with about 50 - 100 ml of medium, a 500 ml container with about 100 - 200 ml of medium, etc. Alternatively, the culture can be large-scale, such as in a 1000 ml container with about 300 - 1000 ml of medium, a 3000 ml container with about 500 ml - 3000 ml of medium, an 8000 ml container with about 2000 ml - 8000 ml of medium, and a 15000 ml container with about 4000 ml - 15000 ml of medium. Cultivation for production can contain 10,000 L of medium or more. Large-scale cell culture, such as for the clinical production of proteinaceous therapeutic substances, is typically maintained for several days or weeks while the cells produce the desired protein. During this period, samples of the culture can be taken and tested for the presence of biological contaminants.

[0039] Production of therapeutic proteins Cell cultures that are monitored for biological contamination may be used to produce proteins or other biological molecules of interest, such as therapeutically effective antibodies or other biopharmaceutical raw materials. Protein products (proteins of interest) can be, inter alia, antibodies, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies, or tetra-bodies, Fab fragments or F(ab')2 fragments, IgA antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. In one aspect, the antibody is an IgG1 antibody. In one aspect, the antibody is an IgG2 antibody. In one aspect, the antibody is an IgG4 antibody.

[0040] The protein of interest can be a recombinant protein (e.g., an Fc fusion protein) containing an Fc portion and another domain. The Fc fusion protein can be a receptor Fc fusion protein containing one or more of one or more extracellular domains of a receptor bound to the Fc portion. In some cases, the Fc portion includes a hinge region followed by the CH2 and CH3 domains of IgG. In some cases, the receptor Fc fusion protein contains two or more individual receptor chains that bind to either a single ligand or multiple ligands. For example, the Fc fusion protein can be a capturer such as an IL-1 capturer (e.g., rilonacept, which contains an IL-1RAcP ligand-binding region fused to the extracellular region of Il-1R1 fused to the Fc domain of hIgG1; see U.S. Patent No. 6,927,004), or a VEGF capturer (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc domain of hIgG1; see U.S. Patents Nos. 7,087,411 and 7,279,159).

[0041] The present invention is not limited to any particular type of cell or cell line for protein production. Examples of cell types suitable for protein production include mammalian cells such as CHO-derived cells like EESYR® (registered trademark), insect cells, avian cells, bacterial cells, and yeast cells. The cells can be stem cells, or recombinant cells transformed with vectors for recombinant gene expression, or cells transfected with a virus to produce viral products. The cells can contain a recombinant heterologous polynucleotide construct encoding the protein of interest. This construct can be episomal (such as an extrachromosomal plasmid or fragment) or physically integrated into the genome of the cell. The cells can also produce the protein of interest without encoding the protein on a heterologous polypeptide construct. In other words, cells such as B cells that produce antibodies can naturally encode the protein of interest. Methods and vectors for genetically modifying a cell or cell line to express a protein of interest are well known to those skilled in the art. For example, various techniques are described in Current Protocols in Molecular Biology , Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly revisions); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture , 1990, pp.15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial suppliers.

[0042] The cells may also be primary cells such as chicken embryo cells, or primary cell lines. Examples of useful cells include BSC cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, chicken embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293 cells, Per.C6 cells, and CHO cells. In various embodiments, the cell line is a CHO cell derivative such as CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, CHO lec mutant strain, or EESYR™ cell line.

[0043] In one particular scenario, the cells are CHO cell derivatives such as EESYR™ cells that heterologously (heterogeneously) express a protein. The protein includes an immunoglobulin heavy chain region such as the CH1, CH2, or CH3 region. In one embodiment, the protein includes the human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein includes the human or rodent immunoglobulin CH1, CH2, and CH3 regions. In one embodiment, the protein includes the hinge region, as well as the CH1, CH2, and CH3 regions. In a specific embodiment, the protein includes an immunoglobulin heavy chain variable domain. In a specific embodiment, the protein includes an immunoglobulin light chain variable domain. In a specific embodiment, the protein includes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In a specific embodiment, the protein is an antibody such as a human antibody, a rodent antibody, or a chimeric human / rodent antibody (e.g., human / mouse, human / rat, or human hamster).

[0044] The protein production phase of cell culture can be carried out in cultures of any scale, from individual flasks and shake flasks or wave bags, to 1-liter bioreactors and up to large-scale industrial bioreactors. Large-scale processes can be carried out at volumes of about 100 liters to 20,000 liters or more. Protein production may be controlled using one or more of several means, such as temperature shift or chemical induction. The cell growth phase can occur at a higher temperature than the production phase in which the protein is expressed and / or secreted. For example, the growth phase can occur at a first temperature of about 35°C to 38°C, and the production phase can occur at a second temperature of about 29°C to 37°C, optionally about 30°C to 36°C or about 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylenebisacetamide (HMBA), can be added simultaneously with, before, or after the temperature shift. If the inducers are added after the temperature shift, they can be added 1 hour to 5 days after the temperature shift, such as 1 to 2 days after the temperature shift. The production cell culture can be carried out as a continuous feed culture system such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec; 17(6):1032-41), or according to a fed-batch (batch-fed) process (Huang, 2010).

[0045] Therapeutic protein products As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably throughout and refer to a molecule containing two or more amino acid residues joined together by peptide bonds. Peptides, polypeptides, and proteins can also include modifications such as glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Peptides, polypeptides, and proteins can be of scientific or commercial interest, such as protein-based drugs. Peptides, polypeptides, and proteins include, among others, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

[0046] As used herein, an "antibody" refers to an immunoglobulin molecule consisting of two heavy (H) chains and two light (L) chains, which are four polypeptide chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes references to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, made, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. The term "antibody" also includes bispecific antibodies, which are heterotetrameric immunoglobulins that can bind to more than one epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527, which is incorporated herein by reference.

[0047] The term "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) the Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, consisting of the VH and CH1 domains; (iv) the Fv fragment, consisting of the VL and VH domains of a single arm of an antibody; (v) the dAb fragment, consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) isolated CDRs; and (vii) the scFv, consisting of two domains of the Fv fragment, VL and VH, joined by a synthetic linker to form a single polypeptide chain in which the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also included under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0048] An antibody or antigen-binding portion thereof can be part of a larger immune adhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immune adhesion molecules include the use of the streptavidin core region to create a tetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), as well as the use of cysteine residues, a marker peptide, and a C-terminal polyhistidine tag to create a bivalent and biotinylated scFv molecule (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions such as Fab and F(ab')2 fragments can be prepared from the whole antibody using conventional techniques such as papain or pepsin digestion of the whole antibody. Furthermore, antibodies, antibody portions, and immune adhesion molecules can be obtained using standard recombinant DNA techniques (see Sambrook et al., 1989).

[0049] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention can contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutation in vivo) in, for example, the CDRs, particularly CDR3. However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into a human framework sequence.

[0050] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, made, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal that is transgenic with respect to a human immunoglobulin gene (e.g., a mouse; see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, made, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic with respect to human Ig sequences is used, in vivo chromosomal mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody, while derived from and related to human germline VH and VL sequences, may be sequences that do not naturally occur within the human antibody germline repertoire in vivo.

[0051] "Fc fusion protein" includes some or all of two or more types of proteins, one of which is the Fc portion of an immunoglobulin molecule and which are not found together in nature other than as a fusion protein. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of an antibody-derived polypeptide (including the Fc domain) is described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", Current Protocols in Immunology, Suppl. 4, pp.10.19.1-10.19.11, 1992. "Receptor Fc fusion protein" includes one or more extracellular domains of a receptor bound to the Fc portion, which in some embodiments includes the hinge region followed by the CH2 and CH3 domains of the immunoglobulin. In some embodiments, the Fc fusion protein contains two or more individual receptor chains that bind to one or more ligands. For example, the Fc fusion protein can be a capturer such as an IL-1 capturer (e.g., rilonacept, which contains an IL-1RAcP ligand-binding region fused to the extracellular region of IL-1R1 fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004), or a VEGF capturer (e.g., aflibercept, which contains an Ig domain 2 of VEGF receptor Flt1 fused to Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patents Nos. 7,087,411 and 7,279,159).

[0052] Biological contaminants As used herein, the term "biological contaminant" means any unwanted, undesirable, harmful, or potentially harmful biological entity. Such entities include, among others, prions (the etiological cause of bovine / spongiform encephalopathy), virions, viruses, mycoplasmas, other bacteria, adventitious mammalian cells, DNA, RNA, transposons, other transposable elements, yeast, other fungi, algae, protozoa, and other exogenous and endogenous agents. Particular concern has been raised with respect to adventitious viruses associated with cell or media or raw materials used in the production process of biopharmaceuticals using rodent cells (such as CHO cells and derivatives of CHO cells). Contamination of cell culture bulk processing materials and the resulting drug products poses both a direct risk to patients as well as an indirect risk that can impede the supply of medicine.

[0053] An exemplary list of exogenous and / or endogenous agents that can infect CHO cell cultures includes single-stranded (-) RNA viruses such as Cache Valley virus, influenza A / B virus, parainfluenza 1 / 2 / 3, simian virus 5, mumps virus, bovine respiratory syncytial virus, and vesicular stomatitis virus; single-stranded (+) RNA viruses such as bovine coronavirus, Bes virus 2117, encephalomyocarditis virus, coxsackievirus B-3, Semliki Forest virus, and Sindbis virus; double-stranded RNA viruses such as bluetongue virus, epidemic hemorrhagic disease virus, and reovirus 1 / 2 / 3; single-stranded DNA viruses such as porcine circovirus 1 and parvoviruses of particular concern including murine minute virus (also known as mouse minute virus); and double-stranded DNA viruses such as adenovirus and pseudorabies virus. Among these potential exogenous agents, four viruses predominate in bulk harvest samples from CHO cell cultures across various manufacturers. Those viruses are reovirus type 2, Cache Valley virus, epidemic hemorrhagic disease virus, and rodent parvovirus murine minute virus. For a detailed overview of adventitious virus contamination of CHO cell cultures, see Andreas Berting et al., Virus Susceptibility of Chinese Hamster Ovary (CHO) Cells and Detection of Viral Contaminations by Adventitious Agent Testing, 106(4) Biotechnology and Bioengineering 598-607 (2010), and Andrew Kerr & Raymond Nims, Adventitious Viruses Detected in Biopharmaceutical Bulk Harvest Samples over a 10 Year Period, 64(5) PDA Journal of Pharmaceutical Science & Technology 481-485 (2010).

[0054] Exogenous agent tests are roughly classified into two categories. The first is the classical virological method using in vitro virus assays. Here, the test sample is applied to an indicator cell line, the cells are incubated and passaged for 14 to 28 days, and then evaluation items such as the cytopathic effect or hemagglutination reaction are measured (Berting, 2010). The second is the PCR-based assay, which measures the presence of nucleic acids associated with exogenous or endogenous agents in real time. See, for example, Zhan et al., Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance during the Purification of Mammalian Cell Substrate Derived Biotherapeutics, 30(4) Biologicals 259-270 (2002).

[0055] Mouse minute virus (also known as MMV, murine minute virus, or MVM) presents special problems for the manufacture of biopharmaceuticals. Both the US Food and Drug Administration (FDA) and the European Medicines Agency require specific testing for MVM. This virus is a member of the Parvoviridae family (parvoviruses) and is commonly found in mice. It is excreted in urine and feces, is hardy, and persists in the environment. It can easily be introduced into the biopharmaceutical manufacturing process. See Moody et al., Mouse Minute Virus (MMV) Contamination - A Case Study: Detection, Root Cause Determination, and Corrective Actions, 65(6) PDA Journal of Pharmaceutical Science and Technology 580-288 (2011). Other rodent parvoviruses can have a negative impact on cell culture-based biopharmaceutical production. In addition to the prototype MVM strains, those rodent parvoviruses include, among others, the MVM immunosuppressive and Cutter strains, mouse parvovirus 1a (MPV-1a), MPV-1b, MPV-1c, hamster parvovirus, Toolan's parvovirus (parvovirus H-1), Kilham rat virus, rat parvovirus 1a, rat minute virus, and the Umass strain of rat virus L. See S.F. Cotmore & P. Tattersal, The Autonomously Replicating Parvoviruses of Vertebrates, 33 Advances in Virus Research 91-174 (1987), and Jacoby et al., Rodent Parvovirus Infections, 46(4) Lab Anim Sci. 370-80 (1996).

[0056] These parvoviruses share a conserved nucleic acid sequence called NS-1 (NS1) that encodes a large non-structural protein involved in amplification of the viral genome. The conserved NS1 nucleotide sequence from MVM is depicted in SEQ ID NO:9. Nucleotides 875 to 956 of that sequence are at least 97% conserved across diverse rodent parvovirus NS1 sequences and thus serve as good target sequences for PCR-based assays of exogenous and endogenous agents. PCR-based assays for rodent parvoviruses (as well as other exogenous and endogenous agents) can be performed on raw materials, pre-harvest culture media at various points along the bulk processing purification of biologic molecules, as well as at the formulation and packaging stages. Detection of contaminants can necessitate remediation steps such as disposal of contaminated materials, replacement of raw materials, and decontamination of facilities.

[0057] In addition to testing for exogenous agents, endogenous agents, and other biological contaminants that enable corrective and preventive actions (CAPA) during manufacturing, special manufacturing and bulk processing steps (i.e., unit operations) may be employed to exclude, reduce, or inactivate viral contaminants. Chemical inactivation, virus retention filtration, and chromatography have all been shown to be effective in reducing herpesvirus, retrovirus, and parvovirus in harvested or partially purified cell culture fluids. The most frequently used chemical inactivation step is low pH treatment, which some in the art believe is due to the denaturation of viral envelope proteins. Protein A, hydroxyapatite, cation exchange, and anion exchange chromatography steps have all been shown to remove virus to some extent. See, for example, Miesegaes et al., Analysis of Viral Clearance Unit Operations for Monoclonal Antibodies, 106(2) Biotechnology and Bioengineering 238-246 (2010), and Liu et al., Recovery and Purification Process Development for Monoclonal Antibody production, 2(5) mAbs 480-499 (2010).

[0058] Q-PCR test: Positive and negative controls Tests for biological contaminants should be properly managed to ensure accuracy, reliability, and credibility. As used herein, a "negative control" includes most or all of the experimental reagents and conditions, but does not include the test sample. The test sample can be replaced with a buffer or mock culture medium known to not contain the biological contaminant of interest. Further, as used herein, a "negative control" should result in a negative result with respect to the biological contaminant. If the negative control results in a positive biological contaminant result, a skilled technician or scientist can infer that a positive result from a parallel test sample may not accurately reflect whether the test sample contains a biological contaminant.

[0059] One or more "positive controls" as used herein are used to evaluate whether the experimental conditions function properly to detect biological contaminants. The positive control is used at any step along the experimental process to ensure that each step is functioning and to determine at which step the process fails.

[0060] In some embodiments, a positive control is used in the nucleic acid extraction step to evaluate whether the intended extraction of any biological contaminant nucleic acid was efficient enough to detect the biological contaminant. Such a positive control is referred to as a "nucleic acid extraction control" or "NEC". In some cases, the NEC is selected to mimic the target biological contaminant from a protein-nucleic acid structure perspective. If the NEC is extracted in a manner sufficient to be detected, the technician can infer that the target biological nucleic acid was also extracted in a manner sufficient to be detected. In one embodiment, the NEC is a single-stranded DNA phage that is not entirely different from parvovirus.

[0061] In a specific embodiment, NEC is M13 bacteriophage, which is composed of circular single-stranded DNA of approximately 6,407 nucleotides. In a more specific embodiment, NEC is the M13K07 strain, which is a commonly available molecular biology reagent used for cloning and other laboratory purposes. See van Wezenbeek et al., Nucleotide Sequence of the Filamentous Bacteriophage M13 DNA Genome: Comparison with Phage fd, 11(1-2) Gene 129-148 (1980). The nucleotide sequence of the M13K07 phage is depicted in SEQ ID NO:8. Although the M13 bacteriophage or the M13K07 bacteriophage strain can be used as NEC in a specific embodiment, the present invention is in no way limited to using that particular reagent as NEC. One of ordinary skill in the art can substitute another reagent as NEC in the practice of the present invention without departing from the scope of the present invention (e.g., MS2 phage for reverse transcriptase PCR assays; see Kothapalli et al., Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template, 109(2) J. Virol. Methods 203-207 (2003)).

[0062] In some aspects of the present invention, a positive control is used in the PCR amplification step to evaluate whether the PCR reagents (including primers) and the PCR steps are sufficient / have been sufficient to detect biological contaminant template nucleic acids. Such a positive control is referred to as a "plasmid amplification control" or "PAC". In some cases, the PAC is selected or designed to match the target biological contaminant nucleic acid sequence and precisely match the forward and reverse oligonucleotide primers of the test sample. In a specific aspect, the PAC further contains a "unique sequence" not found in the target biological contaminant nucleic acid. In a more specific aspect, the unique sequence is not found in nature. The "unique" nucleic acid polymer is designed to specifically anneal to this unique sequence and be incorporated into the amplicon copies of the unique sequence during PCR. The "unique" nucleic acid polymer is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions employed in the performance of the assay. In one aspect, the "unique" nucleic acid polymer contains 17 - 20 nucleotides, where no more than 7 - 10 consecutive internal nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence. In one aspect, the "unique" nucleotide polymer contains 17 - 20 nucleotides, where no more than 7 - 10 consecutive nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence as set forth in SEQ ID NO:9 and 12 - 37.

[0063] This unique sequence enables a skilled artisan to distinguish between the true target biological contaminant sequence and cross - contamination of the test Q - PCR reaction by the PAC plasmid.

[0064] In one aspect, to manage PCR amplification, a PAC (Plasmid as a Control, also known as PAC plasmid) is included in identical but separate Q-PCR reactions. To accurately reflect the amplification of the actual target biological contaminant nucleic acid, the PAC reaction uses oligonucleotide primers and probes that are exactly the same as those used in the Q-PCR reaction of the test sample. The Q-PCR reaction of the test sample and the separate Q-PCR reaction of the PAC plasmid include a target detection probe and a unique sequence probe. When the PAC reaction is functioning properly, it is expected to yield a positive target signal and a positive unique sequence signal. Obtaining a positive target signal and a positive unique sequence signal in the PAC plasmid-containing reaction indicates that whenever the target biological contaminant nucleic acid is present, the reagents and conditions of the test sample Q-PCR are working properly to produce a positive target signal in the test sample.

[0065] Accordingly, the PAC serves as a control for proper PCR amplification. If a negative target signal is obtained in the test sample but the PAC shows a positive target signal, a skilled technician can infer that no detectable target contaminant DNA is present in the test sample. Conversely, if a positive target sequence signal and a positive unique sequence signal are obtained in the test sample, the technician can infer that the test sample has been cross-contaminated by the PAC plasmid and thus the positive target sequence signal may be a false positive.

[0066] Detailed description of several embodiments In some aspects, an improved positive control system (i.e., a composition and method) for detecting biological contaminants by use of the polymerase chain reaction, more specifically Q-PCR, is provided.

[0067] In one aspect, the present invention provides a unique sequence probe (USP) for detecting a positive growth control plasmid (PAC plasmid). The USP is a unique artificial plasmid-specific sequence ( u niquea rtificial p lasmid - specific s An artificial nucleotide sequence capable of hybridizing to a (alias, unique sequence or PACP - unique sequence or UAPS), a fluorophore, and a quencher. In a specific embodiment, the artificial nucleotide sequence capable of hybridizing to UAPS is the nucleic acid sequence of SEQ ID NO:3 including TIFF2025105635000003.tif4128, which is antisense to the UAPS sense strand sequence (e.g., that is TIFF2025105635000004.tif4128).

[0068] As described above, the Q-PCR detection probe contains a fluorophore and a quencher. Quenching can be by contact quenching or by FRET, depending on the fluorophore / quencher pair. In this case, the fluorophore and the quencher are covalently attached to the USP oligonucleotide. In one embodiment, the fluorophore has an excitation wavelength of 495 nm to 680 nm and an emission wavelength of 515 nm to 710 nm. In some embodiments, the fluorophores have an excitation wavelength of 495 nm, 538 nm, or 646 nm, respectively, and an emission wavelength of 520 nm, 554 nm, or 669 nm. In some embodiments, the quencher is a dye having an absorption peak of 430 nm to 672 nm. Examples of useful quenchers include DDQ®-I, Dabcyl, Eclipse®, Iowa Black FQ®, BHQ®-1, QSY®-7, BHQ®-2, DDQ®-II, Iowa Black RQ®, QSY®-21, and DHQ®-3. In a specific embodiment, the fluorophore is fluorescein amidite (FAM; described in U.S. Patent No. 5,583,236, issued December 10, 1996) having an absorption maximum at about 495 nm and an emission maximum at about 520 nm, and the quencher is Black Hole Quencher®-1 (BHQ®-1, Biosearch Technologies, Inc., Petaluma, CA) that absorbs at 480 nm to 580 nm. BHQ®-1 quenches by both FRET and contact quenching. Often, but not always, the quencher is attached to the 3'-hydroxyl group of the oligonucleotide via an ether bond, and the fluorophore is attached to the 5'-phosphate group of the oligonucleotide via an ester bond.

[0069] In another aspect, the present invention provides a mixture of Q-PCR reagents comprising multiple types of oligonucleotides and probes useful for detecting biological contaminants in cell cultures, raw materials, partially purified and purified biological molecules, etc. In one embodiment, the biological contaminant is a DNA virus, more specifically a parvovirus, and even more specifically a rodent parvovirus such as MVM. The parvovirus contains an NS1 gene having a nucleic acid sequence that is at least 88% identical to any of the sequences listed in Table 1. In another embodiment, the parvovirus contains an NS1 gene having a nucleic acid sequence that is at least 97% identical to the sequence shown in SEQ ID NO:9 (i.e., the mouse minute virus (MVM) NS1 gene). In yet another embodiment, the parvovirus contains an NS1 gene comprising the consensus sequence of SEQ ID NO:37.

[0070] (Table 1) Parvovirus NS1 Sequences TIFF2025105635000005.tif110170

[0071] In a specific embodiment, the mixture contains (1) a rodent parvovirus-specific forward oligonucleotide primer; (2) a rodent parvovirus-specific oligonucleotide detection probe; (3) an artificial oligonucleotide detection probe such as USP; and (4) a rodent parvovirus-specific reverse oligonucleotide primer. Such a mixture can be used in both test samples and positive control samples. In a more specific embodiment, the oligonucleotide primer and the parvovirus-specific oligonucleotide detection probe hybridize to an NS1 sequence such as the NS1 sequence shown in SEQ ID NO:9, for example. In a more specific embodiment, (1) the forward primer comprises the sequence of SEQ ID NO:1; (2) the parvovirus-specific oligonucleotide detection probe comprises the sequence of SEQ ID NO:2; (3) the artificial probe is USP and comprises the sequence of SEQ ID NO:3; and (4) the reverse primer comprises the sequence of SEQ ID NO:4.

[0072] As described above, the USP contains a fluorophore and a quencher, and thus this sequence, i.e., the amplicon containing the PAC DNA, can be detected in real time as Q-PCR progresses. In a similar manner, the parvovirus-specific detection probe contains an oligonucleotide with a covalently attached fluorophore and quencher. In fact, to distinguish a true parvovirus positive signal from a false positive caused by contamination of the test sample with the PAC plasmid or other PACPs (e.g., PACP-contaminated amplicons), the emission wavelength of the fluorophore of the parvovirus detection probe should be different from that of the USP. Thus, in embodiments where the USP fluorophore is FAM, the fluorophore attached to the parvovirus detection probe oligonucleotide should have an emission wavelength other than about 520 nm. In a specific embodiment, the fluorophore attached to the rodent parvovirus-specific oligonucleotide detection probe is the VIC™ dye (Life Technologies, Inc., Carlsbad, CA) having an absorption maximum at 538 nm and an emission maximum at about 554 nm. Here, the quencher can be a FRET quencher or a contact quencher. In one embodiment, the quencher is a minor groove binding non-fluorescent quencher (MGBNFQ) (see Sylvain et al., Rapid Screening for HLA-B27 by a TaqMan-PCR Assay Using Sequence-Specific Primers and a Minor Groove Binder Probe, a Novel Type of TaqMan™ Probe, 287(1-2) Journal of Immunological Methods 179-186 (2004)).

[0073] The mixture of primers and probes described above is used to test for and distinguish between positive amplification control constructs and authentic parvovirus contaminants. In some embodiments, the mixture of primers also includes a set of primers and probes that detect a nucleic acid extraction control (NEC). In certain embodiments, the NEC is M13 bacteriophage, such as the M13K07 strain (SEQ ID NO:8). Thus, in some embodiments, in addition to parvovirus primers and probes and the UPS probe, the mixture of primers and probes contains (5) an M13-specific forward oligonucleotide primer; (6) an M13-specific oligonucleotide detection probe; and (7) an M13-specific reverse oligonucleotide primer.

[0074] In some embodiments, the M13 primer and probe hybridize to the sequence of SEQ ID NO:8. In a specific embodiment, the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; the M13-specific oligonucleotide detection probe comprises the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7. The NEC probe contains a fluorophore that emits light in a non-overlapping spectrum, as seen in the case of a non-overlapping fluorophore emission spectrum for the parvovirus probe and USP. In a specific embodiment, the fluorophore conjugated to the M13-specific oligonucleotide detection probe is Cy5 of a cyanine dye having an absorption maximum at about 650 nm and an emission maximum at about 670 nm (see Southwick et al., Cyanine Dye Labeling Reagents: Carboxymethylindocyanine Succinimidyl Esters, 11 Cytometry, 418-430 (1990)). In this case, the quencher can function by FRET or contact quenching. In one embodiment, the quencher is Black Hole Quencher®-2 (BHQ®-2, Biosearch Technologies, Inc., Petaluma, CA) having a maximum absorption at about 579 nm and quenching in the range of about 550 nm to about 650 nm.

[0075] In another aspect, the present invention provides a method for detecting biological contaminants in a production process of biological molecules. The biological contaminants more specifically include parvoviruses, even more specifically include rodent parvoviruses, and most specifically include parvoviruses that share at least 97% identity with the MVM NS1 gene. In some embodiments, the NS1 gene includes the sequence of SEQ ID NO:9. In one embodiment, the biological molecule production process is a mammalian cell culture process for manufacturing an antibody, a capture molecule, or another therapeutic antibody. A test sample is taken from a cell culture (or a bulk component) and extracted for nucleic acids. In some cases, a nucleic acid extract control (NEC), such as M13 (e.g., SEQ ID NO:8), which serves as a control for proper nucleic acid extraction, is added to the test sample prior to Q-PCR. The method includes: (1) mixing (a) a nucleic acid sample extracted from a test sample, (b) oligonucleotide primers and probes (described above), and (c) a DNA polymerase, preferably a thermostable DNA polymerase having 5'-exonuclease activity, such as Taq polymerase; (2) subjecting the combination to a polymerase chain reaction (PCR); and (3) monitoring the production of various amplicons by the magnitude of fluorescence emission.

[0076] The formation of specific amplicons correlates with the presence and amount of various template nucleic acids in the test sample. Specific amplicons include (1) a target amplification polynucleotide (TAP), such as a rodent parvovirus sequence (e.g., a biological contaminant containing an NS1 sequence), (2) a nucleic acid extraction control (NEC), such as an M13 (e.g., M13K07) polynucleotide (NECP), and (3) a plasmid amplification control polynucleotide (PACP), such as a unique artificial plasmid-specific sequence (UAPS). If TAP and NECP are produced and PACP is not produced, it can be concluded that the test sample contains a biological contaminant and does not contain a cross-contaminated positive amplification control plasmid. However, if both TAP and PACP (i.e., UAP) are produced in the test sample Q-PCR reaction, it can be concluded that the test sample is cross-contaminated with the PAC plasmid and the TAP result may be false positive.

[0077] In a specific embodiment, the primers and probes include (1) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO:1, (2) a rodent parvovirus-specific oligonucleotide detection probe comprising the sequence of SEQ ID NO:2, a VIC fluorophore, and an MGBNFQ quencher, (3) an artificial oligonucleotide detection probe, such as a USP labeled with 6-FAM and BHQ®-1 and comprising the sequence of SEQ ID NO:3, (4) a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO:4, (5) an M13-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO:5, (6) an M13-specific oligonucleotide detection probe labeled with Cy5 and BHQ®-2 and comprising the sequence of SEQ ID NO:6, and (7) an M13-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO:7. The production of TAP is monitored at about 533 nm to about 580 nm, the production of PACP is monitored at about 465 nm to about 510 nm, and the production of NECP is monitored at about 618 nm to about 660 nm.

[0078] In one aspect, the test sample is taken from a production CHO cell culture, such as a production culture of EESYR® cells transformed with a nucleic acid encoding a protein of interest, 96 to 72 hours prior to culture harvest. If TAP and NECP are detected in the test sample but PACP is not detected in the test sample, a confirmatory test can be performed on a second test sample obtained from the same production cell culture 48 hours prior to harvest. If TAP and NECP are detected again in the second test sample but PACP is not detected in the test sample, the cell culture will be considered contaminated and will not be further processed. Alternatively, if the second test is not performed, the cell culture will be considered contaminated and the culture will not be further processed.

[0079] In one aspect, the nucleic acid is extracted from a test sample taken from a production cell culture. In this case, 1 milliliter of the test sample is subjected to cell lysis, proteolysis, and heat denaturation, followed by mixing the sample with a nucleic acid extraction control (NEC) sample, and then extracting the nucleic acid from the sample. In one aspect, the nucleic acid is extracted from the test sample (or NEC addition test sample) using an automated nucleic acid extraction system such as a QIAsymphony® instrument (Qiagen, Inc., Valencia, CA) (see Lee et al., Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMerieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory, 52(4) J. Clin. Virol. 339-43 (2011)).

[0080] In one aspect, prior to performing PCR, uracil-N-glycosylase (UNG) enzyme is added to the Q-PCR reaction mixture to selectively degrade contaminating amplicons (see Taggart et al., Use of heat labile UNG in an RT-PCR assay for enterovirus detection, 105(1) J. Virol. Methods. 57-65 (2002)). The reaction mixture is incubated at 50 °C for at least 2 minutes, more specifically for 2 minutes or 5 minutes in some cases.

[0081] In a specific aspect, after any UNG treatment, the reaction mixture is incubated at 95 °C for 2 minutes, and then eight cycles of (1) denaturation at 95 °C for 10 seconds, followed by (2) annealing and extension for 30 seconds are carried out, such that the initial annealing temperature is 70 °C and the annealing temperature decreases by 1 °C per cycle, with the eighth annealing being at 62 °C. After the first eight cycles, 40 cycles of DNA amplification are carried out, including (1) a denaturation step at 95 °C for 10 seconds, followed by (2) an annealing and extension step at 62 °C for 30 seconds. In certain aspects, the rate of temperature change from the denaturation temperature to the annealing temperature is about 4.4 °C per second, and from the annealing temperature to the denaturation temperature is about 2.2 °C per second.

[0082] In some aspects, a method for detecting biological contaminants in a production cell culture medium or its product includes performing an external positive amplification control (PAC) assay that is conducted separately from the test sample assay, as well as performing an external negative control assay that is conducted separately from the test sample assay and the PAC assay. If either the negative control or the positive control fails, the results obtained from the test sample assay are rejected.

[0083] In one aspect, the external positive control involves: (1) among other things and without test samples, (a) in a specific aspect, a positive amplification control (PAC) plasmid containing the sequence of SEQ ID NO:11, (b) a rodent parvovirus-specific forward oligonucleotide primer containing the sequence of SEQ ID NO:1, (c) a rodent parvovirus-specific oligonucleotide detection probe containing the sequence of SEQ ID NO:2 labeled with VIC and MGBNFQ, (d) an artificial oligonucleotide detection probe such as USP containing the sequence of SEQ ID NO:3 labeled with 6-FAM and BHQ®-1, (e) a rodent parvovirus-specific reverse oligonucleotide primer containing the sequence of SEQ ID NO:4, and (f) a DNA polymerase, preferably a thermostable DNA polymerase with 5'exonuclease activity such as Taq polymerase, mixing step; (2) subjecting the positive control mixture to a polymerase chain reaction (PCR) of the positive control; and (3) during PCR, (a) monitoring the production of the target amplification polynucleotide (TAP), (b) the nucleic acid extraction control amplification polynucleotide (NECP), and (c) the plasmid amplification control polynucleotide (PACP). The production of TAP is monitored at about 533 nm to about 580 nm, the production of PACP is monitored at about 465 nm to about 510 nm, and the production of NECP is monitored at about 618 nm to about 660 nm.

[0084] The PCR reaction of the positive amplification control is carried out in the same manner as the PCR reaction of the test sample (described above). When TAP and PACP are produced in the reaction of the positive control, it can be concluded that the PCR amplification procedure is functioning properly. If TAP is not produced in the reaction of the positive amplification control, any negative TAP in the test sample will be ignored as a failed PCR reaction. In one aspect, NEC (i.e., for example, M13K07) is included in the positive amplification control. A properly functioning control should also show a positive NECP signal (see Table 1).

[0085] In one aspect, the external negative control is (1) among other things and without test sample and without PAC plasmid, (a) a buffer that mimics the test sample buffer system, or a blank that can be simply water, (b) a rodent parvovirus-specific forward oligonucleotide primer containing the sequence of SEQ ID NO:1, (c) a rodent parvovirus-specific oligonucleotide detection probe containing the sequence of SEQ ID NO:2 labeled with VIC and MGBNFQ, (d) an artificial oligonucleotide detection probe such as USP containing the sequence of SEQ ID NO:3 labeled with 6-FAM and BHQ®-1, (e) a rodent parvovirus-specific reverse oligonucleotide primer containing the sequence of SEQ ID NO:4, and (f) a DNA polymerase, preferably a thermostable DNA polymerase having 5'-exonuclease activity such as Taq polymerase, the step of mixing; (2) the step of subjecting the positive control mixture to a polymerase chain reaction (PCR) of the positive control; and (3) during PCR, (a) monitoring the production of the target amplification polynucleotide (TAP), (b) the nucleic acid extraction control amplification polynucleotide (NECP), and (c) the plasmid amplification control polynucleotide (PACP). The production of TAP is monitored at about 533 nm to about 580 nm, and the production of PACP is monitored at about 465 nm to about 510 nm.

[0086] The PCR reaction for the negative control is performed identically to the PCR reaction of the test sample (described above). If TAP and PACP are produced in the negative control reaction, it can be concluded that the PCR reagent is contaminated with the PAC plasmid. If TAP is produced in the negative control reaction but PACP is not, it can be concluded that the PCR reagent is contaminated with parvovirus. In both cases, the test sample results are discarded. However, if the production of TAP and PACP is negative in the negative control reaction, the production of TAP and PACP is positive in the positive amplification control, and the production of TAP (and optionally NECP) is positive and the production of PACP is negative in the reaction of the test sample, the technician may conclude that the test sample is contaminated (see Tables 2 and 3).

[0087] In other aspects, the present invention provides a positive amplification control plasmid (PAC plasmid) and a mixture of positive control reagents containing the positive control plasmid. In one aspect, the PAC plasmid comprises (1) a parvovirus nucleic acid sequence, (2) an M13K07 nucleic acid sequence, and (3) an artificial nucleic acid sequence unique to the plasmid (also known as UAPS or "unique" sequence). In a specific aspect, the parvovirus nucleic acid sequence comprises the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; the M13K07 nucleic acid sequence comprises the sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; and the unique sequence comprises the antisense sequence of SEQ ID NO:3. In a more specific aspect, the nucleotide sequence of the PAC plasmid consists of the sequence set forth in SEQ ID NO:11.

[0088] In some embodiments, the mixture of positive control reagents includes, among other things, the PAC plasmid described above, a rodent parvovirus-specific forward oligonucleotide primer, a rodent parvovirus-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe (i.e., USP), a rodent parvovirus-specific reverse oligonucleotide primer, an M13-specific forward oligonucleotide primer, an M13-specific oligonucleotide detection probe, and an M13-specific reverse oligonucleotide primer, as well as a buffer. The mixture optionally contains dNTP and Taq polymerase.

[0089] (Table 2) Assay Controls and Test Session Status TIFF2025105635000006.tif33170

[0090] (Table 3) Test Session Status TIFF2025105635000007.tif31170

[0091] In a specific embodiment, the rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1, the rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2, the USP comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3, the rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4, the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5, the M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6, and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7.

Example

[0092] Example 1: Oligonucleotides and nucleic acid reagents Rodent parvovirus, M13K07, and artificial unique oligonucleotides (oligos) were obtained from various suppliers in various scales and in various forms as described in Table 4. A three-year expiration date was assigned to all oligos upon receipt from the suppliers.

[0093] The oligonucleotides were reconstituted with water to a concentration of 100 μM to prepare a master stock. Before preparing the qPCR reaction, the master stock was further diluted to prepare a 10× stock solution. Table 5 shows the 10× and 1× concentrations of the qPCR oligonucleotides (primers and probes).

[0094] (Table 4) Primers and Probes for Parvovirus, M13, and Unique Artificial Sequences TIFF2025105635000008.tif134170

[0095] (Table 5) Oligonucleotide Concentrations TIFF2025105635000009.tif58170

[0096] Example 2: Positive amplification control plasmid The Parvo-M13 positive amplification control (PAC) plasmid was prepared with the pUC57-Kan plasmid (GeneWiz, Inc., South Plainfield, NJ). This PAC plasmid consists of the sequence shown in SEQ ID NO:11. The parvovirus-M13 PAC plasmid has the following formula: [number = (amount * number / mol) / (bp * ng / g * bp of g / mol)]; where amount = ng, number / mol = 6.022×10 23 , bp = 4372, ng / g = 1×10 9 , and bp of g / mol = 650, and contains 2.1×10 8 copies / ng. The concentration of the plasmid was calculated and expressed as ng / μL, and 10 2It was serially diluted to a 10× concentration of 2 50 μL aliquots were prepared from the 10

[0097] Example 3: Preparation of M13K07 phage Copy / μL dilution and stored in 2 mL sterile screw cap tubes. All aliquots were stored at ≤ -60 °C. A 10-fold serial dilution of M13K07 phage was performed using MEM as a diluent to obtain M13K07 at a titer of 100 pfu / μL. 200 μL aliquots were prepared, stored at ≤ -60 °C, and given a 3-year shelf life.

[0098] Example 4: Preparation of Q-PCR reaction reagents The rodent parvovirus real-time PCR detection assay is a fully automated TaqMan® PCR process consisting of automated DNA purification using QIAsymphony® (Qiagen), followed by nucleic acid amplification, and real-time PCR product detection on a LightCycler® 480 instrument (Roche Diagnostic). Phage M13K07 was automatically added as an internal control (IC) to each test substance to evaluate the presence of PCR inhibitors. Rodent parvovirus primer oligonucleotides were designed to hybridize within a highly conserved region (NS-1 region) of the rodent parvovirus genome to ensure broad detection. The assay was performed in a duplex (i.e., two-target) format, and the primers for rodent parvovirus and M13K07 generated PCR products of approximately 110 and 97 bp, respectively. PCR products were detected in real time by cleavage of two types of probes labeled with different reporter dyes, VIC fluorophore for rodent parvovirus and Cy5 fluorophore for M13K07 phage. A positive amplification control (PAC) plasmid was used at a concentration of 100 copies / μL. This plasmid contains a unique (Flag) sequence (USP) that discriminates the plasmid from wild-type parvovirus using a specific probe labeled with the fluorophore FAM.

[0099] The master mix was aliquoted into 2 mL tubes according to Table 6 in an amount sufficient for at least three times the number of test substances including the control. The tubes were stored at 2 - 8 °C. Negative amplification control (NAC) vials were prepared by adding 50 μL of water to the 2 mL tubes.

[0100] (Table 6) Reaction mixture TIFF2025105635000010.tif46170

[0101] Example 5: Preparation of test samples Since all samples have the potential to contain or be contaminated with exogenous agents, aseptic practices were strictly adhered to for all sample pretreatment steps. The following steps were performed within a clean biosafety cabinet. The test substance (sample) was obtained from an EESYR® cell culture producing an antibody or capture molecule and was either frozen or used directly.

[0102] Approximately 1000 μL of PBS was aliquoted into 2 mL tubes to serve as a negative extraction control (NEC). When PCR was used as an evaluation item for the cell culture step, positive and negative controls for the cell culture were used as a positive extraction control and a negative extraction control, respectively.

[0103] Each test substance was thawed at room temperature. Their samples in 60 mL bags were transferred to 50 mL Falcon tubes and then divided. 1000 μL of each sample was pipetted into 2 mL tubes for pretreatment. 400 μL of lysis buffer (Charge Switch® Lysis Buffer L13) (Invitrogen, catalog number CS11202 or equivalent, Carlsbad, CA) was added to each tube and vortexed for at least 10 seconds. Then, 20 μL of proteinase K (in 40% glycerol (v / v) containing 10 mM Tris-HCl, pH 7.5 with 1 mM calcium acetate, ≥10 mg / mL, ≥800 units / mL; SAFC, catalog number P4850-5ML, Sigma Aldrich, St. Louis, MO) was added to each sample and vortexed for at least 10 seconds. Then, the samples were incubated at 65 °C for 30 minutes. After incubation, the samples were vortexed and centrifuged at 17,000×g for 10 minutes.

[0104] Simultaneously, positive amplification control (PAC; in 2 mL tubes, at least 50 uL of 10 2 copies / μL plasmid) and M13K07 (10 2 PFU / mL) internal control (IC; at least 150 μL / 12 samples) were thawed.

[0105] Example 6: Nucleic acid extraction An internal control (IC) was required for the programmed extraction protocol on the QIASymphony® instrument (Qiagen, Valencia, CA). The instrument automatically added 120 μL of reconstituted IC to each sample. For every 12 samples, 1.8 mL of IC (i.e., 1 vial) was required by the instrument. The IC vial was prepared by adding 1650 μL of AVE buffer (RNase-free water containing 0.04% sodium azide) to 150 μL of M13K07 IC.

[0106] Each prepared sample was placed on the sample carrier of the instrument within a biosafety cabinet (BSC). The vials of ICs were placed on separate (dedicated) sample carriers within the BSC at a ratio of 1 vial of IC per 12 samples. All drawers were closed and the instrument was operated according to the manufacturer's recommended protocol (see QIAsymphony DNA Handbook, 09 / 2010, available at http: / / www.algimed.by / download / EN-QIAsymphony-DNA-Handbool.pdf).

[0107] The reagent prep cartridge was prepared with the QIAsymphony® DSP Virus / Pathogen Kit, which contains all the reagents required for extraction (see QIAsymphony® DSP Virus / Pathogen Kit Instructions for Use (Handbook, April 2013, available at https: / / www.qiagen.com / us / resources / download.aspx?id=f8bc0b3c-0aff-46ee-8807-5ed145f9e969&lang=en)). The reagent prep cartridge contained proteinase K and had a shelf life of approximately two weeks.

[0108] To facilitate integration with Q-PCR, nucleic acid extraction was performed in a 96-well reaction plate. After the automated nucleic acid extraction procedure was completed and passed the status check, the reaction plate was cooled and sealed with LightCycler® 480 Sealing Foil (Roche, Branchburg, NJ). The 96-well plate was placed in a plate spinner balanced with an appropriate counterweight (e.g., another 96-well plate) and spun for 30 - 60 seconds. The wells were checked for bubbles and the spin was repeated if necessary.

[0109] Example 7: Q-PCR Q-PCR was performed on a LightCycler® 480 instrument (Roche, Branchburg, NJ) using either one of two programs: TaqMan Triplex and / or Veriquest Triplex. The TaqMan Triplex protocol used TaqMan Fast Advance Custom Master Mix without reference dye (ROX). Three fluorescence channels (FAM, VIC, and CY5) were selected, and the UNG step time was 2 minutes. The reaction parameters used are summarized in Table 7.

[0110] Similarly, when using VeriQuest® Master Mix without ROX, a 5-minute UNG step time was used. The reaction parameters used are summarized in Table 8.

[0111] (Table 7) Steps of the TaqMan Triplex program for rodent parvovirus PCR TIFF2025105635000011.tif51170

[0112] The crossing point (Cp) fluorescence signals for each of the M13 internal control, rodent parvovirus, and positive amplification control plasmid were determined by one or both of two algorithms. The first algorithm is the automatic second derivative method. This method requires no user input and generally provides greater consistency and was thus considered the preferred method. The second algorithm is the Fit Point method. This method requires the user to set a threshold line in the case of a scattered background. The point at which the logarithmic linear curve exceeds that threshold line is the crossing point. The fluorescence signals were monitored using the following filter combinations: 533 - 580 nm (VIC signal) for rodent parvovirus (subset "sample - parvovirus"); 618 - 660 nm (Cy5 signal) for the internal control M13K07 (subset "sample - M13"); and 465 - 510 nm (FAM signal) for the positive amplification control plasmid (subset "sample - FAM"). In the case of an unclear fluorescence signal, the Fit Point analysis was used to determine the fluorescence background level. The acceptable background fluorescence signal was considered to be ≤ 1 unit on the amplification plot scale. Any fluorescence signal ≤ 1 unit was within the acceptable background and was thus considered negative.

[0113] (Table 8) Steps of the VeriQuest Triplex program for rodent parvovirus PCR TIFF2025105635000012.tif51170

[0114] Example 8: Conditions for a valid test session For a test session to be considered valid, the following conditions must be met. The negative amplification control (NAC, i.e., water) must be negative for the fluorescence signals in all three channels. The negative extraction control (NEC, i.e., PBS) or the cell culture negative control flask must be negative for the fluorescence signal in the [533 - 580] channel (i.e., the rodent parvovirus probe VIC signal), negative for the fluorescence signal in the [465 - 510] channel (i.e., the positive amplification control [PAC] antisense Flag probe - FAM signal), and positive for the fluorescence signal in the [618 - 660] channel (i.e., the M13K07 probe - CY5 signal). The PAC should be positive for the fluorescence signals in all three channels. The Cp value of M13K07 in the NEC was used as a reference for evaluating the presence of inhibitory substances in the samples.

[0115] When using PCR as an evaluation item for the test substance from the cell culture step together with the rodent parvovirus positive control, the positive virus control must be positive for the fluorescence signal in the [533 - 580] channel (i.e., the rodent parvovirus probe VIC signal), positive for the fluorescence signal in the [618 - 660] channel (i.e., the M13K07 probe - CY5 signal), and negative for the fluorescence signal in the [465 - 510] channel (i.e., the PAC antisense Flag probe - FAM signal). The Cp value of M13K07 in the cell culture positive control sample was expected to be within the range of ±4 cycles of the Cp value of NEC - M13.

[0116] For all reactions of the PAC plasmid - containing controls, the fluorescence signals must be positive in all three channels.

[0117] Example 9: Conditions for an invalid test session The assay was considered invalid if any one or more of the following conditions were met: (1) a very low amplification curve (less than 1 unit on the fluorescence scale) for PAC, (2) a determinant error (mechanical, software, or human error) was confirmed, (3) NAC was positive for amplification in any of the three channels, (4) NEC was positive for amplification in the [533 - 580] VIC channel, or positive for amplification in the [465 - 510] FAM channel, or negative for amplification in the [618 - 660] Cy5 channel, and (5) PAC was negative for the amplification signal in any of the three channels. Whenever the assay was determined to be invalid, an investigation and retest of the test sample were always performed.

[0118] Example 10: Conditions for negative sample results in a valid test For a valid negative parvovirus test result, all of the following conditions must be met. The sample must be positive for the M13K07 DNA amplification signal in the M13 [618 - 660] channel within the predicted Cp value range of NEC - M13 Cp ± 4 cycles, which suggests the absence of PCR inhibitors. The sample must be negative for the parvovirus DNA amplification signal in the parvovirus [533 - 580] channel. Fluorescence signals below 1 unit on the fluorescence scale were reported as negative for parvovirus DNA amplification, regardless of whether the Cp value was considered within the acceptable fluorescence background level. The sample must be negative for the PAC amplification signal in the antisense Flag [465 - 510] channel. Note that fluorescence signals below 1 unit on the fluorescence scale were reported as negative for PAC plasmid DNA amplification, regardless of whether the Cp value (automatically provided by the instrument) was considered within the acceptable fluorescence background level.

[0119] Example 11: Conditions for "no sample" results in a valid test session The "no sample" result occurred when any one or more of the following conditions were met. When the sample was negative for the M13K07 DNA amplification signal in the Cy5 channel [618 - 660], negative for the parvovirus DNA amplification signal in the parvovirus channel [533 - 580], and negative for the PAC amplification in the antisense Flag FAM channel [465 - 510], the sample or PCR reagents were always investigated according to standard procedures. This condition suggests the presence of PCR inhibitors or failure of proper nucleic acid extraction. To overcome inhibition, the sample can be diluted (1:2, 1:5, 1:10). Cp values of the fluorescence signal in the M13 channel [618 660] outside the range (NEC M13 Cp ± 4 cycles) indicated partial inhibition of PCR or an error in phage addition and required repeat testing. Dilution of the sample (1:2, 1:5, 1:10) may be considered to overcome any inhibition of PCR. Any evidence of systematic error or an unexpectedly very low fluorescence signal (< 1 unit on the fluorescence scale) observed in the M13 channel [618 - 660] that did not allow a definitive assessment of sample suitability was considered a "no sample result" and suggested a failure during amplification or DNA extraction. This required repeat testing.

[0120] Example 12: Conditions for out-of-specification (OOS) sample results in a valid test session A sample was considered iOOS if it was (i) positive for parvovirus DNA amplification signal (i.e., rodent parvovirus probe VIC signal) in the parvovirus channel [533 - 580] with a fluorescence signal greater than 1 unit on the fluorescence scale (for at least one of the two two - well pairs), and (ii) negative for PAC amplification in the antisense Flag FAM channel [465 - 510] (indicating no cross - contamination by PAC). As a result, the technician (i) initiated GLIF (General Laboratory Investigation Form), (ii) notified the department that the sample was sent to QC (Virology) for testing, (iii) initiated NOE, (iv) stored the amplification tubes (frozen at - 20°C) for further investigation (e.g., Flag sequence screening or sequencing), and (v) had to repeat the assay and retest the sample.

[0121] Example 13: Plan for repeats and retesting Whenever the assay was invalid or the sample result was considered "no result", a repeat test was initiated. The retest was performed to confirm the iOOS event (i.e., the initial sample result was positive for the DNA amplification signal in the parvovirus channel [533 - 580]). The retest or repeat test had to be performed using fresh reagent aliquots (i.e., master mix reagent, extraction kit).

[0122] When NAC was positive for fluorescence in any channel, the entire test session starting from the amplification (PCR) step using the already purified DNA sample with freshly prepared master mix was repeated. When NEC was negative for fluorescence in the M13 channel [618 - 660], the entire test session starting from the DNA extraction step was repeated. When NEC was positive for fluorescence in the parvovirus channel [533 - 580] or the antisense Flag channel [465 - 510], the entire test session starting from the DNA extraction step was repeated. When PAC was negative for fluorescence in any channel, the test session was repeated starting from the amplification (PCR step) using the same purified DNA sample with freshly prepared master mix.

[0123] For samples with "no sample" results, the test session starting from the DNA extraction step was repeated for the affected samples. To demonstrate the presence of inhibitors, as part of the investigation and problem - solving process, the sample can be diluted (1:2, 1:5, or 1:10) before DNA extraction (in addition to the undiluted sample).

[0124] Retesting to confirm the initial positive result (iOOS) was performed using the following four separate aliquots: two aliquots of the test substance from the original sample collection event (e.g., one day after the last nutrient supply), and two aliquots of the test substance from a different sample collection event (if possible, e.g., two days after the last nutrient supply) or from a different sample bag. If any of the additional tests for the four aliquots yielded a positive signal without evidence of a determinate error (demonstrated by investigation), the lot was considered to have failed the requirement for the absence of rodent parvovirus genomic material. An infectious assay was required for the final disposition of such positive Q - PCR results. CHO - K1 cells were used as the indicator cell line to determine the infectious state of the detected nucleic acid.

[0125] Whenever the retest result is negative, a confirmatory test is required at the time of different sample collection events (e.g., 3 days after the last nutrient supply) to confirm the absence of rodent parvovirus genomic material.

[0126] Example 14: Retest plan for other sample types with OOS Samples types that had an initial positive result (iOOS), such as untreated bulk material, end-of-production cells, cells at the limit of in vitro life, and soybean raw materials, were retested as follows using four separate aliquots.

[0127] Two aliquots of the test substance from the original sample container (i.e., a bag, for example) and two aliquots from different sample containers were retested according to standard operating procedures. One sample aliquot of the test substance from the original sample container was inoculated into CHO-K1 indicator cells. The inoculated indicator cells CHO-K1 were harvested after 1 - 3 days under culture according to standard operating procedures to determine the infectious state of the detected nucleic acid. A standard curve for quantification of the re-extracted nucleic acid may be used.

[0128] Whenever any of the additional PCR tests for the four aliquots was positive without evidence of a clear error (demonstrated by investigation), an infectivity assay determines the final disposition of the test substance. Nucleic acid sequencing and transmission electron microscopy (TEM) may be considered to identify the microorganism and rule out any laboratory error upon confirmation of the initial OOS.

[0129] An infectivity assay using CHO-K1 as the indicator cell for the suspected contaminated material is required to determine the infectious state of the detected nucleic acid. Whenever the investigation fails to support the possibility of viral contamination by parvovirus and all of the additional PCR tests and CHOK1 cultures for the four aliquots of the test substance are negative, the lot was considered to meet the requirement for the absence of infectious rodent parvovirus virus.

[0130] Example 15: Parvovirus test during recombinant protein production As an important in-process control for testing the cell culture fluid, i.e., the untreated bulk material from a production bioreactor containing CHO cell derivatives containing the heavy and light chain constructs of the heterologous antibody, the Q-PCR procedure described above was performed. Each heterologous monoclonal antibody (mAb) binds to a different target or epitope. In a large-scale bioprocess production facility, Good manufacturing process (GMP) tests were performed by QC (virology) scientists. Sixteen of those tests are listed in Table 9. In each case, the test session was valid, the assay system suitability criteria were met, and no false positive detections were observed. In test sessions #13 and #14, false negative detections occurred, which suggested the presence of PCR inhibitors or failure of nucleic acid extraction.

[0131] (Table 9) Rodent Parvovirus PCR Tests TIFF2025105635000013.tif120168UPB = Untreated bulk material; EA = Early alert; Rodent Parvo PCR = Q-PCR procedure described above

[0132] Sequence Information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. <120> COMPOSITIONS AND METHODS FOR DETECTING A BIOLOGICAL CONTAMINANT <150> US 62 / 139,321 <151> 2015-03-27 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 24 <212> DNA <213> Parvovirus <400> 1 tgcataaaag agtaacctca ccag 24 <210> 2 <211> 18 <212> DNA <213> Parvovirus <400> 2 actggatgat gatgcagc 18 <210> 3 <211> 19 <212> DNA <213> Parvovirus <400> 3 tgtcgatggc gaatggcta 19 <210> 4 <211> 18 <212> DNA <213> Parvovirus <400> 4 ccacctggtt gagccatc 18 <210> 5 <211> 20 <212> DNA <213> Bacteriophage M13 <400> 5 aagcctcagc gaccgaatat 20 <210> 6 <211> 24 <212> DNA <213> Bacteriophage M13 <400> 6 tatgcgtggg cgatggttgt tgtc 24 <210> 7 <211> 22 <212> DNA <213> Bacteriophage M13 <400> 7 tcagcttgct ttcgaggtga at 22 <210> 8 <211> 6407 <212> DNA <213> Bacteriophage M13 <400> 8 aacgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120 cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180 gttgcatatt taaaacatgt tgagctacag caccagattc agcaattaag ctctaagcca 240 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300 ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420 cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600 ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660 aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720 atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960 aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200 caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta 1260 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560 tttttggaga ttttcaacat gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620 tttttggaga ttttcaacat gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca 1680 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca 1680 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggttgt 1740 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggttgt 1740 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca 1800 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca 1800 tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1860 tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1860 tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1920 tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1920 attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1980 attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1980 aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt 2040 cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact 2100 caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg 2160 tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag 2220 gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat 2280 gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggtgg ctctgagggt 2340 ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt 2400 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat 2460 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2520 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact 2580 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct 2640 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct 2700 tttgtcttta gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta 2760 ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg 2820 tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt 2880 tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttacttttc 2940 ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg 3000 ggcttaactc aattcttgtg ggttatctct ctgatattag cgctcaatta ccctctgact 3060 ttgttcaggg tgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc 3120 tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg 3180 attgggataa ataatatggc tgtttatttt gtaactggca aattaggctc tggaaagacg 3240 ctcgttagcg ttggtaagat tcaggataaa attgtagctg ggtgcaaaat agcaactaat 3300 cttgatttaa ggcttcaaaa cctcccgcaa gtcgggaggt tcgctaaaac gcctcgcgtt 3360 cttagaatac cggataagcc ttctatatct gatttgcttg ctattgggcg cggtaatgat 3420 tcctacgatg aaaataaaaa cggcttgctt gttctcgatg agtgcggtac ttggtttaat 3480 tcctacgatg aaaataaaaa cggcttgctt gttctcgatg agtgcggtac ttggtttaat 3480 acccgttctt ggaatgataa ggaaagacag ccgattattg attggtttct acatgctcgt 3540 acccgttctt ggaatgataa ggaaagacag ccgattattg attggtttct acatgctcgt 3540 aaattaggat gggatattat ttttcttgtt caggacttat ctattgttga taaacaggcg 3600 aaattaggat gggatattat ttttcttgtt caggacttat ctattgttga taaacaggcg 3600 cgttctgcat tagctgaaca tgttgtttat tgtcgtcgtc tggacagaat tactttacct 3660 cgttctgcat tagctgaaca tgttgtttat tgtcgtcgtc tggacagaat tactttacct 3660 tttgtcggta ctttatattc tcttattact ggctcgaaaa tgcctctgcc taaattacat 3720 tttgtcggta ctttatattc tcttattact ggctcgaaaa tgcctctgcc taaattacat 3720 gttggcgttg ttaaatatgg cgattctcaa ttaagcccta ctgttgagcg ttggctttat 3780 gttggcgttg ttaaatatgg cgattctcaa ttaagcccta ctgttgagcg ttggctttat 3780 actggtaaga atttgtataa cgcatatgat actaaacagg ctttttctag taattatgat 3840 actggtaaga atttgtataa cgcatatgat actaaacagg ctttttctag taattatgat 3840 tccggtgttt attcttattt aacgccttat ttatcacacg gtcggtattt caaaccatta 3900 tccggtgttt attcttattt aacgccttat ttatcacacg gtcggtattt caaaccatta 3900 aatttaggtc agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt 3960 aatttaggtc agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt 3960 tgtcttgcga ttggatttgc atcagcattt acatatagtt atataaccca acctaagccg 4020 tgtcttgcga ttggatttgc atcagcattt acatatagtt atataaccca acctaagccg 4020 gaggttaaaa aggtagtctc tcagacctat gattttgata aattcactat tgactcttct 4080 gaggttaaaa aggtagtctc tcagacctat gattttgata aattcactat tgactcttct 4080 cagcgtctta atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat 4140 cagcgtctta atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat 4140 agcgacgatt tacagaagca aggttattca ctcacatata ttgatttatg tactgtttcc 4200 attaaaaaag gtaattcaaa tgaaattgtt aaatgtaatt aattttgttt tcttgatgtt 4260 tgtttcatca tcttcttttg ctcaggtaat tgaaatgaat aattcgcctc tgcgcgattt 4320 tgtaacttgg tattcaaagc aatcaggcga atccgttatt gtttctcccg atgtaaaagg 4380 tactgttact gtatattcat ctgacgttaa acctgaaaat ctacgcaatt tctttatttc 4440 tgttttacgt gctaataatt ttgatatggt tggttcaatt ccttccataa ttcagaagta 4500 taatccaaac aatcaggatt atattgatga attgccatca tctgataatc aggaatatga 4560 tgataattcc gctccttctg gtggtttctt tgttccgcaa aatgataatg ttactcaaac 4620 ttttaaaatt aataacgttc gggcaaagga tttaatacga gttgtcgaat tgtttgtaaa 4680 gtctaatact tctaaatcct caaatgtatt atctattgac ggctctaatc tattagttgt 4740 tagtgcacct aaagatattt tagataacct tcctcaattc ctttctactg ttgatttgcc 4800 aactgaccag atattgattg agggtttgat atttgaggtt cagcaaggtg atgctttaga 4860 tttttcattt gctgctggct ctcagcgtgg cactgttgca ggcggtgtta atactgaccg 4920 cctcacctct gttttatctt ctgctggtgg ttcgttcggt atttttaatg gcgatgtttt 4980 agggctatca gttcgcgcat taaagactaa tagccattca aaaatattgt ctgtgccacg 5040 tattcttacg ctttcaggtc agaagggttc tatctctgtt ggccagaatg tcccttttat 5100 tactggtcgt gtgactggtg aatctgccaa tgtaaataat ccatttcaga cgattgagcg 5160 tcaaaatgta ggtatttcca tgagcgtttt tcctgttgca atggctggcg gtaatattgt 5220 tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat 5280 tactaatcaa agaagtattg ctacaacggt taatttgcgt gatggacaga ctcttttact 5340 cggtggcctc actgattata aaaacacttc tcaagattct ggcgtaccgt tcctgtctaa 5400 aatcccttta atcggcctcc tgtttagctc ccgctctgat tccaacgagg aaagcacgtt 5460 atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 5520 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5580 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5640 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5640 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5700 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5700 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5760 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5760 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5820 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5820 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 5880 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 5880 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 5940 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 5940 tttaaatatt tgcttataca atcttcctgt ttttggggct tttctgatta tcaaccgggg 6000 tttaaatatt tgcttataca atcttcctgt ttttggggct tttctgatta tcaaccgggg 6000 tacatatgat tgacatgcta gttttacgat taccgttcat cgattctctt gtttgctcca 6060 tacatatgat tgacatgcta gttttacgat taccgttcat cgattctctt gtttgctcca 6060 gactctcagg caatgacctg atagcctttg tagacctctc aaaaatagct accctctccg 6120 gactctcagg caatgacctg atagcctttg tagacctctc aaaaatagct accctctccg 6120 gcatgaattt atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg 6180 gcatgaattt atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg 6180 gcctttctca cccttttgaa tctttaccta cacattactc aggcattgca tttaaaatat 6240 gcctttctca cccttttgaa tctttaccta cacattactc aggcattgca tttaaaatat 6240 atgagggttc taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat 6300 atgagggttc taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat 6300 tacagggtca taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc 6360 ttaattttgc taattctttg ccttgcctgt atgatttatt ggatgtt 6407 <210> 9 <211> 2019 <212> DNA <213> Minute virus of mouse <400> 9 atggctggaa atgcttactc tgatgaagtt ttgggagcaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaaa atgttcaact gaatggaaaa 120 gatatcggat ggaatagtta caaaaaagag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacttg ggaccaaagc gaggacatgg aatgggaaac cacagtggat 240 gaaatgacca aaaagcaagt attcattttt gattctttgg ttaaaaaatg tttatttgaa 300 gtgcttaaca caaagaatat atttcctggt gatgttaatt ggtttgtgca acatgaatgg 360 ggaaaagacc aaggctggca ctgccatgta ctaattggag gaaaggactt tagtcaagct 420 caagggaaat ggtggagaag gcaactaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc aactaacacc agctgaaaga attaaactaa gagaaatagc agaagacaat 540 gagtgggtta ctctacttac ttataagcat aagcaaacca aaaaagacta taccaagtgt 600 gttctttttg gaaacatgat tgcttactat tttttaacta aaaagaaaat aagcactagt 660 ccaccaagag acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg agcgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaagtttcta ttaaaactac acttaaagag ctggtgcata aaagagtaac ctcaccagag 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acactaactc tagccagaac caaaacagca 1020 tttgacttaa ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 acaagaacct gcagaatttt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttttaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg caatgttggt 1260 tgctataatg cagccaatgt aaactttcca tttaatgact gtaccaacaa gaacttgatt 1320 tgggtagaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca taccttgcct 1560 ggtgactttg gtttggttga caaaaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc aaaggtgcca actcctataa atttactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 10 <211> 19 <212> DNA <213> Artificial <220> <223> synthetic <400> 10 tagccattcg ccatcgaca 19 <210> 11 <211> 1707 <212> DNA <213> Artificial <220> <223> synthetic <400> 11 aagcctcagc gaccgaatat atcggttatg cgtgggcgat ggttgttgtc attgtcggcg 60 caactatcgg tatcaagctg tttaagaaat tcacctcgaa agcaagctga cacgcctacc 120 gcgatgctga atgacccgga ctagagtggc gaaatttatg gcgtgtgacc cgttatgctc 180 catttcggtc agtgggtcat tgctagtagt cgattgcatt gccattctcc gagtgattta 240 gcgtgacagc cgcagggaac ccataaaatg caatcgtagt ccacctgatc gtacttagaa 300 atgagggtcc ccttttgccc acgcacctgt tcgctcgtcg tttgctttta agaaccgcac 360 gaaccacaga gcataaagag aacctctagc tcctttacaa ggtactggtt ccctttccag 420 cgggatgcct tatctaaacg caatgacaga cgtattcctc aggccacatc gcttcctact 480 tccgctggga tccatcattg gcggccgaag ccgccattcc atagtgagtc cttcgtctgt 540 gtctttctgt gccagatcgt ctagcaaatt gccgatccag tttatctcac gaaactatag 600 tcgtacagac cgaaatctta agtcaaatca cgcgactagg ctcagctcta ttttagtggt 660 catgggtttt ggtccgcccg agcggtgcaa ccgattagga ccatgtaaaa catttgttac 720 aagtcttctt ttaaacacaa tcttcctgct cagtggcgca tgattatcgt tgttgctagc 780 cagcgtggta agtaacagca ccactgcgag cctaatgtgc cctttccacg aacacagggc 840 tgtccgatcc tatattagga ctccgcaatg gggttagcaa gtcgcaccct aaacgatgtt 900 gaagactcgc gatgtacatg ctctggtaca atacatacgt gttccggctg ttatcctgca 960 tcggaacctc aatcatgcat cgcaccagcg tattcgtgtc atctaggagg ggcgcgtagg 1020 ataaataatt caattaagat gtcgttatgc tagtatacgc ctacccgtca ccggccatct 1080 gtgtgcagat ggggcgacga gttactggcc ctgatttctc cgcttctaat accacacact 1140 gggcaatacg agctcaagcc agtctcgcag taacgctcat cagctaacga aagagttaga 1200 ggctcgctaa cggagacgag ttaaagacac gagttcccaa aaccaggcgg gctcgccacg 1260 acggctaatc ctggtagttt acgtgaacaa tgttctgaag aaaatttgtg aaagaaggac 1320 ccgtcaccgc ctacaattac ctacaacggt cggccgcacc ttcgattgtc gtggccaccc 1380 tcggattaca cggcagaggt ggttgtgtcc cgacaggcca gcatattatc ctgaggcgtt 1440 accccaatcg ttctccgtcg gatttgctac agcccctgag cgctacatgc acgaaaccaa 1500 gttatgtatg cactgggccg tcaataggac gtagccttgt agttagcacg tagcccggcc 1560 gcattagtac agtagagcct ccgccggcat cctgtttatt aagttatttc tgcataaaag 1620 agtaacctca ccagaggact ggatgatgat gcagccagac agttagccat tcgccatcga 1680 cattgaaatg atggctcaac caggtgg 1707 <210> 12 <211> 2019 <212> DNA <213> Mvm lymphotropic variant <400> 12 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaaaaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gagtgggtta ctttactcac ttataaacat aagcaaacca aaaaggacta tactaaatgt 600 gttctttttg gaaatatgat tgcttactac tttttaacca aaaagaaaat aagcaccagt 660 ccgccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 ccgccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ttatacactg atgacatgcg gccagaaacg 780 aaagagggcg aacgccatct agtgagcaaa ttatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctagtgcata aaagagtaac ctcaccagaa 900 gaggtttcta ttaaaaccac acttaaagag ctagtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acgctaactc tagccagaac aaaaacagca 1020 aacctgctga aaaatacgct agagatttgt acgctaactc tagccagaac aaaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatttt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 acaagaacct gcaagatttt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcacggacca 1200 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcacggacca 1200 gccagtacag gcaaatctat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 gccagtacag gcaaatctat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagctaatgt gaactttcca tttaatgact gtaccaacaa gaacttgatt 1320 tgctataatg cagctaatgt gaactttcca tttaatgact gtaccaacaa gaacttgatt 1320 tgggtagaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 tgggtagaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacaaattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaattag agacagaatg ctcaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caagaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgcgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc aaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcaat aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 13 <211> 2019 <212> DNA <213> Mouse parvovirus 4b <400> 13 atggctggaa atgcttactc tgatgaagtt ttgggagcaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtattctc atttgttttt aaaaatgagg atgttcaact gaatggaaaa 120 gatattggat ggaatagcta caaaaaagag ctacaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtaacca aaaagcaagt attcattttt gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat agctcctagt gatgttaatt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctatcacc agctgaaaga attaaactaa gagaaatagc agaagacaat 540 gagtgggtta gcttgctcac ttataagcat aagcaaacca aaaaggacta tactaagtgt 600 gttctttttg gcaacatgat tgcttactac tttttaacca aaaagaaaat aagcactagt 660 ccaccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcacaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ctaacttttc actgccggac 1080 acaagaacct gcaagatttt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttttaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt aaactttcca ttcaatgact gtaccaacaa gaacttgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agttcaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcagaa taggctgtga agaaagacca 1500 gagcacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgtg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 14 <211> 2042 <212> DNA <213> Mouse parvovirus 4a <400> 14 atggctggaa atgcttactc tgatgaagtt ttgggagcaa ccaactggtt aaaggaaaaa 60 Met Ala Gly Met Leu Thr Leu Asp Glu Val Phe Gly Ser Asn Pro Thr Gly Lys Gly Lys Lys aataatcagg aagtgttctc atttgttttt aaaaatgagg atgttcaact gaatggaaaa 120 Asn Asn Gln Glu Val Phe Ser Phe Val Phe Lys Asn Glu Asp Val Gln Leu Met Gly Lys gatatcggat ggaatagtta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 Asp Ile Gly Met Gly Ile Val Thr Lys Arg Leu Gln Glu Asp Glu Leu Lys Ile Phe Thr Gln cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tgcagtggat 240 Arg Glu Arg Glu Asn Tyr Leu Gly Pro Lys Ala Glu Asp Met Glu Trp Glu Ser Ala Val Asp gaagtgacca aaaagcaagt attcattttt gactctttag ttaaaaaatg tttgtttgaa 300 Glu Val Thr Lys Ser Ser Ile His Phe Phe Asp Ser Leu Val Lys Asn Val Phe Glu gtgcttaaca caaagaacat agctcctgct gatgttaatt ggtttgtgca gcatgaatgg 360 Val Leu Thr Gln Glu His Ser Pro Ala Asp Val Ile Trp Phe Cys Gln His Glu Trp ggaaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 Gly Lys Asp Gln Gly Gly His Ala Met Tyr Leu Ile Gly Gly Lys Asp Leu Ser Lys Ala caaggaaagt ggtggagaag gcagctaaat gtttactgga gcagatggtt agtaacagcc 480 Gln Gly Lys Val Gly Glu Lys Ala Ala Asn Val Tyr Gly Gln Asp Gly Val Ser Asn Ser Pro tgtaatgtac agctatcacc agctgaaaga attaaactaa gagaaatagc agaagacaat 540 Cys Asn Val Gln Ala Ile Thr Ser Glu Arg Ile Lys Leu Glu Glu Ile Ala Glu Asp Asn gagtgggtta ccttgctcac ttataagcat aagcaaacca aaaaggacta tactaagtgt 600 Glu Trp Val Thr Leu Ala His Leu Ile Ser Ile Ser Lys Pro Lys Arg Thr Thr Ser Val gttctttttg gcaacatgat tgcttactac tttttaacca aaaagaaaat aagcactagt 660 Val Phe Phe Gly Thr Met Ile Ala Tyr Tyr Phe Thr Lys Glu Asn Lys His Ser Val ccaccaaggg acggaggcta ttttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatatactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcaa ttaaaaccac acttaaagag ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acactaactc tagccagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa caaacttttc actgcctgat 1080 acaagaacct gcaagatttt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttttaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatctat cattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacatcaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcaacga gccgctgaac ttggactaag gtacgatggc gcctccagct 2040 aa 2042 <210> 15 <211> 2019 <212> DNA <213> Mouse parvovirus 1b <400> 15 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaagaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gagtgggtta ctttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttactac tttttaacca agaagaaaat aagcactagt 660 ccgccaaggg acggaggcta ttttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcaaaa taggctgcga ggagagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaagtgag tggcccatga tctgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 16 <211> 2019 <212> DNA <213> Mouse parvovirus minute virus immunosuppressive variant <400> 16 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaaaaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gagtgggtta ctttactcac ttataaacat aagcaaacca aaaaggacta tactaaatgt 600 gttctttttg gaaatatgat tgcttactac tttttaacca aaaagaaaat aagcaccagt 660 ccgccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ttatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctagtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acgctaactc tagccagaac aaaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatttt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcacggacca 1200 gccagtacag gcaaatctat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagctaatgt gaactttcca tttaatgact gtacgaacaa aaacttgatt 1320 tgggtagaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacaaattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaattag agacagaatg ctcaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caagaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgcgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 17 <211> 2019 <212> DNA <213> Mouse parvovirus 1 <400> 17 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaagaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gagtgggtta ctttacttac ttataaacat aagcaaacca aaaaggacta tactaaatgt 600 gttctttttg gaaatatgat tgcttactac tttttaacca aaaaaaaaat aagcaccagt 660 ccgccaagag acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg accagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 18 <211> 2019 <212> DNA <213> Mouse parvovirus 5a <400> 18 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgaag atgttcaact gaatggaaaa 120 gatattggat ggaataatta cagaaaggag ctgcaagagg acgagctaaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtacttagca caaagaacat agctcctagt gatgttaatt ggtttgtgca gcatgaatgg 360 ggaagagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaagt gtttactgga gcagatggtt ggtaacagct 480 tgtaatgtac agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gaatgggtta ccttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttctttttg gaaacataat tgcttactac tttttaacta aaaagaaaat aagcaccagt 660 ccgccaagag acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcaa ttaaaaccac acttaaagag ctggtgcata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacctgctaa aaaatacgct agagatttgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 acaagaacct gcaaaatttt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aagagaaata ctgttttatt tcatggacca 1200 gccagcacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 19 <211> 2019 <212> DNA <213> Mouse parvovirus UT <400> 19 atggctggaa acgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta cagaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcag aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat atctcctggt gatgttaatt ggtttgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagccaagct 420 caaggaaagt ggtggagaag gcagctaagt gtttactgga gcagatggtt agtaacagcc 480 tgtaatgtgc agctatcacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gagtgggtta ccttgctcac ttataagcat aagcaaacca aaaaagacta tactaagtgt 600 gttctttttg gcaacataat tgcttactac tttttaacca agaagaaaat aagcactagt 660 ccgccaaggg acggaggcta ttttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaagtttcta ttaaaaccac acttaaagaa ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctaa aaaatacgct agagatttgt acgctaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ctaacttttc actgcctgac 1080 acaagaacct gcaagatttt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcaaaa taggctgcga ggagagacca 1500 gaacacactc aaccaataag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaagtgag tggcccatga tctgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 20 <211> 2009 <212> DNA <213> Mouse parvovirus 1e <400> 20 atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa agtaaccagg 60 aagtgttctc atttgttttt aaaactgaag atattcaact gaatggaaaa gatattggat 120 ggaataatta cagaaaggag ctgcaagagg acgagctaaa atctttacaa cgaggagcgg 180 aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat gaagtgacca 240 aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa gtgcttaaca 300 caaagaacat atctcctggt gatgttaatt ggtttgtgca gcatgaatgg ggaagagacc 360 aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct caaggaaagt 420 ggtggagaag gcagctaagt gtttactgga gcagatggtt ggtaacagcc tgtaatgtac 480 agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt gaatgggtta 540 ccttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt gttctttttg 600 gcaacataat tgcttactac tttttaacca agaagaaaat aagcaccagt ccgccaaggg 660 acggaggcta ttttcttagt agtgactctg gctggaaaac taacttttta aaagagggcg 720 aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg gttgaaacca 780 cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa gaggtttcta 840 ttaaaaccac acttaaagag ctagtgcata agagagtaac ctcaccagaa gactggatga 900 tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa aacctgctga 960 aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca tttgacttga 1020 ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac acaagaacct 1080 gcaagatctt tgcttttcat ggctggaact acattaaagt ttgccatgct atttgctgtg 1140 ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca gccagtacag 1200 gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt tgctataatg 1260 cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt tgggtggaag 1320 aagctggtaa ctttggacaa caagtaaacc agtttaaagc catttgctct ggtcaaacaa 1380 ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca gtcatcatga 1440 ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca gaacacactc 1500 aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct ggtgactttg 1560 gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag aatggttacc 1620 aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg acggaaaact 1680 gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc tcaccattca 1740 cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt gcatcggatc 1800 tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg ggcactgcag 1860 aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa ctgagcccaa 1920 cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg ttgaagagag 1980 acttcagcga gccgctgaac ttggactaa 2009 <210> 21 <211> 2019 <212> DNA <213> Mouse parvovirus 1c <220> <221> misc_feature <222> (609)..(609) <223> n is a, c, g, or t <220> <221> misc_feature <222> (1590)..(1590) <223> n is a, c, g, or t <400> 21 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgaag atgttcaact aaatggaaaa 120 gatattggat ggaataatta cagaaaggag ctgcaagagg acgagctaaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat atctcctggt gatgttaatt ggtttgtgca gcatgaatgg 360 ggaagagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaagt gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtac agctaacacc agctgaaaga attaaactaa gagaaatagc agaagacagt 540 gaatgggtta ccttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttcttttng gcaacataat tgcttactac tttttaacca agaagaaaat aagcaccagt 660 ccgccaaggg acggaggcta ttttcttagt agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctagtgcata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgttataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaacatgan tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgaaag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 22 <211> 2019 <212> DNA <213> Hamster parvovirus <400> 22 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaagagaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaag atgttcagct caatggaaaa 120 gatatcggat ggaatagtta caaaaaggag ctgcaagagg aagagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtggat 240 gaactaacca aaaagcaagt attcattttt gactctttag ttaaaaaatg tttgtttgaa 300 gtgctgagta caaagaacat agcacctagt gatgttactt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgta ctaattggag gcaaggactt tagccaagct 420 caaggaaaat ggtggagaag gcagttaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtagtgtgc agctattacc agctgaaaga attaagctga gagagatagc ggaagaccaa 540 gaatgggtca ctttgcttac ttataagcat aagcaaacca aaaaagacta taccaagtgt 600 aatgggtca ctttgcttac ttataagcat aagcaaacca aaaaagacta taccaagtgt 600 gtttgctttg gaaatatagt tgcttactac tttttatcca agaagaaaat atgcaccagt 660 gtttgctttg gaaatatagt tgcttactac tttttatcca agaagaaaat atgcaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtctcta ttaaaaccac acttaaagag ctggtgcata agagagtaac ctcaccagaa 900 gaggtctcta ttaaaaccac acttaaagag ctggtgcata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acactaactc tagccagaac aaaaacagca 1020 aacctgctga aaaatacgct agagatttgt acactaactc tagccagaac aaaaacagca 1020 tttgacttaa ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 tttgacttaa ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact atattaaagt ttgccatgct 1140 acaagaacct gcaagatctt tgcttttcat ggctggaact atattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca tttaatgact gcaccaacaa aaacctgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgtga agaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaacatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 23 <211> 2019 <212> DNA <213> Mouse parvovirus 3 <400> 23 atggctggaa acgcttactc tgatgaagtt ttaggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaag atgttcaact gaatggaaaa 120 gatatcggat ggaataatta cagaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tgcagtggat 240 gaactaacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgctgagta caaagaacat agctcctagt gatgttactt ggtttgtacg gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgtg ctcattggag gcaaggactt tagccaagct 420 caaggaaaat ggtggagaag gcagttaaat gtttactgga gcagatggtt agtaacagcc 480 tgtaatgtgc agttatcacc agctgaaaga attaagctga gagagatagc ggaagaccaa 540 gaatgggtca ctttgcttac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttctttttg gaaatatagt tgcttactac tttttaacca agaagaaaat aagcaccagt 660 ccaccaaggg acggagacta ttttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg accagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaagaaa 840 gaggtctcta ttaaaaccac acttaaagag ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acactaactc tagccagaac aaaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact atattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gcaccaacaa aaacctgatt 1320 tgggtagaag aagctggtaa ctttggacag caagtaaacc aatttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgtga agaaagacca 1500 gaacacactc aaccaatcag agatagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaacatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 24 <211> 2019 <212> DNA <213> Mouse minute virus <400> 24 atggctggaa atgcttactc tgatgaggtt ttgggagcaa ccaactggtt aaaggaaaaa 60 agtaaccagt tagtattctc atttgttttt aaaaatgaag atgttcaatt gaatggaaaa 120 gatatcggat ggaatagtta cagaaaggag ctgcaagagg acgagctaaa atctttacaa 180 cgaggagcgg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtggat 240 gaactaacca caaagcaagt attcattttt gactctttag ttaaaaagtg tttatttgaa 300 gtgctaagta caaagaacat agctcctagt gatgttaatt ggtatgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagccaagct 420 caaggaaagt ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgcagtgtgc agctatcacc agccgaaaga attaagctga gagaaatagc ggaagaccaa 540 gaatgggtca ctttgcttac ttataagcat aagcaaacca aaaaagacta taccaaatgt 600 aatgggtca ctttgcttac ttataagcat aagcaaacca aaaaagacta taccaaatgt 600 gtttgctttg gaaatatgat tgcttactac tttttaacca agaagaaaat atgcactagt 660 gtttgctttg gaaatatgat tgcttactac tttttaacca agaagaaaat atgcactagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata agagagtaac ctcaccagaa 900 gaggtttcta ttaaaaccac acttaaagag ctggtgcata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcagcc aggtggagaa 960 gactggatga tgatgcagcc agacagttac attgaaatga tggctcagcc aggtggagaa 960 aacctgctta aaaatacgct agagatctgt acgctaactc tagctagaac caaaacagcc 1020 aacctgctta aaaatacgct agagatctgt acgctaactc tagctagaac caaaacagcc 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 acaagaacct gtaagatttt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 acaagaacct gtaagatttt tgcttttcat ggctggaact acattaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat cattgcacaa gccatagcac aggcagttgg taatgttggt 1260 gccagtacag gcaaatccat cattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gcaccaacaa aaacctgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc aatttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaattag agacagaatg ctcaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 25 <211> 2019 <212> DNA <213> MVM strain M <400> 25 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaagagaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaag atgttcagct caatggaaaa 120 gatatcggat ggaatagtta caaaaaggag ctgcaagagg aagagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtggat 240 gaactaacca aaaagcaagt attcattttt gactctttag ttaaaaaatg tttgtttgaa 300 gtgctgagta caaagaacat agctcctagt gatgttactt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaagctggca ctgtcatgta ctaattggag gcaaggactt tagccaagct 420 caaggaaaat ggtggagaag gcagttaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtagtgtgc agctatcacc agctgaaaga attaagctga gagagatagc ggaagaccaa 540 gaatgggtca ctttgcttac ttataagcat aagcaaacca aaaaagacta taccaagtgt 600 gtttgctttg gaaatatagt tgcttactac tttttatcca agaagaaaat atgcaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca caataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtctcta ttaaaaccac acttaaagag ctggtacata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctta aaaatacgct agaaatctgt acgctaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca tttaatgact gcaccaacaa aaacctgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta tccgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcagaa taggctgcga ggagagacca 1500 gagcacactc aaccaatcag agacagaatg ctcaacattc atctgacaca tacattgcct 1560 ggtgactttg gtttggttga caagaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt acctgattgg 1680 tcagaaaact gggcagagcc gaaggtaccg actcctataa attcactagg ttcagcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 26 <211> 1966 <212> DNA <213> Parvovirus LuII <400> 26 atggctggaa acgcgtactc tgatgaagtt ttgggaacaa ctaactggtt gaaggataag 60 agcaaccagg aagtattctc atttgttttt aaaaatgagg atgttcagct caatggaaaa 120 aatatcggat ggaacagtta cagaaaggag ctgcaagagg aggagctgaa atctttacaa 180 cgaggagctg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtggat 240 gaactgacca aaaagcaagt attcattttt gactctttag ttaaaaagtg tctctttgaa 300 gtactgagca caaagaacat agctcctagt gatgttactt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgtg ctcattggag gcaagaactt tagccaggct 420 caaggaaaat ggtggaggag acaattaaat gtttactgga gtagatggtt ggtaacagcc 480 tgtagcgtgc agctatcacc agctgaaaga attaaactaa gagaaatagc agaagaccaa 540 gaatgggtta ctctgcttac ttataagcat aagcaaacca aaaaagacta tactaagtgt 600 gtttgctttg gaaatatggt tgcttactac tttttaacca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttctcagt agtgactctg gctggaaaac taactttttg 720 ccaccaaggg acggaggcta ttttctcagt agtgactctg gctggaaaac taactttttg 720 aaagaaggcg aacgccatct agtgagcaaa ctatatactg atgacatgcg gccagaaacg 780 aaagaaggcg aacgccatct agtgagcaaa ctatatactg atgacatgcg gccagaaacg 780 gttgagacca cagtaaccac agcgcaggaa actaagcgcg gcagaattca aactaagaag 840 gttgagacca cagtaaccac agcgcaggaa actaagcgcg gcagaattca aactaagaag 840 gaagtctcta ttaagactac acttaaagag ctggtacata agagagtaac ctcaccagaa 900 gaagtctcta ttaagactac acttaaagag ctggtacata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc agggggagaa 960 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc agggggagaa 960 aacctactta agaatacgct agagatctgt acgctgactc tagccagaac caaaacagcc 1020 aacctactta agaatacgct agagatctgt acgctgactc tagccagaac caaaacagcc 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttt actggctgat 1080 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttt actggctgat 1080 acaagaacct gtagaatctt tgcttttcat ggctggaact acatcaaagt ctgtcatgct 1140 acaagaacct gtagaatctt tgcttttcat ggctggaact acatcaaagt ctgtcatgct 1140 atttgttgtg tcttgaacag acagggaggc aaaagaaata ctgttctgtt tcatggacca 1200 atttgttgtg tcttgaacag acagggaggc aaaagaaata ctgttctgtt tcatggacca 1200 gccagtacag gcaaatcaat cattgcacag gccatagcac aggcagttgg taatgttggt 1260 gccagtacag gcaaatcaat cattgcacag gccatagcac aggcagttgg taatgttggt 1260 tgttataacg cagccaatgt gaactttcca tttaatgact gtaccaacaa gaacttaatc 1320 tgttataacg cagccaatgt gaactttcca tttaatgact gtaccaacaa gaacttaatc 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgttct 1380 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgttct 1380 ggtcagacca ttcgcattga ccaaaaagga aaaggcagca aacagattga accaacacca 1440 gtgatcatga ccacaaatga aaacatcaca gtggtcaaaa tagggtgtga agagagacca 1500 gaacacactc aaccaatcag agacagaatg ttaaacattc atctgacaca tacattgcct 1560 ggtgactttg gtttggttga taaaaacgaa tggcctatga tatgtgcttg gttggtaaag 1620 aacggttacc aatcgaccat ggcaagttac tgtgctaaat ggggcaaagt tcctgattgg 1680 acagaaaact gggcggagcc aaaagtaacg actgaaataa attcggtagg ttcaaccaac 1740 tcaccatctc cgaaaagtac gcctctcagc cagaactacg cactaactcc gtcggatctc 1800 gaggacctgg ctctggagcc ttggagcaca ccaagtactc ctgttgtggg cactgtcaaa 1860 accccgaaca ctggggaaac tggttcaaca gcctgtcaag aagctcaacg gagcccaact 1920 tggtccgaga tcgaggagga tttgagagcg tgcttcagtt cggaac 1966 <210> 27 <211> 2019 <212> DNA <213> Rat parvovirus UT <400> 27 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccaag aggtgttctc atttgttttt aaaaatgaga acgtccagct aaatgggaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggagcgg aaaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaaatg tttgtttgaa 300 gtgctcagca caaagaacat aactcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctaattggag gcaaagactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga ataaaactga gagaaatagc agaggacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttttaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaag ctgtatactg atgagatgaa accagaaacg 780 gttgagacca cagtgaccac agcacaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcta ttaaaaccac acttaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagca 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctaac 1080 accagaacct gtagaatctt tgctgaacat ggctggaact atattaaagt ctgtcatgcc 1140 atctgttgtg tgctaaatag acaaggaggc aaaaggaaca ctgtgctctt tcatggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttataatg ctgccaatgt gaactttcca tttaatgact gcaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaagact gggcggagcc gaagctagag actcctataa attcactagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgtg 1860 ggcactgcag caagccagaa cactggggag gctggtttca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgacc ttggactaa 2019 <210> 28 <211> 2019 <212> DNA <213> Kilham rat virus <400> 28 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaagtg tttgtttgaa 300 gtgctcagca caaagaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaagacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttctaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctatatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctaaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aaggagttgg taatgttggt 1260 tgttataatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagag actcctataa tttcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccagaa cactggggag gctggtttca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgaca ttggactaa 2019 <210> 29 <211> 2024 <212> DNA <213> Rat minute virus 1c <400> 29 atggctggaa acgcttactc cgatgaagtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aagtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gacatcggtt ggaatagtta cagaaaagag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaagtg tctgtttgaa 300 gtgctcagca caaagaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt agtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaggacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttctaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatttgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tactaaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aaggagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga gaacattaca gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctaa tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agccctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccagaa cactggggag gctggtttca cagcctgtca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgacc ttggactaag gtac 2024 <210> 30 <211> 2019 <212> DNA <213> Rat minute virus 1b <400> 30 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctgcaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaagtg tctgtttgaa 300 gtgctcagca caaagaacat agctcctagt gatgttactt ggtttgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaggacagt 540 gaatgggtga ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttctaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttt catggctaac 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgtcatgcc 1140 atctgttgtg tgctaaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aaggagttgg taatgttggt 1260 tgttataatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcaaaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggatc tcgcggacct ggcactggaa ccttggagca caccaaatac tcctgttgtg 1860 gacactgtac aaaccccgaa cactggggag gctggtttca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgacc ttggactaa 2019 <210> 31 <211> 1997 <212> DNA <213> Rat minute virus 1a <400> 31 atgaggtttt gggagcaacc aactggctaa aggacaaaag tagccaggag gtgttctcat 60 ttgtttttaa aaatgagaac gtccaactaa atgggaagga catcggttgg aatagttaca 120 gaaaggagct acaagatgac gagctgaagt ctttgcaacg aggggcggag accacttggg 180 accaaagcga ggacatggaa tgggagagcg cagtggatga catgaccaaa aagcaagtat 240 tcatttttga ttctttggtt aaaaagtgtc tgtttgaagt gctcagcaca aagaacatag 300 ctcctagtga tgttacttgg ttcgtgcagc atgaatgggg aaaggaccaa ggctggcact 360 gtcatgtgct gattggaggc aaggacttta gtcaacctca aggaaagtgg tggagaaggc 420 agctaaatgt gtactggagt agatggttgg tgactgcctg taatgttcaa ctaacaccag 480 ctgaaagaat taaactgaga gaaatagcag aggacagtga atgggtcact ttgcttacct 540 ataagcataa acacaccaag aaggactata ccaagtgtgt tttttttgga aacatgattg 600 cttattactt tctaagcaaa aagaaaatat gtaccagtcc accaagggac ggaggctatt 660 ttcttagcag tgactctggc tggaaaacta actttttgaa agagggcgag cgccatctag 720 tgagcaaact gtatactgat gagatgaaac cagaaacggt cgagaccaca gtgaccactg 780 cgcaggaagc taagcgcggc agaattcaaa ctagaaagga ggtctcgatt aaaaccacac 840 tcaaagagtt ggtgcataaa agagtaacct caccagaaga ctggatgatg atgcagccag 900 acagttacat tgaaatgatg gctcaaccag gtggagaaaa cttgcttaaa aatacactag 960 agatctgtac actgactcta gcaagaacca aaacagcctt tgacttgatt ctagaaaaag 1020 ctgaaaccag caaactagcc aacttttcca tggctaacac cagaacctgt agaatctttg 1080 ctgagcatgg ctggaactat attaaagtct gccatgccat ctgttgtgtg ctgaatagac 1140 aaggaggcaa aaggaacact gtgctctttc acggaccagc cagcacaggc aaatctatca 1200 ttgcacaagc catagcacaa ggagttggta atgttggttg ttacaatgct gccaatgtga 1260 actttccatt taatgactgt accaacaaaa acttgatttg ggtggaagaa gctggtaact 1320 ttggccagca agtaaaccaa ttcaaagcta tttgttctgg ccaaaccata cgcattgatc 1380 aaaaaggaaa aggcagcaaa cagattgaac caacaccagt tatcatgacc accaacgaga 1440 acattaccgt ggtcagaata ggctgtgagg aaaggccaga acacactcaa ccaatcagag 1500 acagaatgct caacattcac ctgacacgta cactgcctgg tgactttggt ctggtggata 1560 agcacgaatg gcctctgatc tgtgcttggt tggtgaagaa tggttaccaa tctaccatgg 1620 cttgttactg tgctaaatgg ggcaaagttc ctgattggtc agaggactgg gcggagccga 1680 agctagagac tcctataaat tcgctaggtt caatgcgctc accatctctg actccgagaa 1740 gtacgcctct cagccagaac tacgctctta ctccacttgc atcggacctt gcggacctag 1800 ctctagagcc ttggagcaca ccaaatactc ctgttgcggg cactgcagca agccagaaca 1860 ctggggaggc tggtttcaca gcctcccaag gtgctcaacg gagcccaacc tggtccgaga 1920 tcgaggcgga tctgagagcg tgcttcagcc aggaacagct ggagaaagac ttcagcgatt 1980 cactgacctt ggactaa 1997 <210> 32 <211> 2019 <212> DNA <213> H-1 parvovirus <400> 32 atggctggaa acgcttactc cgatgaggtt ttgggagtaa ccaactggct gaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaaa acgtccaact aaatggaaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt atttattttt gattctttgg ttaagaagtg tttgtttgaa 300 gtgctcagca caaagaacat agctcctagt aatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaacct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaggacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttttaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcacaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctgaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttattatga ccaccaacga gaacattacc gtggttagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactacct 1560 ggtgactttg gtttggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagac actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaaa actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccaaaa cactggggag gctggttcca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggagctgacc ttggactaa 2019 <210> 33 <211> 2019 <212> DNA <213> Rat minute virus isolate NTU1 <400> 33 atggctggaa acgcttactc cgatgaagtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaagtg tttgtttgaa 300 gtgctcagca caaagaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ttgattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaggacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttctaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcacaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatatgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc tatggctaac 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctaaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctacaat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 aatggttacc aatctacaat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaagact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcagaagact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctggag ccttggagca caccaaatac tcctgttgcg 1860 gcatcggacc ttgcggacct agctctggag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccagaa cactggggag gctggtttcg cagcctgcca aggtgctcaa 1920 ggcactgcag caagccagaa cactggggag gctggtttcg cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatagaagca gacttgagag cttgcttcag tcaagaacag 1980 cggagcccaa cctggtccga gatagaagca gacttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggaactgacc ttggactaa 2019 ttggagagcg acttcaacga ggaactgacc ttggactaa 2019 <210> 34<210> 34 <211> 2019 <211> 2019 <212> DNA <212> DNA <213> Parvovirus h-1 <213> Parvovirus h-1 <400> 34 <400> 34 atggctggaa acgcttactc cgatgaggtt ttgggagtaa caaactggct gaaggacaaa 60 atggctggaa acgcttactc cgatgaggtt ttgggagtaa caaactggct gaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaaa acgtccaact aaatggaaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt atttattttt gattctttgg ttaagaagtg tttgtttgaa 300 gtgctcagca caaagaacat agctcctagt aatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc caggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaacct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaggacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttttaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcacaggaa gctaagcgcg gcagaattca aactagagag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctgaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttattatga ccaccaacga gaacattacc gtggttagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactacct 1560 ggtgactttg gtttggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagac actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaaa actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccaaaa cactggggag gctggttcca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggagctgacc ttggactaa 2019 <210> 35 <211> 1997 <212> DNA <213> Rat minute virus isolate NTU2 <400> 35 atgaggtttt gggagcaacc aactggctaa aggacaaaag tagccaggag gtattctcat 60 ttgtttttaa aaatgagaac gtccaactaa atgggaagga catcggttgg aatagttaca 120 gaaaggagct acaagatgac gagctgaagt ctctacaacg aggggcggag accacttggg 180 accaaagcga ggacatggaa tgggagagcg cagtggatga catgaccaaa aagcaagtat 240 tcatttttga ttctttggtt aagaagtgtc tgtttgaagt gctcagcaca aagaacatag 300 ctcctagtga tgttacttgg ttcgtgcagc atgaatgggg aaaggaccaa ggctggcact 360 gtcatgtgct gattggaggc aaggacttta gtcaagctca aggaaaatgg tggagaaggc 420 agctaaatgt gtactggagt agatggttgg tgactgcctg taatgttcaa ctaacaccag 480 ctgaaagaat taaactaaga gaaatagcag aggacagtga atgggtcact ttgcttacct 540 ataagcataa gcacaccaag aaggactata ccaagtgtgt tctttttgga aacatgattg 600 cttattactt tctaagcaaa aagaaaatat gtaccagtcc accaagggac ggaggctatt 660 ttcttagcag tgactctggc tggaaaacta actttttgaa agagggcgag cgccatctag 720 tgagcaaact gtatactgat gagatgaaac cagaaacggt cgagaccaca gtgaccactg 780 cacaggaagc taagcgcggc agaattcaaa ctagaaagga ggtctcgatt aaaaccacac 840 tcaaagagtt ggtacataaa agagtaacct caccagaaga ctggatgatg atgcagccag 900 acagttatat tgaaatgatg gctcaaccag gtggagaaaa cttgcttaaa aatacactag 960 agatatgtac actgactcta gcaagaacca aaacagcctt tgacttgatt ctggaaaaag 1020 ctgaaaccag caaactagcc aacttttcta tggctagcac cagaacctgt agaatctttg 1080 ctgagcatgg ctggaactat attaaagtct gccatgccat ctgttgtgtg ctaaatagac 1140 aaggaggcaa aaggaacact gtgctctttc acggaccagc cagcacaggc aaatctatca 1200 ttgcacaagc catagcacaa gcagttggta atgttggttg ttacaatgct gccaatgtga 1260 actttccatt taatgactgt accaacaaaa acttgatttg ggtggaagaa gctggtaact 1320 ttggccagca agtaaaccaa ttcaaagcta tttgttctgg ccaaaccata cgcattgatc 1380 aaaaaggaaa aggcagcaaa cagattgaac caacaccagt tatcatgacc accaacgaga 1440 acattaccgt ggtcagaata ggctgtgagg aaagaccaga acacactcaa ccaatcagag 1500 acagaatgct caacattcac ctgacacgta cactgcctgg tgactttggt ctggtggata 1560 agcacgaatg gcctctgatc tgtgcttggt tggtgaagaa tggttaccaa tctaccatgg 1620 cttgttactg tgctaaatgg ggcaaagttc ctgattggtc agaagactgg gcggagccga 1680 agctagagac tcctataaat tcgctaggtt caatgcgctc accatctctg actccgagaa 1740 gtacgcctct cagccagaac tacgctctta ctccacttgt atcggacctt gcggacctag 1800 ctctggagcc ttggagcaca ccaaatactc ctgttgcggg cactgcagca agccagaaca 1860 ctggggaggc tggtttcgca gcctgtcaag gtgctcaacg gagcccaacc tggtccgaga 1920 tagaagcaga cttgagagct tgcttcagtc aagaacagtt ggagagcgac ttcaacgagg 1980 agctgacctt ggactaa 1997 <210> 36 <211> 2019 <212> DNA <213> Rat minute virus 2a <400> 36 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaagtg tctgtttgaa 300 gtgctcagca caaagaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaaactga gagaaatagc agaggacagt 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttctaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttcttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatatgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgttgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tactaaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga aaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctacaat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaagact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctggag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgtag caagccagaa cactggggag gctggtttcg cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatagaagca gacttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggaactgacc ttggactaa 2019 <210> 37 <211> 192 <212> DNA <213> Artificial <220> <223> consensus sequence <400> 37 ctarrrarga rgtytcdatt aaaacyacac tyaaagaryt rgtrcataar agagtaacct 60 caccagarga ctggatgatg atgcagccag acagttayat tgaaatgatg gctcarccag 120 gkggagaaaa cytrctdaar aatacrctag aratytgtac rctractcta gchagaacma 180 aaacagcmtt tg 192

Claims

1. A composition comprising an artificial nucleotide sequence, a fluorophore, and a quencher, wherein the artificial nucleotide sequence comprises, at its 3'-end, 5 or fewer consecutive nucleic acids identical to any one of the sequences of SEQ ID NO: 9 and 12-37.

2. The composition according to claim 1, wherein the fluorophore is selected from the group of fluorophores having an excitation wavelength in the range of 495 nm or more and 680 nm or less, and an emission wavelength in the range of 515 nm or more and 710 nm or less.

3. The composition according to claim 2, wherein the quencher is selected from the group of dyes having an absorption peak in the range of 430 nm or more and 672 nm or less.

4. The composition according to claim 1, wherein the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm.

5. The composition according to claim 4, wherein the fluorophore is FAM.

6. The composition according to claim 5, wherein the quencher is BHQ-1.

7. The composition according to claim 1, wherein the fluorophore is bound to the 5'-end of the artificial nucleotide sequence, and the quencher is bound to the 3'-end of the artificial nucleotide sequence.

8. The artificial nucleotide sequence comprises the nucleic acid sequence of.

9. a. A rodent parvovirus-specific forward oligonucleotide primer; b. A rodent parvovirus-specific oligonucleotide detection probe; c. An artificial oligonucleotide detection probe; d. A rodent parvovirus-specific reverse oligonucleotide primer; e. An M13-specific forward oligonucleotide primer; f. An M13-specific oligonucleotide detection probe; and g. An M13-specific reverse oligonucleotide primer comprising.

10. The composition according to claim 9, wherein the oligonucleotide sequences of a, b, and d each comprise the NS-1 sequence of a rodent parvovirus selected from the group consisting of the murine minute virus prototype strain (MVMp), the murine minute virus immunosuppressive strain (MVMi), the murine minute virus Cutter strain (MVMc), murine parvovirus 1b (MPV-1b), murine parvovirus 1a (MPV-1a), murine parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat minute virus, and the Umass strain of rat virus L (RV-Umass).

11. The composition according to claim 9, wherein the artificial oligonucleotide detection probe comprises a nucleotide sequence having, at its 3'-end, 5 or fewer consecutive nucleic acids identical to any one of the sequences of SEQ ID NOs: 9 and 12 to 37.

12. The composition according to claim 10, wherein the oligonucleotide sequences of a, b, and d hybridize to SEQ ID NO:

37.

13. The composition according to claim 9, wherein each of the detection probes of b, c, and f comprises a fluorophore and a quencher such that the fluorophores for each detection probe emit light at different wavelengths.

14. The composition according to claim 13, wherein the fluorophore of each probe is selected from the group of fluorophores having an excitation wavelength in the range of 495 nm or more and 680 nm or less, and an emission wavelength in the range of 515 nm or more and 710 nm or less.

15. The composition according to claim 14, wherein the quencher is selected from the group of dyes having an absorption peak in the range of 430 nm or more and 672 nm or less.

16. The composition according to claim 13, wherein the artificial oligonucleotide detection probe (c) comprises the fluorophore FAM and the quencher BHQ-1; the rodent parvovirus-specific oligonucleotide detection probe (b) comprises the fluorophore VIC and a minor groove-binding non-fluorescent quencher (MGBNFQ); and the M13-specific oligonucleotide detection probe comprises the fluorophore Cy5 and the quencher BHQ2.

17. The composition according to claim 12, wherein the rodent parvovirus-specific forward oligonucleotide primer (a) comprises the nucleic acid sequence of SEQ ID NO:1; the rodent parvovirus-specific reverse oligonucleotide primer (d) comprises the nucleic acid sequence of SEQ ID NO:4; and the rodent parvovirus-specific oligonucleotide detection probe (b) comprises the nucleic acid sequence of SEQ ID NO:

2.

18. The composition according to claim 11, wherein the artificial oligonucleotide detection probe (c) comprises the nucleic acid sequence of SEQ ID NO:

3.

19. The composition according to claim 9, wherein the M13-specific forward oligonucleotide primer (e) comprises the nucleic acid sequence of SEQ ID NO:5; the M13-specific oligonucleotide detection probe (f) comprises the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer (g) comprises the nucleic acid sequence of SEQ ID NO:

7.

20. a. A step of mixing a plurality of components to prepare a reaction mixture, the components comprising (i) a nucleic acid sample derived from a test sample, (ii) an oligonucleotide, and (iii) a DNA polymerase; b. A step of subjecting the reaction mixture to a polymerase chain reaction (PCR); c. A step of monitoring the production of (i) a target amplification polynucleotide (TAP), (ii) a nucleic acid extraction control amplification polynucleotide (NACP), and (iii) a plasmid amplification control polynucleotide (PACP) during PCR; and d. A step of comparing the production of TAP with the production of NACP and PACP A method for detecting a biological contaminant in a test sample, comprising the presence of TAP produced during PCR, the presence of NACP, and the absence of PACP, indicating that the test sample contains a biological contaminant and does not contain a positive amplification control plasmid.

21. The method according to claim 20, wherein the oligonucleotide comprises (a) a murine parvovirus-specific forward oligonucleotide primer, (b) a murine parvovirus-specific oligonucleotide detection probe, (c) an artificial oligonucleotide detection probe, (d) a murine parvovirus-specific reverse oligonucleotide primer, (e) an M13-specific forward oligonucleotide primer, (f) an M13-specific oligonucleotide detection probe, and (g) an M13-specific reverse oligonucleotide primer.

22. The method according to claim 20, wherein the test sample is obtained from a mammalian cell culture or a purified fraction thereof.

23. The method according to claim 20, wherein M13K07 phage is added to the test sample.

24. The method according to claim 20, wherein the nucleic acid sample is prepared by subjecting about 1 mL of the test sample to lysis, proteolysis, and heat denaturation, followed by mixing the sample with an extraction control sample and then extracting the nucleic acid from the sample.

25. The method according to claim 24, wherein the extraction control sample is M13K07 phage.

26. (a) The murine parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1; (b) The murine parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding non-fluorescent quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2; (c) The artificial oligonucleotide detection probe comprises a FAM fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3; (d) The murine parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4; (e) The M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; (f) The M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:

7. The method according to claim 25.

27. The method according to claim 20, wherein the component comprises uracil-N-glycosylase (UNG).

28. The method according to claim 20, comprising the step of incubating the reaction mixture at 50 °C for at least 2 minutes.

29. The PCR step (b) is (i) incubating the reaction mixture at 95 °C for 2 minutes; followed by (ii) (1) 8 cycles of denaturation at 95 °C for 10 seconds, followed by (2) annealing at 30 seconds, wherein the annealing temperature of the first cycle of the 8 cycles is 70 °C, the annealing temperature decreases by 1 °C per cycle, and the annealing temperature of the last cycle of the 8 cycles is 62 °C; followed by (iii) at least 40 cycles of DNA amplification comprising (1) a denaturation step at 95 °C for 10 seconds, followed by (2) an annealing step at 62 °C for 30 seconds, wherein the rate of temperature change from the denaturation temperature to the annealing temperature is about 4.4 °C per second, and from the annealing temperature to the denaturation temperature is about 2.2 °C per second, said DNA amplification The method according to claim 28, comprising

30. (a) monitoring the production of TAP by measuring fluorescence at 533 - 580 nm in each amplification cycle; (b) monitoring the production of NACP by measuring fluorescence at 618 - 660 nm in each amplification cycle; and (c) monitoring the production of PACP by measuring fluorescence at 465 - 510 nm in each amplification cycle, the method according to claim 26.

31. The method according to claim 20, comprising comparing the results obtained from the method performed on a test sample with an external positive control and an external negative control, and if the negative control or the positive control fails, the results obtained from the method performed on the test sample are rejected.

32. The external positive control is a. a step of mixing a plurality of positive control components to prepare a positive control mixture, said positive control components comprising (i) a positive amplification control (PAC) plasmid, and (ii) a positive control oligonucleotide cocktail, and (iii) not containing a test sample, said step; b. subjecting the positive control mixture to a polymerase chain reaction (PCR) of the positive control; and c. monitoring the production of (i) a target amplification polynucleotide (TAP), (ii) a nucleic acid extraction control amplification polynucleotide (NACP), and (iii) a plasmid amplification control polynucleotide (PACP) during PCR The method according to claim 31, comprising the presence of TAP, NACP, and PACP produced during PCR, indicating that the PCR is functioning properly, and the absence of any one or more of TAP, NACP, or PACP indicating that the positive control has failed.

33. The method according to claim 32, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid.

34. The method according to claim 33, wherein (i) the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO:37; (ii) the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO:8; and (iii) the artificial nucleic acid sequence unique to the plasmid comprises the sequence of SEQ ID NO:

10.

35. The method according to claim 34, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:

11.

36. The positive control oligonucleotide cocktail comprises a rodent parvovirus-specific forward oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:1; a rodent parvovirus-specific oligonucleotide detection probe comprising a VIC fluorophore, an MGBNFQ quencher, and the nucleic acid sequence of SEQ ID NO:2; an artificial oligonucleotide detection probe comprising a FAM fluorophore, a BHQ quencher, and the nucleic acid sequence of SEQ ID NO:3; a rodent parvovirus-specific reverse oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:4; an M13-specific forward oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:5; an M13-specific oligonucleotide detection probe comprising a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and an M13-specific reverse oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:7 The method according to claim 33.

37. The positive control PCR comprises a. incubating the positive control mixture at 95°C for 2 minutes; followed by b. Eight cycles of (i) denaturation at 95°C for 10 seconds, followed by (ii) annealing for 30 seconds, where the initial annealing temperature of the eight cycles is 70°C, the annealing temperature decreases by 1°C per cycle, and the final annealing temperature of the eight cycles is 62°C; and subsequent c. DNA amplification in 40 cycles, including (i) a denaturation step at 95°C for 10 seconds, followed by (ii) an annealing step at 62°C for 30 seconds The method according to claim 36, comprising the above.

38. In positive control PCR, the production of TAP is monitored by measuring fluorescence at 533 - 580 nm in each amplification cycle, the production of NACP in positive control PCR is monitored by measuring fluorescence at 618 - 660 nm in each amplification cycle; and the production of PACP in positive control PCR is monitored by measuring fluorescence at 465 - 510 nm in each amplification cycle. The method according to claim 36.

39. a. Positive amplification control (PAC) plasmid; b. Rodent parvovirus - specific forward oligonucleotide primer; c. Rodent parvovirus - specific oligonucleotide detection probe; d. Artificial oligonucleotide detection probe; e. Rodent parvovirus - specific reverse oligonucleotide primer; f. M13 - specific forward oligonucleotide primer; g. M13 - specific oligonucleotide detection probe; h. M13 - specific reverse oligonucleotide primer; and i. Buffer A composition comprising the above.

40. The composition according to claim 39, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid.

41. The composition according to claim 40, wherein the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO:37, the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO:8, and the unique nucleic acid sequence comprises the sequence of SEQ ID NO:

10.

42. The composition according to claim 41, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:

11.

43. The rodent parvovirus - specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1; The rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2; The artificial oligonucleotide detection probe comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3; The rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4; The M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; The M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and The M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7, The composition according to claim 42.

Images