Serological assay for the detection of swine virus

Abstract

This disclosure provides a novel serological method for detecting zoonotic viral infections, such as porcine cytomegalovirus (PCMV) infection or porcine lymphotropic herpesvirus (PLHV) infection, in subjects at any stage of disease development. More specifically, this disclosure provides an automated multiplexed immunoassay for the rapid and simultaneous detection of one or more anti-PCMV antibodies or anti-PLHV antibodies in the biological fluid of subjects suspected of being infected with PCMV or PLHV.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 459,490, filed on April 14, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure generally relates to novel serological assays for detecting viremic or latent zoonotic viral infections, such as porcine cytomegalovirus (PCMV) infection or porcine lymphotropic herpesvirus (PLHV) infection, in a subject at any stage of onset.

Background Art

[0003] Porcine cytomegalovirus (PCMV) is an enveloped DNA virus belonging to the family Herpesviridae and the subfamily Betaherpesviridae. PCMV infection was previously known as "inclusion body rhinitis" based on the histopathological features of the disease. PCMV is an endemic in almost all pig populations worldwide, including in North America, and the seropositivity rate approaches 100% in most regions. However, PCMV is not considered a pathogen that causes production risks in the agricultural environment. PCMV infection has not been confirmed in humans. However, there are concerns about transmission through xenotransplantation. For example, PCMV infection has been suggested to affect the prognosis of xenotransplantation in non - human primate studies.

[0004] Polymerase chain reaction (PCR)-based methods have been developed by academic and diagnostic laboratories and are primarily used to detect PCMV infection in various animals. These methods are limited to detecting PCMV infection when the virus is in the active viremia stage. After the virus enters the incubation period, PCR-based assays tend to produce false-negative results. Furthermore, PCR-based assays are cumbersome. To date, no diagnostic laboratory offers a serological assay for PCMV.

[0005] Therefore, there is a need for a serological assay for PCMV that can be performed in any veterinary diagnostic laboratory (VDL) under Good Laboratory Practice (GLP) conditions to reliably detect PCMV infection at any stage of infection (e.g., during the incubation period relative to the viremia phase) and in any subject. This disclosure addresses this need. [Overview of the project]

[0006] One aspect of the present disclosure provides a method for detecting porcine cytomegalovirus (PCMV) in a biological sample derived from a subject, comprising, or essentially comprising, the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with a PCMV antigen for a sufficient time to allow binding between the corresponding antibody in the diluted serum sample and the PCMV antigen; and detecting the presence or absence of an antibody from the diluted serum sample that reacts with the PCMV antigen using Western blot analysis (e.g., a size-based automated serological Western blot such as WES®, Jess®, Abby®, Peggy Sue®, Sally Sue® (Bio-techne.com), or similar).

[0007] Another aspect of the present disclosure provides a method for detecting anti-porcine cytomegalovirus (PCMV) antibodies in a biological sample of interest, comprising, or essentially consisting of, the following steps: (a) introducing one or more operationalized PCMV antigens, a biological sample of interest, one or more anti-immunoglobulin antibodies, and one or more chemiluminescent molecules into one or more capillaries of a microfluidic device; (b) separating one or more operationalized PCMV antigens electrophoretically; (c) immobilizing one or more electrophoretically separated operationalized PCMV antigens onto the capillary wall; (d) contacting one or more immobilized operationalized PCMV antigens with a biological sample of interest; (e) incubating one or more immobilized operationalized PCMV antigens with the biological sample for about 10 minutes to about 120 minutes; and (f) detecting the binding of one or more operationalized PCMV antigens to the biological sample by immunodetection and / or chemiluminescence detection. In some embodiments, the presence of an immunodetection signal and / or chemiluminescence signal indicates the presence of one or more anti-PCMV antibodies in the biological sample.

[0008] Another aspect of the present disclosure provides an automated serological method (e.g., size-based automated serological Western blot) for detecting anti-porcine cytomegalovirus (PCMV) antibodies in porcine animals, comprising, or essentially consisting of, the following steps: (a) contacting one or more manipulated PCMV antigens with a biological sample derived from a porcine animal; (b) incubating one or more manipulated PCMV antigens with the biological sample for about 10 minutes to about 120 minutes; and (c) detecting the binding of one or more manipulated PCMV antigens to the biological sample by immunodetection and / or chemiluminescence detection (e.g., immunodetection signal and / or chemiluminescence signal).

[0009] In some embodiments of the automated serological methods described herein (e.g., automated serological Western blotting based on size), prior to step (a), one or more operationalized PCMV antigens are separated electrophoretically by mass and immobilized on a solid support. In some embodiments, the solid support is a capillary wall of a microfluidic device. In some embodiments, the method is carried out in a closed-loop automated capillary immunoassay system.

[0010] In some embodiments of any of the methods described herein, one or more operationalized PCMV antigens are incubated together with a biological sample for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

[0011] In some embodiments of any of the methods described herein, the anti-PCMV antibody is an IgM antibody or an IgG antibody. In some embodiments, one or more manipulated PCMV antigens are selected from the group consisting of envelope glycoprotein B (gB), envelope glycoprotein H (gH), envelope glycoprotein L (gL), envelope glycoprotein M (gM), envelope glycoprotein N (gN), major tegument phosphorylated protein 1 (U54A), major tegument phosphorylated protein 2 (U54B), and U100p. In some embodiments, one or more manipulated PCMV antigens include glycoprotein B (gB).

[0012] In some embodiments of any of the methods described herein, one or more operationalized PCMV antigens include an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs. 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, or 32.

[0013] In some embodiments, one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, 32, or a combination thereof. In some embodiments, one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NOs: 3 or 32. In some embodiments, one or more operationalized PCMV antigens include the amino acid sequences of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, and 32.

[0014] In some embodiments of any of the methods described herein, one or more operationalized PCMV antigens are encoded by polynucleotide sequences selected from SEQ ID NOs: 1, 5, 6, 7, 10, 11, or combinations thereof.

[0015] In some embodiments of any of the methods described herein, one or more operationalized PCMV antigens comprise glycoprotein gH and / or glycoprotein gL. In some embodiments, one or more operationalized PCMV antigens comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 18 or 22. In some embodiments, one or more operationalized PCMV antigens comprise the amino acid sequence of SEQ ID NO: 18 and / or 22.

[0016] In some embodiments, one or more operationalized PCMV antigens are encoded by polynucleotide sequences selected from SEQ ID NOs: 17, 21, or combinations thereof.

[0017] In some embodiments of any of the methods described herein, one or more operationalized PCMV antigens comprise U100p(gQ).

[0018] In some embodiments of any of the methods described herein, the subject is a mammal selected from primates, non-human primates, humans, or pigs.

[0019] Another aspect of the present disclosure provides a method for detecting porcine lymphotropic herpesvirus (PLHV) in a biological sample derived from a subject, comprising, or comprising, the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with PLHV antigen for a sufficient time to allow binding between the corresponding antibody in the diluted serum sample and the PLHV antigen; and detecting, by Western blotting, the presence or absence of an antibody from the diluted serum sample that reacts with the PLHV antigen.

[0020] Another aspect of the present disclosure provides a method for detecting anti-porcine lymphotropic herpesvirus antibodies in a biological sample of interest, comprising, or essentially consisting of, the following steps: (a) introducing one or more operationalized PLHV antigens, a biological sample of interest, one or more anti-immunoglobulin antibodies, and one or more chemiluminescent molecules into one or more capillaries of a microfluidic device; (b) separating one or more operationalized PLHV antigens electrophoretically; (c) immobilizing one or more electrophoretically separated operationalized PLHV antigens onto the capillary wall; (d) contacting one or more immobilized operationalized PLHV antigens with a biological sample of interest; (e) incubating one or more immobilized operationalized PLHV antigens with the biological sample for about 10 minutes to about 120 minutes; and (f) detecting the binding of one or more operationalized PLHV antigens to the biological sample by immunodetection and / or chemiluminescent detection (e.g., an immunodetection signal and / or a chemiluminescent signal).

[0021] In some embodiments, the presence of an immunodetection signal and / or a chemiluminescent signal indicates the presence of one or more anti-PLHV antibodies (e.g., anti-PLHV-1 antibodies) in the biological sample.

[0022] Another aspect of the present disclosure provides an automated serological method (e.g., an automated serological Western blot based on size) for detecting anti-porcine lymphotropic herpesvirus antibodies in porcine animals, comprising, or essentially comprising, the following steps: (a) contacting one or more operationalized PLHV antigens with a biological sample derived from porcine animals; (b) incubating one or more operationalized PLHV antigens with the biological sample for about 10 minutes to about 120 minutes; and (c) detecting the binding of one or more operationalized PLHV antigens to the biological sample by immunodetection and / or chemiluminescence detection.

[0023] In some embodiments of the automated serological methods described herein, prior to step (a), one or more engineered PLHV antigens are electrophoretically separated by mass and immobilized on a solid support. In some embodiments, the solid support is the capillary wall of a microfluidic device.

[0024] In some embodiments of the methods described herein, porcine lymphotropic herpesvirus (PLHV) is selected from PLHV-1, PLHV-2, PLHV-3, or a combination thereof.

[0025] In some embodiments, the method is performed in a closed-loop automated capillary-based immunoassay system. In some embodiments, one or more engineered PLHV antigens are incubated with a biological sample for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes. In some embodiments, the anti-PLHV antibody is an IgM antibody or an IgG antibody. [[ID=​​​​​​​​​In some embodiments of the methods described herein, one or more engineered PLHV antigens comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, or a combination thereof.

[0029] In some embodiments, one or more engineered PLHV antigens comprise the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, or a combination thereof.

[0030] In some embodiments, one or more engineered PLHV antigens are one or more of: (a) a PLHV-1 antigen comprising the amino acid sequence of SEQ ID NO: 34, 35, 36, or a combination thereof; (b) a PLHV-2 antigen comprising the amino acid sequence of SEQ ID NO: 37, 38, 39, or a combination thereof; and / or (c) a PLHV-3 antigen comprising the amino acid sequence of SEQ ID NO: 40, 41, 42, or a combination thereof.

[0031] In some embodiments of the methods described herein, one or more engineered PLHV antigens comprise the amino acid sequences of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, and 42. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] [Figure 1]Figure 1 shows a schematic diagram illustrating the steps of the serological Western blot assays for porcine cytomegalovirus (PCMV) and porcine lymphotropic herpesvirus (PLHV) disclosed herein. The serological Western blot assay may consist of approximately six steps. In step 1, approximately 1-2 ml of whole blood (e.g., a biological sample) is collected from the subject (e.g., a pig); in step 2, serum is separated from the whole blood by centrifugation; in step 3, the separated serum from the subject is dispensed into small Eppendorf tubes; in step 4, the sample is prepared for automated capillary electrophoresis (CE) Western (e.g., Simple Western®, ProteinSimple's Jess®, or Wes®) and loaded onto a CE Western (e.g., Wes®) plate; in step 5, the CE Western (e.g., Wes®) plate is mounted on a CE Western instrument (e.g., a Simple Western® instrument); and in step 6, the results obtained by the Simple Western® instrument are analyzed using relevant software (e.g., Compass software) to identify, characterize, and quantify anti-PCMV or anti-PLHV antibodies in the sample collected from the subject. [Figure 2]Figure 2 shows Western blot results prepared with Compass software illustrating the efficacy of the serological Western blot assay for porcine cytomegalovirus (PCMV) disclosed herein, demonstrating the detection of anti-PCMV antibodies in pigs housed outside the biosecure barrier facility (samples #4, #5, and #6; infected) compared to their littermates housed inside the barrier facility (samples #1, #2, and #3) (uninfected). The operationalized PCMV antigen tested had two isoforms: a first isoform with a molecular weight (MW) of approximately 17 kDa and a second isoform with a MW of approximately 40 kDa. The two isoforms were detected using rabbit anti-histidine (His) primary antibody and anti-porcine IgG HRP conjugate. Positive controls contained rabbit anti-His primary antibody and were used to determine the expected molecular weight of the PCMV antigen. Negative controls did not contain serum. [Figure 3] Figure 3 shows the Western blot results of the serological Western blot assay for porcine cytomegalovirus (PCMV) disclosed herein, demonstrating the detection of anti-PCMV antibodies in pigs raised outside the biosecure barrier facility (samples #4, #5, and #6) (infected) compared with their littermates raised inside the biosecure barrier facility (samples #1, #2, and #3) (uninfected). [Figure 4] Figure 4 shows the Western blot results of the serological Western blot assay for porcine cytomegalovirus (PCMV) disclosed herein, demonstrating the detection of anti-PCMV antibodies in pigs raised outside the biosecure barrier facility (samples #4, #5, and #6) (infected) compared with their littermates raised inside the biosecure barrier facility (samples #1, #2, and #3) (uninfected). [Figure 5]Figure 5 shows Western blot results prepared with Compass software illustrating the efficacy and sensitivity of the porcine cytomegalovirus (PCMV) serological Western blot assay disclosed herein, demonstrating the detection of anti-PCMV antibodies at different infection stages (e.g., 12–103 days) in pigs housed outside a biosecure barrier facility. Positive controls contained serum from pigs known to be PCMV-positive by PCR analysis from an independent veterinary diagnostic laboratory. Positive controls were used to determine the expected molecular weight of the PCMV antigen. Negative controls did not contain serum. At day 12, the serological Western blot assay for PCMV showed a weak positive signal. Stronger positive signals were detected at days 83, 88, and 103 (see also Table 1). [Figure 6] Figure 6 shows Western blot results prepared with Compass software illustrating the efficacy and sensitivity of the porcine cytomegalovirus (PCMV) serological Western blot assay disclosed herein, demonstrating the detection of anti-PCMV antibodies during the incubation period of PCMV infection in two of 21 pig animals (samples #12 and #19) housed in a biosecure barrier facility. All 21 animals received negative PCMV PCR results from an independent veterinary diagnostic laboratory. Positive controls contained serum from pigs known to be PCMV-positive by PCR analysis from an independent veterinary diagnostic laboratory and were used to determine the expected molecular weight of the PCMV antigen. Two negative controls were used: the first contained serum from a pure isolation group pig, and the second did not contain serum. [Figure 7]Figure 7 shows Western blot results prepared with Compass software illustrating the efficacy and sensitivity of the serological Western blot assay for porcine lymphotropic virus (PLHV)-1 disclosed herein, demonstrating the detection of anti-PLHV-1 antibodies in porcine animals housed outside the biosecure barrier facility (sample #3; infected) or porcine animals housed inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. The PLHV-1 antigen tested had a molecular weight of approximately 31 kDa. The operationalized PLHV-1 antigen was detected using rabbit anti-histidine (His) primary antibody (anti-6×His) and anti-porcine IgG HRP conjugate. Positive controls included rabbit anti-His primary antibody and were used to determine the expected molecular weight of the PLHV-1 antigen. Negative controls did not include serum (e.g., buffer only). [Figure 8] Figure 8 shows Western blot results prepared with Compass software illustrating the efficacy and sensitivity of the serological Western blot assay for porcine lymphotropic virus (PLHV)-2 disclosed herein, demonstrating the detection of anti-PLHV-2 antibodies in porcine animals housed outside the biosecure barrier facility (sample #3; infected) or porcine animals housed inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. The PLHV-2 antigen tested had a molecular weight of approximately 41 kDa. The manipulated PLHV-2 antigen was detected using rabbit anti-histidine (His) primary antibody and anti-porcine IgG HRP conjugate. Positive controls contained rabbit anti-His primary antibody and were used to determine the expected molecular weight of the PLHV-2 antigen. Negative controls did not contain serum. [Figure 9]Figure 9 shows Western blot results prepared with Compass software illustrating the efficacy and sensitivity of the serological Western blot assay for porcine lymphocytotropic virus (PLHV)-3 disclosed herein, demonstrating the detection of anti-PLHV-3 antibodies in porcine animals housed outside the biosecure barrier facility (sample #3; infected) or porcine animals housed inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. The PLHV-3 antigen tested had a molecular weight of approximately 40 kDa. The manipulated PLHV-3 antigen was detected using rabbit anti-histidine (His) primary antibody and anti-porcine IgG HRP conjugate. Positive controls contained rabbit anti-His primary antibody and were used to determine the expected molecular weight of the PLHV-3 antigen. Negative controls did not contain serum. [Modes for carrying out the invention]

[0033] 1. Overview A. Novel PCMV serological assays In xenotransplantation of porcine organs or tissues, donor pigs must be screened for a detailed panel of potential zoonotic microorganisms before transplantation of those organs in non-human primates (NHPs) or humans. One particularly challenging virus is PCMV, as it can cross the placenta in pregnant sows. PCMV can also remain in the incubation stage of infection for extended periods, thereby evading detection by standard screening methods (e.g., polymerase chain reaction (PCR) assays). For example, in the case of a 57-year-old patient with end-stage heart disease who received a genetically modified porcine heart for the first time, the donor pig was screened by PCR in a certified diagnostic laboratory and found to be negative for PCMV in four consecutive tests. Griffith et al., N. Engl. J. Med. 387(1): 35-44 (2022). The donor animal was from a porcine endogenous retrovirus (PERV)-C negative strain. This animal was tested every three months using PCR for pathogens affecting porcine or human health, including PERV-A, PERV-B, PERV-C, porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV). However, the pig was ultimately found to be potentially infected with PCMV at a later date. Cell-free DNA of the PCMV microorganism was finally found in the donor heart 20 days after xenotransplantation. There is a need for a serological assay for PCMV that can be performed in any veterinary diagnostic laboratory (VDL) under Good Laboratory Practice (GLP) conditions to reliably detect PCMV infection at any stage of infection (e.g., during the incubation period versus the viremia phase) and in any subject prior to xenotransplantation.

[0034] Novel serological assays are disclosed herein. These assays are highly sensitive and consistently produce detection of both active and latent PCMV infections.

[0035] B. Novel PLHV serological assays Porcine lymphotropic herpesvirus (PLHV) is a gamma herpesvirus that affects pig populations. Three distinct species: PLHV-1, PLHV-2, and PLHV-3 are recognized. However, these herpesviruses are not considered a threat to pig health because pigs are thought to be their natural host. Some data suggest that early weaning of piglets to a high herd health environment (e.g., a biosecure barrier) may potentially avoid PLHV infection. Although PLHV does not exist in humans, its detection and analysis are important public health issues because PLHV is linked to Epstein-Barr virus (EBV), which causes complications in organ transplantation. Experimental preclinical data have shown that porcine organs infected with PLHV-1 cause post-transplant lymphoproliferative disorder (PTLD) when they are transplanted into PLHV-negative animals (e.g., miniature pigs). While there is currently no data demonstrating PLHV-1, PLHV-2, and PLHV-3 transmission from pigs to humans (or non-human primates, NHPs), monitoring donor pigs for PLHV infection may result in better xenotransplant outcomes. To date, no reliable serological assays for PLHV exist. Therefore, this disclosure provides a high-speed, high-throughput PLHV serological Western blotting technique using the Simple Western® automated Western blot system (Simple Wes®; Bio-techne) for detecting anti-IgG antibodies to PLHV-1, PLHV-2, and PLHV-3 in biological samples from a subject.

[0036] C. Novel serological assay for reliable detection of PCMV or PLHV antibodies in samples Immunological methods such as enzyme-linked immunosorbent assays (ELISA) and Western blotting can serve as reliable alternatives to PCR-based methods. Western blotting can detect infection at both the viremia stage and the incubation stage. However, no diagnostic laboratory offers a serological assay for PCMV. Furthermore, all published serological assays for PCMV are cumbersome, insensitive, and unreliable.

[0037] The methods described herein provide a fast, high-throughput serological Western blotting technique for PCMV or PLHV using SimpleWes® instruments for detecting anti-IgG antibodies against PCMV or PLHV in samples derived from a target organism. Figure 1 illustrates the process used in the PCMV and PLHV serological Western blotting assays disclosed herein. Figures 2–4 further show Western blotting results generated using the PCMV serological Western blotting assays disclosed herein. In particular, Figures 2–4 demonstrate the detection of anti-PCMV antibodies in pigs raised outside the biosecure barrier facility (samples #4, #5, and #6) (e.g., infected) compared with their littermates raised inside the biosecure barrier facility (samples #1, #2, and #3) (uninfected). As used herein, “barrier” or “biosecure barrier” refers to a Source Animal Facility (SAF) for producing designated pathogen-free (DPF) pigs that serve as viable organ, tissue, or cell donors for xenotransplantation into clinical subjects.

[0038] The sensitivity of the porcine cytomegalovirus (PCMV) serological Western blot assays disclosed herein is further shown in Figure 5 and Table 1. In particular, the PCMV serological Western blot assays were as sensitive as PCR-based assays. In some embodiments, the PCMV serological Western blot assays were more sensitive than PCR-based assays. For example, the PCMV serological Western blot assays detected maternal antibodies in animals at 12 days of age, animals with latent infection, and animals with early viremia. Table 1 shows a comparison of the sensitivity of well-established PCR-based assays over time with the sensitivity of the novel PCMV serological Western blot assays described herein.

[0039] Table 1 shows the improved and remarkable sensitivity of the PCMV serological Western blot assay for detecting maternal antibodies and antibodies produced during initial viremia.

[0040] [Table 1]

[0041] Serum samples were collected from animals between 12 and 103 days of age and tested for PCMV by PCR-based assays and / or serological Western blot assays of PCMV in an independent veterinary diagnostic laboratory. As shown in Table 1, at day 12, PCR results from the independent veterinary diagnostic laboratory were negative, but the serological Western blot assay for PCMV showed a weak positive signal (Figure 5). Thus, the serological Western blot assay for PCMV can detect passively transmitted maternal antibodies in biological samples. At days 83 and 88, both PCR results from the independent veterinary diagnostic laboratory and the serological Western blot assay for PCMV showed positive signals. See also Figure 5. At day 103, the serological Western blot assay for PCMV continued to show positive results. These results demonstrate the ability of the serological Western blot assay for PCMV to detect antibodies during early PCMV viremia.

[0042] Figure 6 shows that the serological Western blot assay for PCMV described herein can also detect anti-PCMV antibodies during the incubation period of PCMV infection. Surprisingly, the serological Western blot assay for PCMV detected anti-PCMV antibodies in animals that had received negative PCMV PCR results from an independent veterinary diagnostic laboratory. Each of the 21 animals tested in Figure 6 had received negative PCMV PCR results from an independent veterinary diagnostic laboratory. Nevertheless, Figure 6 shows that two of the 21 pig animals were positive for anti-PCMV antibodies using the serological Western blot assay for PCMV described herein. These two animals were from different age groups. Sample #12 was approximately >3 months old, and sample #19 was approximately 14 months old. Thus, the serological Western blot assay for PCMV described herein reliably detected PCMV infection in the incubation stage throughout the lifespan of pig animals, despite previous negative PCR results.

[0043] The serological Western blot assays described herein also reliably detected PLHV-1, PLHV-2, and PLHV-3 antibodies in the samples. Figure 7 shows that anti-PLHV-1 antibodies were detected in pigs housed outside the biosecure barrier facility (sample #3; infected), but not in pigs housed inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. Figure 8 shows that anti-PLHV-2 antibodies were detected in pigs housed outside the biosecure barrier facility (sample #3; infected), but not in pigs housed inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. Figure 9 shows that anti-PLHV-3 antibodies were detected in pigs raised outside the biosecure barrier facility (sample #3; infected), but not in pigs raised inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result.

[0044] The serological Western blot assay described herein distinguished between PLHV-1, PLHV-2, and PLHV-3 (see Figures 7, 8, and 9). For example, the tested PLHV-1 antigen had a molecular weight of approximately 31 kDa (Figure 7). The tested PLHV-2 antibody had a molecular weight of approximately 41 kDa (Figure 8). The tested PLHV-3 antigen had a molecular weight of approximately 40 kDa (Figure 9). In contrast, a PCR-based assay performed by an independent veterinary diagnostic laboratory did not distinguish between PLHV-1, PLHV-2, and PLHV-3. These results further demonstrate the sensitivity of the novel serological Western blot assay over methods used in the art for the detection of PLHV-1, PLHV-2, and PLHV-3.

[0045] In some embodiments, the PCMV-specific and PLHV-specific automated serological assays described herein are based on an optimized Wes® protocol. The optimized Wes® protocol includes processes (e.g., incubation time settings), reagents, dilutions, and antigen concentrations that are specific to porcine serum. The same protocol can be used with other size-based automated Western blot systems such as Abby® and Jess®. The PCMV-specific and PLHV-specific automated serological assays disclosed herein enable reliable detection of anti-PCMV and anti-PLHV antibodies (e.g., anti-IgG antibodies) in any subject.

[0046] In some embodiments, the assays described herein can process at least about 24 samples in a time required of 4–5 hours to produce a final result, compared to at least 8–24 hours for existing methods. In some embodiments, the time required may be longer than the traditional Wes® protocol. The novel assays disclosed herein can be used to detect anti-PCMV antibodies and anti-PLHV antibodies (e.g., anti-IgG antibodies) at various life stages of any subject (e.g., pigs). For example, the methods disclosed herein can detect maternal anti-PCMV antibodies and / or anti-PLHV antibodies. In some embodiments, the methods disclosed herein can detect anti-PCMV antibodies and / or anti-PLHV antibodies present in neonatal piglets. In some embodiments, the methods disclosed herein can detect anti-PCMV antibodies and / or anti-PLHV antibodies in potentially infected young piglets (e.g., about 2–3 months old). In some embodiments, the methods disclosed herein can detect anti-PCMV antibodies and / or anti-PLHV antibodies in potentially infected donor pigs (e.g., >4 months old) designated for use in xenotransplantation. The methods disclosed herein can also detect anti-PCMV antibodies and / or anti-PLHV antibodies in potentially infected young sows (e.g., >7 months old). Alternatively, the methods disclosed herein can detect anti-PCMV antibodies and / or anti-PLHV antibodies in potentially infected boars (e.g., >12 months old) and / or mature breeding sows (e.g., >12 months old).

[0047] In some embodiments, the novel assays described herein utilize porcine serum to enable consistent and reliable detection of PCMV or PLHV antibodies (e.g., IgG antibodies), and leverage the application of specific serum dilutions and antigen concentrations to the Wes® protocol, as well as proprietary optimizations. The combination of novel antigen designs and processes (e.g., increased incubation time) with Simple WES® or similar protocols such as Abby® and Jess® provides a novel serological system that can be performed in any veterinary diagnostic laboratory (VDL) under Good Laboratory Practice (GLP) conditions.

[0048] Accordingly, one aspect of the present disclosure provides a method for detecting porcine cytomegalovirus (PCMV) or porcine lymphotropic herpesvirus (PLHV) in a biological sample derived from a subject, comprising the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with a PCMV antigen or PLHV antigen for a time sufficient to allow binding between the corresponding antibody in the diluted serum sample and the PCMV antigen or PLHV antigen; and detecting the presence or absence of an antibody from the diluted serum sample that reacts with the PCMV antigen or PLHV antigen using Western blot analysis (e.g., an automated Western blot system; or automated capillary electrophoresis (CE) Western, e.g., Simple Western®, or ProteinSimple's Jess® or Wes®).

[0049] Another aspect of this disclosure provides a method for detecting anti-porcine cytomegalovirus (PCMV) antibodies or anti-porcine lymphotropic herpesvirus (PLHV) antibodies in a biological sample derived from a subject. The method may include the steps of introducing one or more manipulated PCMV antigens or one or more manipulated PLHV antigens, a biological sample derived from the subject, one or more anti-immunoglobulin antibodies, and one or more chemiluminescent molecules into one or more capillaries of a microfluidic device; separating one or more manipulated PCMV antigens or one or more manipulated PLHV antigens by electrophoresis; immobilizing one or more electrophoretically separated manipulated PCMV antigens or PLHV antigens onto the capillary wall; contacting one or more immobilized manipulated PCMV antigens or PLHV antigens with a biological sample derived from the subject; incubating one or more immobilized manipulated PCMV antigens or PLHV antigens with the biological sample for about 10 minutes to about 120 minutes; and detecting the binding of one or more manipulated PCMV antigens or PLHV antigens to the biological sample by immunodetection and / or chemiluminescence detection. The presence of an immunodetection signal and / or chemiluminescence signal may indicate the presence of one or more anti-PCMV antibodies or anti-PLHV antibodies in the biological sample.

[0050] In another embodiment, the Disclosure provides an automated serological method for detecting anti-porcine cytomegalovirus (PCMV) antibodies or anti-porcine lymphotropic herpesvirus (PLHV) in porcine animals, comprising the steps of: contacting one or more manipulated PCMV antigens or PLHV antigens with a biological sample derived from porcine animals; incubating one or more manipulated PCMV antigens or PLHV antigens with the biological sample for about 10 minutes to about 120 minutes; and detecting the binding of one or more manipulated PCMV antigens or PLHV antigens to the biological sample by immunodetection and / or chemiluminescence detection.

[0051] In any embodiment disclosed herein, the subject may be a pig, a primate, a non-human primate, or a human. The pig may be a wild-type pig or a transgenic pig. In some embodiments, the human was transplanted with cells, tissues, or organs derived from a pig (e.g., wild-type or transgenic). In some embodiments, the porcine lymphotropic herpesvirus is porcine lymphotropic herpesvirus-1 (PLHV-1), porcine lymphotropic herpesvirus-2 (PLHV-2), and / or porcine lymphotropic herpesvirus-3 (PLHV-3).

[0052] One of the advantages of the automated serological Western blot assay described herein in relation to xenotransplantation is that it is serum / blood-based. Therefore, the assay described herein can be used with samples (e.g., blood / serum) that are readily available / obtained from the donor animal before transplantation. This is particularly important for detecting latent viruses that may be hiding in lymph nodes or other tissues (such as the spleen) that are difficult to biopsy and cannot be readily obtained before organ harvesting and subsequent transplantation.

[0053] Therefore, the novel serological assays for the detection of porcine viruses disclosed herein can be applied to the detection of any virus. In some embodiments, the methods disclosed herein can be used to detect antibodies against any porcine virus, any zoonotic porcine virus, or any latent porcine virus. In those embodiments, porcine viruses may be a concern for xenotransplantation. The methods disclosed herein are particularly important for this type of assay as pre-transplant screening due to the type of sample (serum / blood) used for detection. The methods described herein are more sensitive than PCR screening assays currently used in the art. Therefore, the methods disclosed herein can be used as diagnostic and prognostic methods, thereby enabling those skilled in the art to eliminate potentially infected donor animals before or prior to future transplants, rather than before or after transplantation.

[0054] II. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the art pertains.

[0055] Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise clearly indicated by the context. For example, a reference to “a cell” includes a combination of two or more cells. In general, the nomenclature used herein, as well as the experimental procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, nucleic acid chemistry, and hybridization described below, are well known and commonly used in the art.

[0056] Whenever the terms “at least,” “greater than,” or “greater than or equal to” are placed before the first number in a set of two or more numbers, those terms apply to each of the numbers in that set. For example, “greater than or equal to 1, 2, or 3” is equivalent to 1 or greater, 2 or greater, or 3 or greater.

[0057] Whenever the terms “no more than,” “less than,” or “less than or equal to” are placed before the first number in a set of two or more numbers, those terms apply to each of the numbers in that set. For example, “less than or equal to 3, 2, or 1” is equivalent to “3 or less,” “2 or less,” or “1 or less.”

[0058] Certain ranges are presented herein with the term “approximately” preceding the number. The term “approximately” is used herein to provide a literal basis for the exact number it precedes, in addition to a number that is close to or approximately the preceding number. In determining whether a number is close to or approximately the specifically enumerated number, a close or approximate unenumerated number may be a number that, in the context in which it is presented, provides a substantial equivalent of the specifically enumerated number. Where the degree of approximation is not evident from the context, “approximately” includes the provided value and, in all cases, means either within plus or minus 10% of the provided value, or rounded to its nearest significant figure. In some embodiments, the term “approximately” indicates up to ±10%, ±5%, or ±1% of the specified value.

[0059] As will be understood by those skilled in the art, for all purposes, and in particular with respect to providing written explanations, all scopes disclosed herein also encompass all possible partial scopes and combinations thereof. Any listed scope can be readily recognized as being sufficiently described and made possible to be broken down into at least two, three, four, five, ten, and so on. As a non-limiting example, each scope discussed herein can be readily broken down into a lower third, a middle third, and an upper third, and so on. Similarly, as will be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” includes the listed number and refers to a scope that can be broken down into the partial scopes discussed above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group with 1 to 5 cells refers to a group with 1, 2, 3, 4, or 5 cells, and so on.

[0060] Items, such as (a), (b), or (i), are provided solely to facilitate reading of this specification and the claims. The use of items in this specification or the claims does not require that the steps or elements be carried out in alphabetical or numerical order, or in the order in which they are presented.

[0061] The use of ordinal terms such as “first,” “second,” and “third” in the claims to modify the claimed elements does not in itself imply any priority, precedence, or order of one claimed element over another claimed element, nor does it imply any chronological order in which the actions of the method are performed. Rather, it is merely used as a marker to distinguish one claimed element having a particular name from another element having the same name (if the ordinal terms were not used). Similarly, the use of these terms herein does not in itself imply any required priority, precedence, or order.

[0062] As used herein, the term "analyte" means a biological molecule. Analytes include, but are not limited to, DNA analytes, RNA analytes, oligonucleotides, reporter molecules, reporter molecules formed to directly link with proteins, reporter molecules formed to indirectly link with proteins, reporter molecules formed to directly link with metabolites, and reporter molecules formed to indirectly link with metabolites.

[0063] As used herein, the term “animal” refers to a mammal. The animals in this disclosure may be “genetically modified” or “transgenic.” A transgenic animal may be a genetically modified animal that includes, typically, one or more exogenous transgenes or other foreign DNA added or incorporated into at least one germline cell of the animal, so as to mediate a genotypic or phenotypic effect in at least one cell of the animal, or one or more modified (e.g., targeted, recombined, interrupted, removed, disrupted, replaced, repressed, enhanced, or otherwise altered) endogenous genes. An animal may have an introduced gene incorporated into one allele of its genome (heterozygous transgenic). Alternatively, an animal may have an introduced gene on two alleles (homozygous transgenic).

[0064] As used herein, the term "electrophoresis" refers to the movement of suspended or dissolved molecules through a fluid or gel under the action of an electromotive force applied to an electrode in contact with the fluid.

[0065] As used herein, the term “immobilization” means substantially reducing or eliminating the motion of molecules in a fluid conduit. Immobilization may be via covalent bonding or non-covalent means such as hydrophobic or ionic interactions. In some embodiments, the analyte to be elucidated present in the sample (e.g., anti-PCMV antibody or anti-PLHV antibody) is immobilized in the fluid conduit by isoelectric focusing (e.g., according to the Wes® system specifications).

[0066] As used herein, the term “sample” means the biological sample of interest. A biological sample may contain any number of polymers, such as cellular polymers. A sample may be a cell sample. A sample may be a cell line or cell culture sample. A sample may contain one or more cells. A sample may contain one or more microorganisms. A biological sample may be a nucleic acid sample or a protein sample. A biological sample may also be a carbohydrate sample or a lipid sample. A biological sample may be derived from another sample. A sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine-needle aspirate. A sample may be a skin sample. A sample may be a cheek swab. A sample may be a plasma or serum sample. A sample may be a cell-free or cell-avoid sample. A cell-free sample may contain extracellular polynucleotides. Extracellular polynucleotides can be isolated from a physical sample, which may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excrement, sputum, feces, and tears.

[0067] A sample may be any substance that contains or is presumed to contain one or more analytes of interest (e.g., antibodies, particularly antibodies against PCMV or PLHV). A sample may be of natural or synthetic origin and may be obtained by any means known to those skilled in the art. A sample may also be synthetic, including, but not limited to, in vitro cell culture components, including conditioned media, recombinant cells, and cellular components. A sample may be a tissue, cell, organ, or liquid isolated from the subject. Liquids may include, but are not limited to, plasma, serum, whole blood, cerebrospinal fluid (CSF), semen, amniotic fluid, lymph, synovial fluid, urine, or tears.

[0068] In some embodiments of the methods disclosed herein, the sample may be a blood product used for transfusion or treatment. The sample may be any liquid found within the body of an organism that may contain components of PCMV or PLHV, or immune system components that can react with one or more PCMV or PLHV antigens. For example, the immune system component may be an antibody that specifically binds to the PCMV or PLHV antigen.

[0069] In some embodiments of the methods disclosed herein, the sample is a body fluid, which may include, for example, blood, plasma, serum, urine, saliva, tears, synovial fluid, and / or lymph. In some embodiments, the sample is blood or whole blood. In some embodiments, the sample includes plasma or serum. The sample may be serum.

[0070] Therefore, in some embodiments of the methods disclosed herein, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, seminal plasma, cerebrospinal fluid, and saliva. Alternatively, the biological sample is whole blood or serum.

[0071] As used herein, the term “subject” means any organism, not limited to mammals. For example, mammals may be agricultural animals, domestic animals, pets, primates, or non-human primates. In some embodiments, mammals may be humans, rhesus macaques, crab-eating macaques, dogs, cats, mice, rats, foxes, deer, ferrets, guinea pigs, rabbits, pigs, goats, cattle, baboons, monkeys, or horses. In preferred embodiments, the subject is a human or a pig (e.g., a pig). In some embodiments, the mammal is a female pig that has given birth at least once. In certain embodiments, the mammal is a non-human primate (e.g., a monkey or a baboon).

[0072] As used herein, the terms “porcine,” “porcine animal,” “pig,” and “swine” are general terms referring to animals of the same type, regardless of sex, size, or breed.

[0073] As used herein, the terms “operatably linked,” “conjugated,” or “fused” mean, with respect to the operationalized antigen sequences described herein, that when transcribed and translated, one or more sequences are located at the N-terminus or C-terminus, generating an additional polypeptide bound to the enzyme amino acid sequence, thereby resulting in a conjugation or fusion of one or more polypeptides from a single expression vector.

[0074] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources. Antibodies may be the immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this disclosure may exist in various forms, e.g., polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, in addition to single-chain antibodies (scFv) and humanized antibodies. In some embodiments, an antibody refers to such an assembly (e.g., an intact antibody molecule, immunoadhesin, or a variant thereof) that has significant known specific immunoreactive activity against an antigen of interest (e.g., a virus-associated antigen). Antibodies and immunoglobulins include light and heavy chains, with or without interchain covalent bonds between the light and heavy chains. The basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0075] As used herein, the terms “antigen” or “Ag” are defined as molecules that induce an immune response. For example, macromolecules or proteins expressed by PCMV or PLHV. This immune response may include antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can act as an antigen. Furthermore, antigens may be recombinant or derived from genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that induces an immune response, therefore, encodes an “antigen” as used herein. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent, though not limited, that this disclosure includes the use of partial nucleotide sequences of more than one gene (e.g., the PCMV gene or PLHV gene), and that these nucleotide sequences may be arranged in various combinations to induce a desired immune response or binding affinity. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded by a “gene.” Antigens can be prepared, synthesized, or derived from biological samples (e.g., manipulated PCMV or PLHV antigens). Such biological samples may include, but are not limited to, tissue samples, tumor samples, xenografts, cells, or biological fluids.

[0076] As used herein, the terms “immunoglobulin” or “Ig” define a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucosal secretions of the respiratory and genitourinary tracts. IgG is the most commonly found circulating antibody. IgM is the major immunoglobulin produced in the primary immune response to most targets. It is the most efficient immunoglobulin in agglutination, complement binding, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate-type hypersensitivity reactions by triggering the release of mediators from mast cells and basophils upon exposure to allergens. In some embodiments of this disclosure, the immunoglobulin is IgG or IgM. In some embodiments, the immunoglobulin is IgG.

[0077] As used herein, the term “organ” refers to a collection of tissues that are joined together as structural units to perform a common function. Organs can be solid organs. Solid organs are internal organs that have a firm, dense tissue and are neither hollow (e.g., organs of the gastrointestinal tract) nor fluid (e.g., blood). Examples of solid organs include the heart, kidneys, liver, lungs, pancreas, spleen, and adrenal glands.

[0078] As used herein, the term “primate” refers to a variety of mammals of the order Primates, consisting of lemurs, lorises, tarsiers, New World monkeys, Old World monkeys, apes, and humans. Primates are characterized by their fingernails and toenails, short noses, and large brains. In certain embodiments, primates are non-human primates. In other embodiments, primates are humans.

[0079] As used herein, the terms “detection,” “to detect,” and “to detect” mean to discover the presence or existence of one or more analytes (e.g., anti-PCMV antibody or anti-PLHV antibody).

[0080] As used herein, the terms “identify,” “to identify,” and “to identify” mean to recognize exposure to a specific pathogen or active substance in a sample derived from the subject.

[0081] As used herein, the term “incubating” encompasses maintaining or providing conditions favorable to or leading to a desired reaction, such as, for example, providing reaction components or reagents. It is recognized that conditions favorable to or leading to one reaction may differ from conditions favorable to another. Suitable conditions for various reactions and reaction types are known in the art.

[0082] As used herein, the term “to bring into contact” means to mix, blend, or incubate one or more components for the purpose of stimulating interaction and / or bonding of two or more of those components. In particular, to bring into contact may also mean to bring one or more components into very close proximity to trigger interaction (e.g., bonding) between those two or more components.

[0083] As used herein, the term “identical” in relation to two nucleic acid sequences or polypeptide sequences refers to residues in two sequences that are identical when aligned for maximum match, as measured using a sequence comparison algorithm. Sequence comparison algorithms are known to those skilled in the art (see, for example, ebi.ac.uk / Tools / msa / clustalo / ).

[0084] As used herein, the term “sequence identity” refers to the percentage of nucleotide / amino acid residues in a target sequence that are identical to those in a reference sequence, after aligning sequences to achieve maximum percentage sequence identity between them and introducing gaps where necessary. Pairwise and multiple sequence alignments for the purpose of determining the percentage sequence identity between two or more amino acid sequences or nucleic acid sequences can be achieved in various forms known to those skilled in the art, using publicly available computer software such as Clustal Omega (Soding, J., Bioinformatics 21, 951-960 (2005)), T-coffee (Notre dame et al., J. Mol. Biol. 302, 205-217 (2000)), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics, 6(298) (2005)), and MAFFT (Katoh and Standley, Molecular Biology and Evolution, 30(4) 772-780 (2013)). When using such software, default parameters (e.g., gap penalty and elongation penalty) are preferably used.

[0085] III. PCMV Serological Assay Porcine cytomegalovirus (PCMV) infection is caused by a herpesvirus found in tissues throughout the body, including the nose of newborn piglets, which causes inflammation (rhinitis). PCMV is present worldwide and is present in most pig populations tested to date. However, most infections are asymptomatic. Clinical disease is rare, at least so far. Serological analysis of several pig populations has shown that over 90% of the herds are exposed to the infection. Rhinitis caused by this virus is rare and mainly occurs in newborn piglets. In most herds, the infection is not significant and does not have a major impact on the pigs' health, except that it sometimes causes mild sneezing.

[0086] Despite the relatively minor nature of PCMV infection in pigs, for xenotransplantation, donor pigs must be screened for a detailed panel of potential zoonotic microorganisms before their organs are used for non-human primate (NHP) or human transplantation. PCMV, for example, poses a significant challenge because it can cross the placenta in pregnant sows and may remain in a latent stage of infection that evades detection by standard screening methods (e.g., polymerase chain reaction (PCR) assays). Therefore, the method of this disclosure was developed to enable those skilled in the art to perform high-throughput serological screening to diagnose multiple infections in subjects (e.g., humans or pigs) at all stages of PCMV infection.

[0087] Therefore, the novel serological assays for the detection of porcine viruses disclosed herein can be applied to the detection of any virus. In some embodiments, the methods disclosed herein can be used to detect antibodies against any porcine virus, any zoonotic porcine virus, or any latent porcine virus. In those embodiments, the porcine virus may be a concern in relation to xenotransplantation. The methods disclosed herein are particularly important for this type of assay as pre-transplant screening thanks to the type of sample (serum / blood) used for detection. The methods described herein are more sensitive than PCR screening assays currently used in the art. The methods disclosed herein can be used as diagnostic and prognostic methods, thereby enabling those skilled in the art to eliminate latently infected donor animals before or prior to future transplants, rather than before or after transplantation.

[0088] Optimization method for PCMV antibody detection This disclosure provides an optimization of Wes® for PCMV antibody detection in subjects suspected of having PCMV infection, preferably latent PCMV infection. Wes® is an automated Western blotting method capable of producing results similar to those obtained using traditional Western blotting assays. See, for example, bio-techne.com / p / simple-western / wes_004-600. Western blotting assays are the gold standard for protein detection and characterization. However, Wes® is an improvement over this gold standard, as it is a gel-free, blot-free, hands-free alternative assay for protein size classification and quantitative immunodetection. Wes® is an automated system that integrates and automates all manual operations associated with Western blotting. The entire Wes® process is also faster, simpler, more sensitive, more accurate, and more automatable than traditional Western blotting analysis techniques. For example, using Wes®, analytes can be analyzed in approximately 50 minutes, compared to up to 24 hours for traditional Western blotting. With respect to Wes®, the time required for the initial separation of cell material may be approximately 5 minutes or less. The time required for subsequent immobilization may be approximately 2 minutes or less. The detection agent can be ligated with the separated sample within 10 minutes of the start of separation, and one or more analytes can be analyzed within 30 minutes of the separation step. See, for example, U.S. Patent No. 7,935,489. Thus, this disclosure provides a fast and highly sensitive serological assay for determining whether a subject is infected with PCMV using a PCMV serological assay based on Wes® optimization for PCMV. The assay described herein can be performed using a conventional Western blot assay, but the Wes® method is generally preferred.

[0089] While Wes® is illustrated, the assays described herein can be used with any automated capillary electrophoresis (CE) Western system (e.g., Simple Western® or ProteinSimple's Jess® or Wes®) to identify, characterize, and quantify targeted antiviral antigen antibodies (e.g., anti-PCMV antibodies or anti-PLHV antibodies). CE Western includes a protein size separation step (e.g., size distribution profiling), a protein immobilization step, and a protein detection step using fluorescence detection and / or chemiluminescence detection. Thus, CE Western technique can be a highly sensitive method for characterizing, qualitatively, and quantitatively assessing antiviral antigens with enhanced resolution and size separation range.

[0090] In addition, CE (Chemical Emission) may be more sensitive than enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of target proteins in serum samples derived from immunized animals. While ELISA can only provide protein binding information, CE Western techniques can provide additional information about the target protein, such as molecular weight estimation (e.g., as size and size distribution) and protein integrity (e.g., isomers, fragments, and aggregates).

[0091] In some embodiments, CE is Jess® (or its equivalent). Therefore, in step 4 of Figure 1, the sample can be prepared for Jess® (or its equivalent) and loaded onto a Jess® plate. Specifically, one or more serum samples containing one or more viral antigens (e.g., PCMV or PLHV) can be placed in a single well together with a serum diluent or buffer, a primary antibody targeting one or more viral antigens (e.g., PCMV or PLHV), and / or a secondary antibody conjugated with a chemiluminescent or fluorescent molecule, as well as Jess® reagents (standard equipment for Western assays). Chemiluminescent or fluorescent molecules can be obtained from any known supplier. Jess® reagents can be obtained from its manufacturer.

[0092] Accordingly, one aspect of the present disclosure provides a method for detecting porcine cytomegalovirus (PCMV) in a biological sample derived from a subject, comprising, essentially, or comprising, the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with a PCMV antigen for a sufficient time to allow binding between the corresponding antibody in the diluted serum sample and the PCMV antigen; and detecting the presence or absence of an antibody from the diluted serum sample that reacts with the PCMV antigen using Western blotting, automated capillary electrophoresis (CE) Western, including Wes®, Jess®, or equivalents thereof.

[0093] Another aspect of the present disclosure provides a method for detecting anti-porcine cytomegalovirus (PCMV) antibodies in a biological sample of a subject, comprising the steps of: introducing one or more manipulated PCMV antigens, a biological sample of a subject, one or more anti-immunoglobulin antibodies, and one or more chemiluminescent molecules into one or more capillaries of a microfluidic device. Following this first step, one or more manipulated PCMV antigens are separated electrophoretically, and subsequently, one or more electrophoretically separated manipulated PCMV antigens are immobilized on the capillary wall. Once immobilized, one or more immobilized manipulated PCMV antigens are brought into contact with a biological sample of a subject. The one or more immobilized manipulated PCMV antigens are then incubated with the biological sample for about 10 minutes to about 120 minutes.

[0094] In some embodiments, one or more operationalized PCMV antigens and a biological sample are incubated for approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. This incubation period is much longer than the recommended immobilization step of approximately 2 minutes or less.

[0095] Finally, the binding of one or more operationalized PCMV antigens to a biological sample is detected by immunodetection and / or chemiluminescence detection. In some embodiments, the presence of an immunodetection signal and / or chemiluminescence signal may indicate the presence of one or more anti-PCMV antibodies in the biological sample. In some embodiments, the immunodetection signal is a fluorescent signal.

[0096] A further aspect of the present disclosure provides an automated serological method for detecting anti-porcine cytomegalovirus (PCMV) antibodies in porcine animals, comprising, or comprising, a step of contacting one or more manipulated PCMV antigens with a biological sample derived from porcine animals; followed by a step of incubating the one or more manipulated PCMV antigens with the biological sample for about 10 to about 120 minutes, and a step of detecting the binding of the one or more manipulated PCMV antigens to the biological sample by immunodetection and / or chemiluminescence detection.

[0097] Embodiments of the method described herein are illustrated in Figure 1. Step 1 can begin with the collection of whole blood from the subject. In some embodiments, about 1–2 mL is collected. The subject may be a human or a porcine, as defined herein. In Step 2, serum is separated from the whole blood by centrifugation using any method known to those skilled in the art. In Step 3, the separated serum from the subject may be dispensed into a container (e.g., an Eppendorf tube) for analysis. In Step 4, the sample may be prepared for Wes®, Jess® (or equivalents thereof) and loaded onto a Wes® plate. Specifically, one or more operationalized antigens intended in the present invention (e.g., operationalized PCMV antigen; operationalized PLHV antigen) may be placed in a single well together with porcine serum diluted in a serum diluent, rabbit anti-porcine IgG HRP secondary antibody (available from any known supplier), and Simple Wes® reagent or Jess® reagent (standard equipment for Western assays) (available from the manufacturer). Therefore, the serological assays disclosed herein may be performed using an automated capillary immunoassay system (e.g., Wes®, ProteinSimple instrument). The diluent may be a buffer. The diluent may be an instrument-specific buffer manufactured by the manufacturer. In step 5, the Wes® plate can be mounted on the Simple Wes® instrument. In step 6, the results produced by the Simple Wes® instrument can be analyzed using Compass software. Thus, protein band images and quantitative results can be produced using Compass software and / or chromatogram output data after running one or more samples through a Wes® or Jess® capillary electrophoresis system (ProteinSimple).

[0098] In some embodiments, one or more operationalized PCMV antigens are separated electrophoretically by mass and immobilized on a solid support. The solid support may be a capillary wall of a microfluidic device. The methods disclosed herein can be carried out in a closed-loop automated capillary immunoassay system (e.g., a SimpleWes® instrument).

[0099] Immunodetection In some embodiments of the methods disclosed herein, the method may include the step of detecting the binding of one or more operationalized PCMV antigens present in a biological sample by immunodetection and / or chemiluminescence detection. In those embodiments, the presence of an immunodetection signal and / or chemiluminescence signal indicates the presence of one or more analytes (e.g., anti-PCMV antibody or anti-PLHV antibody) in the biological sample.

[0100] The method includes a detection agent that has the ability to bind to or interact with the analyte to be detected in a biological sample. Non-limiting examples of detection agents include proteins, peptides, antibodies, enzyme substrates, transition state analogs, cofactors, nucleotides, polynucleotides, aptamers, lectins, small molecules, ligands, inhibitors, drugs, and other biomolecules, as well as non-biomolecules, that have the ability to bind to the analyte to be detected. In some embodiments, the detection agent is an antibody.

[0101] In some embodiments, detecting the binding of one or more operationalized PCVM antigens or PLHV antigens involves a biantibody CE Western. A biantibody CE Western may comprise (a) a primary antibody targeting one or more operationalized PCVM or PLHV antigens; (b) a secondary antibody targeting the primary antibody; and (c) a chemiluminescent or fluorescent molecular substrate. In some embodiments, the secondary antibody targeting the primary antibody is operably linked to a chemiluminescent or fluorescent molecule.

[0102] The antibody may be an IgM and / or IgG antibody. In some embodiments, the analyte to be detected in the biological sample is one or more anti-PCMV antibodies. Therefore, in some embodiments of the methods disclosed herein, the anti-PCMV antibody is an IgM antibody or an IgG antibody.

[0103] Contacting the detection agent with one or more analytes (e.g., one or more anti-PCMV antibodies) in a biological sample can be carried out by any method known in the art. However, the selected method should be compatible with the methods described herein (e.g., Wes®, Jess®). The detection agent may include any organic or inorganic molecules capable of binding to and interacting with the analytes to be detected (e.g., one or more anti-PCMV antibodies or anti-PLHV antibodies).

[0104] In some embodiments, the detection agent may include one or more labeled portions. In some embodiments, the detection agent may include two or more labeled portions. In those embodiments, each labeled portion may be the same or different.

[0105] In some embodiments, the labeled portion may include immunodetection labels or chemiluminescent labels (e.g., immunodetection and / or chemiluminescent detection). Chemiluminescent labels may include any entity that provides a light signal and can be used according to the methods and devices described herein. A wide variety of such chemiluminescent labels are known in the art. See, for example, U.S. Patents 6,689,576, 6,395,503, 6,087,188, 6,287,767, 6,165,800, and 6,126,870.

[0106] Suitable labels include enzymes capable of reacting with a chemiluminescent substrate to induce chemiluminescent photon emission. Possible labels include peroxidases, beta-galactosidases, phosphatases, or others, for which chemiluminescent substrates are available. In some embodiments, the chemiluminescent label can be selected from any of the various classes of luminols or isoluminols. In some embodiments, the detection agent comprises one or more chemiluminescent substrates.

[0107] Chemiluminescent detection can be a more sensitive detection method for detecting target molecules that may be present in low concentrations in biological samples. For example, chemiluminescent detection can maximize immunoassay output by detecting proteins present in picogram concentrations in a sample.

[0108] In some embodiments, the methods described herein detect nanograms or picograms (pg) of anti-PCMV or anti-PLHV antibodies in a biological sample. In some embodiments, the methods detect at least about 0.0001 ng to 1.0 ng, and at least about 0.0001 ng to 0.1 ng.

[0109] In some embodiments, the method detects anti-PCMV antibody or anti-PLHV antibody in a biological sample at a concentration of at least about 0.1 pg to 1.0 pg, at least about 0.5 pg to 2.5 pg, at least about 0.1 pg to 10 pg, at least about 5 pg to 50 pg, at least about 10 pg to 100 pg, or more than 100 pg. In some embodiments, the method detects at least about 0.1 pg, at least about 0.2 pg, at least about 0.3 pg, at least about 0.4 pg, at least about 0.5 pg, at least about 0.6 pg, at least about 0.7 pg, at least about 0.8 pg, at least about 0.9 pg, at least about 1.0 pg, at least about 1.1 pg, at least about 1.5 pg, at least about 5.0 pg, at least about 10 pg, at least about 20 pg, at least about 30 pg, at least about 40 pg, at least about 50 pg, at least about 60 pg, at least about 70 pg, at least about 80 pg, at least about 90 pg, or at least about 100 pg of anti-PCMV antibody or anti-PLHV antibody in a biological sample.

[0110] The labeled portion may also contain a bioluminescent compound. As used herein, bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of a chemiluminescent reaction. The presence of a bioluminescent compound is determined by detecting the presence of luminescence. Suitable bioluminescent compounds may include luciferin, luciferase, and aequorin.

[0111] In some embodiments, the labeling portion comprises a fluorescent dye. The fluorescent dye comprises a resonance delocalization system or aromatic ring system that absorbs light of a first wavelength and, in response to the absorption event, emits fluorescence of a second wavelength. A wide variety of such fluorescent dye molecules are known in the art. For example, the fluorescent dye can be selected from any of the various classes of fluorescent compounds, and non-limiting examples include xanthenes, rhodamine, fluorescein, cyanine, phthalocyanine, squaline, body pea dyes, coumarin, oxazine, and carvopyronine. In some embodiments, for example, if the detection agent contains fluorophores such as fluorescent dyes, their fluorescence is detected by exciting them with a suitable light source and monitoring their fluorescence with a detector sensitive to their characteristic fluorescence emission wavelength. In some embodiments, the detection agent comprises a fluorescent dye-labeled antibody.

[0112] In some embodiments, immunodetection and / or chemiluminescent detection comprises a horseradish peroxidase (HRP) conjugate secondary antibody, streptavidin-HRP, luminol-S, peroxide, LacZ, biotin, or luciferase. In some embodiments, the detection agent comprises a chemiluminescent labeled antibody. In some embodiments, immunodetection and / or chemiluminescent detection comprises a horseradish peroxidase (HRP) conjugate anti-IgG or anti-IgM antibody.

[0113] Analyte detection may include detecting the presence or absence of an analyte (e.g., one or more anti-PCMV antibodies or anti-PLHV antibodies), measuring it, and / or characterizing it. Typically, the analyte (e.g., one or more anti-PCMV antibodies or anti-PLHV antibodies) is detected by detecting a signal from a label. Such detection may include, but is not limited to, detecting isotope labels, immunolabels, optical dyes, enzymes, particles, and combinations thereof, such as chemiluminescent and fluorescently labeled antibodies.

[0114] Furthermore, the methods disclosed herein enable those skilled in the art to monitor the signal in real time. Real-time monitoring can enable users to quickly determine whether an analyte is present in a sample and / or the amount or activity of the analyte. In some embodiments, the signal may be measured from at least two different points in time. In some embodiments, the signal may be monitored continuously or at several selected points in time. Alternatively, the signal may be measured at predetermined endpoints. For example, the signal may be measured after a certain amount of time. Alternatively, the signal may be compared to a control signal (e.g., a sample without the analyte; or a sample without biological material), a threshold signal, or a standard curve. The amount of the generated signal may be insignificant and may vary over a wide range. The only requirement may be that the signal is measurable by the detection system being used. In some embodiments, the signal may be at least twice as large as the background signal (e.g., a control signal; a sample without the analyte; or a sample without biological material). In some embodiments, the signal may be 2 to 10 times larger than the background. In some embodiments, the signal may be 10 times larger than the background.

[0115] subject In certain embodiments, the mammal is a pig. In some embodiments of the methods disclosed herein, the pig is about 60 to 323 days old. For example, the pig may be about 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 116, 150, 180, 200, 215, 230, 238, 215, 238, 240, 317, 323, 340, or 350 days old.

[0116] The pig can be of any size. For example, the pig may weigh from about 10 pounds to about 500 pounds. In some embodiments, the pig may weigh more than 500 pounds. The weight of the pig may depend, for example, on the weight and size of the xenograft recipient. The pig may be a wild-type pig or a transgenic pig.

[0117] The novel automated serological assays described herein may include the use of porcine serum, or serum derived from subjects transplanted with porcine cells, tissues, or organs, the application of specific dilutions and antigen concentrations, and proprietary modifications to the Wes® protocol.

[0118] In some embodiments of the methods disclosed herein, the subject is suspected of having latent PCMV or PLHV infection, lysed PCMV or PLHV infection, or active viremia-stage PCMV or PLHV infection. PCMV or PLHV infection can be latent when the presence of PCMV or PLHV in the body does not produce signs of pathological symptoms. This period is similar to the incubation period. Generally, during latent infection, a PCR test will yield a negative result. Latent infection can sometimes occur when infectious material survives in the patient for various periods after infection.

[0119] In some embodiments, PCMV or PLHV infection may be a latent infection if the presence of one or more anti-PCMV antibodies or anti-PLHV antibodies is detected in a biological sample, and a standard PCR assay using the same one or more manipulated PCMV or PLHV antigens is negative. In some embodiments, PCMV or PLHV infection is a latent infection if the presence of one or more anti-PCMV or anti-PLHV antibodies is detected in a biological sample with HRP-conjugate anti-IgG antibodies, but not with HRP-conjugate anti-IgM antibodies.

[0120] sample In some embodiments, the biological sample may be selected from the group consisting of whole blood, serum, plasma, urine, seminal plasma, cerebrospinal fluid, and saliva. The biological sample may also be whole blood or serum.

[0121] In some embodiments, the biological sample is diluted to approximately 1:20; approximately 1:40; approximately 1:50; approximately 1:75, approximately 1:100; approximately 1:150; approximately 1:120; approximately 1:250; approximately 1:300; approximately 1:350; approximately 1:375; approximately 1:400; approximately 1:500; approximately 1:750; approximately 1:800; approximately 1:1000; approximately 1:1100; or approximately 1:1200. In some embodiments, the biological sample is diluted to approximately 1:75, approximately 1:200, or approximately 1:300 (Figures 2-4). In some embodiments, the biological sample is diluted to approximately 1:75.

[0122] In some embodiments, one or more manipulated PCMV antigens or one or more manipulated PLHV antigens may be used at concentrations of approximately 0.1 ug; approximately 0.125 ug; approximately 0.150 ug; approximately 0.2 ug; approximately 0.225 ug; approximately 0.25 ug; approximately 0.3 ug; approximately 0.315 ug; approximately 0.325 ug; approximately 0.350 ug; approximately 0.4 ug; approximately 0.415 ug; approximately 0.425 ug; approximately 0.450 ug; approximately 0.5 ug; approximately 0.515 ug; approximately 0.52 ug; approximately 0.55 ug; or approximately 0.6 ug.

[0123] Engineered PCMV antigen The methods disclosed herein are based on the design of recombinant PCMV antigens. Numerous studies have shown that human serum can react with a wide range of human CMV proteins derived from either purified virus or virus-infected cells. In humans, the majority of antiglycoprotein responses have been observed to be directed towards glycoprotein B of human CMV. Foust et al., J. Virol. 86(13): 7444-7447 (2012). However, the main neutralizing antibody responses observed against human CMV proteins appear to be directed towards non-gB glycoproteins, e.g., the gH / gL / UL128 / UL130 / UL131 complex. It is important to note, however, that many human CMV genes are not conserved within the PCMV genome. In fact, HCMV is not related to PCMV at the molecular level. Following molecular characterization of the PCMV genome, PCMV was renamed roseolovirus by the International Committee on Taxonomy of Viruses. Gu et al., Virology 460-461:165-172 (2014). To date, no correlation has been reported between antibody reactivity to PCMV and antibody reactivity to HCMV. In addition, human CMV antibodies do not cross-react with PCMV. Therefore, it is important to identify and design as many relevant antigens as possible to completely capture the presence of PCMV in biological samples.

[0124] Accordingly, in some embodiments of the methods disclosed herein, one or more operationalized PCMV antigens may comprise one or more operationalized PCMV antigens selected from the group consisting of envelope glycoprotein B (gB), envelope glycoprotein H (gH), envelope glycoprotein L (gL), envelope glycoprotein M (gM), envelope glycoprotein N (gN), major tegument phosphorylated protein 1 (U54A); major tegument phosphorylated protein 2 (U54B); and U100p.

[0125] In some embodiments, one or more manipulated PCMV antigens include glycoprotein B (gB). One or more manipulated PCMV antigens may include glycoprotein B gB, gH, and gL. In some embodiments, one or more manipulated PCMV antigens include gB, gM, and gN. In some embodiments, one or more manipulated PCMV antigens include gB and phosphorylated protein pp65. In some embodiments, one or more manipulated PCMV antigens include gB, gH, gL, gM, gN, and U100(Q1). In some embodiments, one or more manipulated PCMV antigens include gB, gH, gL, gM, gN, U100(Q1), and phosphorylated protein pp65.

[0126] One or more operationalized PCMV antigens intended by this disclosure may contain an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs. 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, or 32.

[0127] In some embodiments, one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, 32, or a combination thereof. In some embodiments, one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, or 31, and further include a tag. In some embodiments, the tag includes the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, one or more operationalized tagged PCMV antigens include the amino acid sequence of SEQ ID NO: 32.

[0128] In some embodiments, one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NO: 2. In some embodiments, one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 32. In some embodiments, one or more operationalized PCMV antigens include the amino acid sequences of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, and 32.

[0129] Sequence ID 2 ACM17140.1 Glycoprotein B [Swine beta-herpesvirus 2]

[0130] [ka]

[0131] In some embodiments, one or more operationalized PCMV antigens include a fragment of the amino acid sequence of SEQ ID NO: 2. For example, the fragment may include the C-terminus of glycoprotein B. In some embodiments, the C-terminal fragment of glycoprotein B includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the C-terminal fragment of glycoprotein B includes the amino acid sequence of SEQ ID NO: 32.

[0132] Sequence ID 3 MNSSSGTAISNTHSYYETDNKQEFENDRKPDTSNAVSEGSANKYSQEDAVCMLMAIKNLGDAYRRKNATKPSPSVL DKIRHLEYQQLSTED

[0133] Sequence ID 8

[0134] [ka]

[0135] Sequence ID 9 AAL47542.1 Glycoprotein B [Porcine beta-herpesvirus 2]; Porcine cytomegalovirus strain (nucleotides 539-3118); Glycoprotein B (gB)

[0136] [ka]

[0137] Sequence ID No. 25 C-terminal fragment - Glycoprotein B (gB) (AF268039.2 nucleotides 2771-3118) MFPYANSSSGTVISNTHSYYETNNKQEFENDRKPDTSNAVSEGSANKYSQEDAVCMLMAIKNLGDAYRRKNATKPSPSVLDKIRHLEYQQLSTEDV

[0138] Sequence ID 26 ACM17140.1 Glycoprotein B MFPYANSSSGTAISNTHSYYETDNKQEFENDRKPDTSNAVSEGSANKYSQEDAVCMLMAIKNLGDAYRRKNATKPSPSVLDKIRHLEYQQLSTEDV

[0139] Sequence ID No. 27 N-terminal fragment - Glycoprotein B (gB) (AF268039.2 nucleotides 539-3118)

[0140] [ka]

[0141] Sequence ID 28 ACM17140.1 Glycoprotein BN-terminal fragment

[0142] [ka]

[0143] Sequence ID 29 ACM17140.1 Glycoprotein B fragment

[0144] [ka]

[0145] Sequence ID 30 AAL47542.1 Glycoprotein B fragment

[0146] [ka]

[0147] Sequence ID 31 ACM17140.1 Glycoprotein B fragment (center)

[0148] [ka]

[0149] Sequence ID 32 [ka]

[0150] In some embodiments of the methods disclosed herein, one or more operationalized PCMV antigens are encoded by a polynucleotide sequence selected from SEQ ID NOs: 1, 5, 6, 7, 10, 11, or a combination thereof.

[0151] Sequence ID No. 1: Porcine cytomegalovirus glycoprotein B gene; GenBank:FJ595497.1

[0152] [ka]

[0153] Sequence ID 4 AF268039.2 Porcine cytomegalovirus strain B6 ORF 40-like protein gene; as well as glycoprotein B(gB) gene and DNA polymerase (pol) gene

[0154] [ka]

[0155] [ka]

[0156] [ka]

[0157] Sequence ID 5 AF268039.2 Porcine cytomegalovirus strain (nucleotides 539-929) glycoprotein B (gB)

[0158] [ka]

[0159] Sequence ID 6 AF268039.2 Porcine cytomegalovirus strain (nucleotides 2771-3118); glycoprotein B (gB)

[0160] [ka]

[0161] Sequence ID 7 AF268039.2 Porcine cytomegalovirus strain (nucleotides 539-3118); glycoprotein B (gB)

[0162] [ka]

[0163] Sequence ID 10 AF268039.2 Porcine cytomegalovirus strain (nucleotides 2309-2771)

[0164] [ka]

[0165] In some embodiments of the methods disclosed herein, one or more operationalized PCMV antigens comprise glycoprotein gH and / or glycoprotein gL. In some embodiments, one or more operationalized PCMV antigens comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 18 or 22. One or more operationalized PCMV antigens may comprise the amino acid sequence of SEQ ID NO: 18 and / or 22. The operationalized PCMV antigen may comprise glycoprotein gL or SEQ ID NO: 22.

[0166] The manipulated PCMV antigen may contain an amino acid sequence that is at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to glycoprotein gH or SEQ ID NO: 22.

[0167] Sequence ID 22 AGT99272.1 Glycoprotein L(U82) [Porcine beta-herpesvirus 2]

[0168] [ka]

[0169] One or more operationalized PCMV antigens may include glycoprotein gH or SEQ ID NO: 18. One or more operationalized PCMV antigens may include an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to glycoprotein gH or SEQ ID NO: 18.

[0170] Sequence ID 18 AGT99240.1 Glycoprotein H(U48) [Porcine beta-herpesvirus 2]

[0171] [ka]

[0172] In some embodiments, one or more operationalized PCMV antigens may comprise the amino acid sequences of SEQ ID NO: 18 and / or 22, as well as a tag. The tag may comprise the amino acid sequences of SEQ ID NO: 33 HHHHHHHHHH. The tag may also be a 10×his tag. The tag may be located at the N-terminus or C-terminus of the operationalized PCMV antigen.

[0173] In some embodiments, one or more operationalized PCMV antigens described herein are encoded by a polynucleotide sequence selected from SEQ ID NOs: 17, 21, or a combination thereof. The glycoprotein gH may be encoded by the polynucleotide sequence of SEQ ID NO: 17.

[0174] Sequence ID 17 KF017583.1:c64486-62438 Porcine cytomegalovirus strain BJ09(U48), whole genome

[0175] [ka]

[0176] The glycoprotein gL can be encoded by the polynucleotide sequence of SEQ ID NO: 21.

[0177] Sequence ID 21 KF017583.1:c106307-105552 Porcine cytomegalovirus strain BJ09(U82), whole genome

[0178] [ka]

[0179] In some embodiments of the methods disclosed herein, one or more operationalized PCMV antigens comprise U100p(gQ). In those embodiments, one or more operationalized PCMV antigens may comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 24. Alternatively, one or more operationalized PCMV antigens may comprise the amino acid sequence of SEQ ID NO: 24.

[0180] Sequence ID No. 24 AGT99280.1 Presumed protein U100p(gQ) [Porcine beta-herpesvirus 2]

[0181] [ka]

[0182] In some embodiments, one or more operationalized PCMV antigens may comprise glycoprotein gN and / or glycoprotein gM.

[0183] In this embodiment, glycoprotein gN may comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 16 or 20. Alternatively, glycoprotein gN may be encoded by the polynucleotide sequence of SEQ ID NO: 15.

[0184] In some embodiments, glycoprotein gM may contain an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 20, or may contain the amino acid sequence of SEQ ID NO: 20. Alternatively, glycoprotein gM may be encoded by a polynucleotide sequence selected for SEQ ID NO: 19.

[0185] Sequence ID 15 KF017583.1:61918-62247 Porcine cytomegalovirus strain BJ09(U46), whole genome

[0186] [ka]

[0187] Sequence ID 16 AGT99239.1 Glycoprotein N(U46) [Porcine beta-herpesvirus 2] MAILLKIDLQYERHRTFLCHIIMTWIVVLCFALFNVIVASEGLSFPSHKIDHNTEFYKQSCQSHVFEVEFTSFTTIWLLINSLFLLSSVGIFLKFWCYKSFAEETVKGY

[0188] Sequence ID 19 KF017583.1:c92555-91524 Porcine cytomegalovirus strain BJ09 (U72; glycoprotein M), whole genome

[0189] [ka]

[0190] Sequence ID 20 AGT99264.1 Glycoprotein M(U72) [Porcine beta-herpesvirus 2]

[0191] [ka]

[0192] One or more operationalized PCMV antigens may also comprise one or more major tegument phosphorylated proteins (pp65). In its embodiment, pp65 may comprise an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12 or 14, or may comprise the amino acid sequence of SEQ ID NO: 12 or 14. Alternatively, pp65 may be encoded by a polynucleotide sequence selected for SEQ ID NO: 11 or 13.

[0193] Sequence ID 11 KF017583.1:c72304-70307 Porcine cytomegalovirus strain BJ09 (major tector phosphorylated protein pp65; U54A), whole genome

[0194] [ka]

[0195] Sequence ID 12 AGT99246.1 Major tetragenic phosphorylated protein pp65;U54A [Porcine beta-herpesvirus 2]

[0196] [ka]

[0197] Sequence ID 13 KF017583.1:c73541-72354 Porcine cytomegalovirus strain BJ09 (U54B), whole genome

[0198] [ka]

[0199] Sequence ID 14 AGT99247.1 Major tetragenic phosphorylated protein pp65;U54B [Porcine beta-herpesvirus 2]

[0200] [ka]

[0201] One aspect of the present disclosure provides an automated serological method for detecting anti-porcine cytomegalovirus (PCMV) antibodies in porcine animals, comprising the steps of: contacting one or more manipulated PCMV antigens having an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs. 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, and 32 with a biological sample derived from a porcine animal; incubating one or more manipulated PCMV antigens with the biological sample for about 10 minutes to about 120 minutes; and detecting the binding of one or more manipulated PCMV antigens to the biological sample by immunodetection and / or chemiluminescence detection.

[0202] The methods disclosed herein may be multi-antibody detection assays. For example, the methods disclosed herein may be used to detect multiple antigens in a sample. The presence of antibodies against multiple active substances or antigens may result from co-infection, asymptomatic infection, or past infection. This represents a significant improvement over traditional standard immunoassays, such as fluorescent bead technology, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), and indirect immunofluorescence assay (IFA), which can only detect a single active substance in a sample.

[0203] In practice, ELISA, Western blotting, and IFA require the separate measurement of each antibody, making them cumbersome and time-consuming. Furthermore, traditional ELISA, Western blotting, and IFA cannot be used for high-throughput analysis of multiple antibodies in a single sample of biological fluid. Such analysis requires a method that can provide parallel and rapid analysis of a single sample containing one or more antibodies against one or more pathogens. While ELISA only provides protein binding information, CE Westernization techniques provide additional information about the target protein, such as molecular weight estimation (e.g., as size and size distribution) and protein integrity (e.g., isomers, fragments, and aggregates). Thus, automated capillary electrophoresis (CE) Westernization may be more sensitive than enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of target proteins in serum samples derived from immunized animals. CE Westernization techniques can be orthogonal methods for analyzing proteins in biological samples. They can complement traditional fluorescence and / or chemiluminescence detection methods such as ELISA methods.

[0204] The identification of pathogenically related antibodies in bodily fluids such as whole blood or serum can also be hindered by the presence of large amounts of native antibodies in the sample. These native antibodies may appear in complex staining patterns. Avrameas S., Immunol. Today 12(5):154-9 (1991). The presence of these native antibodies can also complicate the distinction of disease-related antibodies from a complex background of "autoimmune noise." Furthermore, as mentioned above, certain viruses may remain in the incubation stage and therefore may be undetectable using routine PCR-based assays.

[0205] Therefore, the methods described herein provide a system in which more than one antibody can be detected using a single assay. Accordingly, in some embodiments, one or more operationalized PCMV antigens may comprise glycoprotein B (gB). One or more operationalized PCMV antigens may comprise gB, gH, and gL. In some embodiments, one or more operationalized PCMV antigens may comprise gB, gM, and gN. In some embodiments, one or more operationalized PCMV antigens may comprise gB and phosphorylated protein pp65. In some embodiments, one or more operationalized PCMV antigens may comprise gB, gH, gL, gM, gN, and U100(Q1). In some embodiments, one or more operationalized PCMV antigens may comprise gB, gH, gL, gM, gN, U100(Q1), and phosphorylated protein pp65. In some embodiments, one or more operationalized PCMV antigens may include SEQ ID NOs: 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0206] In some embodiments, one or more operationalized PCMV antigens may contain amino acid sequences that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs. 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, and any combination thereof.

[0207] tag In some embodiments of the methods disclosed herein, one or more operationalized PCMV antigens may be conjugated with a tag protein. The tagged proteins can be selected from histidine tags (his tags), polyhistidine (poly(His)) tags, small molecular weight ubiquitin-like modifier tags (SUMO), VariFlex C-terminal solubility enhancement tags, short peptide C-terminal tags, thioredoxin (Trx) tags, VariFlex C-terminal solubility enhancement tags, solubility-enhancing peptide sequence (SET) tags, protein G IgG domain B1 (GB1) tags, protein A IgG repeat domain ZZ (ZZ) tags, immunoglobulin Fc tags, IgG-Fc tags, glutathione-S-transferase (GST) tags, maltose-binding protein tags (MBP), FLAG tag peptides (FLAG), streptavidin-binding peptide tags (Strep-II; strep), calmodulin-binding protein tags (CBP), mutant dehalogenase tags (Halo tags), biotin, or Staphylococcus aureus protein A (protein A).

[0208] In some embodiments, one or more operationalized PCMV antigens may be conjugated with a polyhistidine (polyhis) tag. The polyhistidine can be selected from 2×his tags, 3×his tags, 4×his tags, 5×his tags, 6×his tags, 7×his tags, 8×his tags, 9×his tags, or 10×his tags. The polyhis tag may be a 10×his tag or may contain the amino acid sequence of SEQ ID NO: 33. In some embodiments, the tagged operationalized PCMV antigen is PCMV envelope glycoprotein B (gB). The tagged operationalized PCMV antigen may contain the amino acid sequence of SEQ ID NO: 32. In some embodiments, the tagged operationalized PCMV antigen may contain the amino acid sequence of SEQ ID NO: 33 and an amino acid sequence selected from SEQ ID NOs: 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, or 31. In that embodiment, Sequence ID 33 may be at the N-terminus or C-terminus of Sequence IDs 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, or 31.

[0209] In some embodiments, the tagged operationalized PCMV antigen may include an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NOs. 33 and SEQ ID NOs. 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, and 31. In that embodiment, SEQ ID NO: 33 may be at the N-terminus or C-terminus of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 2, 3, 8, 9, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 29, 30, and 31.

[0210] A high-speed, high-throughput PCMV serological Western blotting technique (e.g., Simple Wes®) using automated capillary electrophoresis (CE) Western blotting has been developed to detect PCMV anti-IgG antibodies in samples derived from target organisms. The novel automated serological assays described herein include the use of porcine serum, or serum derived from target organisms transplanted with porcine cells, tissues, or organs, the application of specific dilutions and antigen concentrations, and proprietary modifications to the Wes® protocol. Collectively, these modifications (e.g., optimizations) have demonstrated consistent and reliable detection of anti-PCMV antibodies (e.g., anti-IgG antibodies) in the samples tested.

[0211] IV. PLHV Serological Assay Another aspect of this disclosure provides a method for detecting porcine lymphotropic herpesvirus (PLHV) in a subject using a novel serological assay disclosed herein. In some embodiments, the porcine lymphotropic herpesvirus (PLHV) is selected from PLHV-1, PLHV-2, PLHV-3, or a combination thereof.

[0212] In some embodiments, one or more operationalized PLHV antigens are selected from the group consisting of envelope glycoprotein B (gB), envelope glycoprotein H (gH), envelope glycoprotein L (gL), envelope glycoprotein M (gM), envelope glycoprotein N (gN), major tegument phosphorylated protein 1 (U54A), major tegument phosphorylated protein 2 (U54B); and U100p.

[0213] One or more operationalized PLHV antigens may contain glycoprotein B(gB). PLHV glycoprotein B(gB) may contain an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, or a combination thereof. In some embodiments, PLHV glycoprotein B(gB) contains the amino acid sequence of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, or a combination thereof.

[0214] For example, one or more operationalized PLHV antigens may be PLHV-1 antigens containing the amino acid sequences of SEQ ID NOs. 34, 35, 36, or combinations thereof.

[0215] Sequence ID 34 AMO03269.1 Glycoprotein envelope protein (full sequence) Porcine lymphotropic herpesvirus 1

[0216] [ka]

[0217] In some embodiments, one or more operationalized PLHV antigens intended by this disclosure are variants of SEQ ID NO: 34. In some embodiments, one or more operationalized PLHV antigens intended by this disclosure include the N-terminus derived from the original GENBANK sequence named AMO03269.1. In some embodiments, one or more operationalized PLHV antigens may include SEQ ID NO: 35.

[0218] Sequence ID 35 PLHV1 antigen sequence - partial

[0219] [ka]

[0220] Sequence ID 36 10xhis tag-PLHV1 antigen sequence - partial

[0221] [ka]

[0222] In another embodiment, one or more operationalized PLHV antigens may be PLHV-2 antigens comprising the amino acid sequences of SEQ ID NOs. 37, 38, 39, or combinations thereof.

[0223] Sequence ID 37 AAO12353.1 Glycoprotein B (full sequence) Porcine lymphotropic herpesvirus 2

[0224] [ka]

[0225] In some embodiments, one or more operationalized PLHV antigens intended by this disclosure are variants of SEQ ID NO: 37. In some embodiments, one or more operationalized PLHV antigens intended by this disclosure include the N-terminus derived from the original GENBANK sequence named AA012353.1. In some embodiments, one or more operationalized PLHV antigens may include SEQ ID NO: 38.

[0226] Sequence ID 38 PLHV2 antigen sequence - partial

[0227] [ka]

[0228] Sequence ID 39 10×his tag-PLHV2 antigen sequence - partial

[0229] [ka]

[0230] In another embodiment, one or more engineered PLHV antigens can be PLHV-3 antigens comprising the amino acid sequences of SEQ ID NO: 40, 41, 42, or combinations thereof. In some embodiments, one or more engineered PLHV antigens can comprise the amino acid sequences of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0231] SEQ ID NO: 40 AAO12300.1 Glycoprotein B (Full sequence) [Porcine lymphotropic herpesvirus 3]

[0232]

Chem.

[0233] In some embodiments, one or more engineered PLHV antigens contemplated by the present disclosure are variants of SEQ ID NO: 40. In some embodiments, one or more engineered PLHV antigens contemplated by the present disclosure comprise the N-terminus from the native GENBANK sequence designated AA012300.1. In some embodiments, one or more engineered PLHV antigens can comprise SEQ ID NO: 41.

[0234] SEQ ID NO: 41 PLHV3 antigen sequence - a portion

[0235]

Chem.

[0236] SEQ ID NO: 42 10×his-tag - PLHV3 antigen sequence - a portion

[0237]

Chem.

[0238] V. Other viral assays and multiplex viral assays Methods for detecting PLHV and PCMV are described herein, but these methods can also be applied to other viruses of interest using the same general techniques and methods, achieving the same advantages, such as the detection of latent infections. For example, the assays described herein can be used to detect any porcine virus.

[0239] In some embodiments, the porcine viruses include enteric alphacoronavirus (SeACoV), porcine epidemic diarrhea virus, African swine fever virus (ASFV), classical swine fever virus (CSFV), foot-and-mouth disease virus, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine circovirus type 4 (PCV4), porcine circovirus-associated disease (PCVAD), porcine hepatitis E virus (porcine HEV), porcine influenza virus (SIV), porcine tolkutenosus virus (TTSuV), porcine sapovirus (porcine SaV), seneca virus A (SVA), highly pathogenic porcine epidemic diarrhea virus (PEDv), influenza virus H1N1pdm09, and porcine influenza virus. Porcine virus, porcine thrombus virus (PToV), porcine saperovirus (PSV), porcine bocavirus (PBoV), porcine cob virus (PKBV), SENECA virus (SVA), atypical porcine pestivirus (APPV), porcine acute diarrhea syndrome (SADS coronavirus); SADS-CoV, influenza D, porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV); classical swine fever virus (CSF); porcine thrombus virus (PToV); porcine reproductive and respiratory syndrome virus (porcine Respiratory reproductive syndrome virus (PRRSV); human influenza A; Nipah virus; atypical swine pestivirus (APPV); porcine epidemic diarrhea coronavirus (PEDV); and porcine delta coronavirus (PDCoV); porcine epidemic diarrhea coronavirus (PED-CoV); coronavirus infectious gastroenteritis virus (TGEV); hepatitis E virus (HEV); swine bokavirus; sapovirus; saperovirus; posavirus-1; swine astrovirus; swine enterovirus-9; cobb virus; swine bokavirus-2; swine enterovirus-9; coronavirus; Po-circo-like or swine bokavirus-4; swine encephalomyelitis virus (different from swine hemagglutinating encephalomyelitis virus (coronavirus)); rotavirus A, B, or C; pseudorabies virus (PHV-1);Porcinis stomatitis virus; porcine parvovirus 1 or 2; porcine adenovirus; porcine respiratory coronavirus; or porcine pneumovirus, including orthopneumovirus (SOV).

[0240] In some embodiments, the virus may belong to the families Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picornaviridae, or Reoviridae.

[0241] The methods can be carried out sequentially or in parallel. For example, in some embodiments, a single assay can be performed to detect more than one virus, if present, such as PLHV and PCMV.

[0242] One aspect of this disclosure provides a method for detecting the presence of multiple viruses in a biological sample derived from a subject, the method which may include the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with antigens for each of the viruses of interest for a time sufficient to allow binding between the corresponding antibody in the diluted serum sample and the antigen; and detecting the presence or absence of antibodies from the diluted serum sample that react with the antigen using automated capillary electrophoresis (CE) Western or Western blot analysis. Methods described herein for detecting corresponding antibodies, if present, using multiple viral antigens may be used by those skilled in the art. Therefore, the methods described herein are not limited to a single virus, nor to PCMV and PLHV. Similarly, those skilled in the art may use multiple antigens for the same virus in a single assay. This is possible in the assays described herein because CE Western techniques can provide additional information about the target protein, such as molecular weight estimation (e.g., as size and size distribution) and protein integrity (e.g., isomers, fragments, and aggregates).

[0243] In some embodiments, automated capillary electrophoresis (CE) Western is a closed-loop automated capillary immunoassay system. In some embodiments, the sample is separated electrophoretically by mass. In some embodiments, the separated proteins are immobilized on a solid support. In some embodiments, the solid support is the capillary wall of a microfluidic device. In some embodiments, electrophoretic separation of proteins from the sample can provide size distribution profiling of the proteins found in the sample, thereby enabling the detection of one or more viral antigens.

[0244] Another aspect of this disclosure provides a kit for detecting anti-porcine cytomegalovirus (PCMV) antibodies in a biological sample derived from a subject. The kit may include: one or more operationalized PCMV antigens as described herein; and a detection entity for detecting interactions between one or more operationalized PCMV antigens and one or more anti-PCMV antibodies present in the biological sample. [Examples]

[0245] The Technology is further illustrated by the following examples, which should not be construed as limiting. The examples herein are provided to illustrate the merits of the Technology and to further assist those skilled in the art in preparing or using compositions and systems of the Technology. The examples should not be construed as limiting the scope of the Technology as defined by the appended claims. The examples may include or incorporate any of the above-described variations, aspects, or embodiments of the Technology. The above-described variations, aspects, or embodiments may also include or incorporate any other variations, aspects, or embodiments of the Technology.

[0246] [Example 1] PCMV Serological Western Blot Assay Numerous PCR-based methods have been developed by academic and diagnostic laboratories to detect PCMV infection in subjects. These methods are cumbersome and can only detect zoonotic virus infections when the virus is in the active viremia stage. After the virus enters the incubation stage, PCR-based assays often tend to produce false-negative results. Immunological methods such as ELISA or Western blotting can serve as reliable alternatives to PCR-based methods. Western blotting can detect infection in both the viremia and incubation stages. However, to date, diagnostic laboratories have neither provided nor offer serological assays for PCMV. Furthermore, all published serological assays for PCMV are cumbersome, insensitive, and unreliable. Therefore, a high-speed, high-throughput PCMV serological Western blotting technique using the SimpleWes™ instrument has been developed to detect PCMV anti-IgG antibodies in subject-derived samples. The novel automated serological assays described herein include the use of porcine serum, or serum derived from subjects transplanted with porcine cells, tissues, or organs, the application of specific dilutions and antigen concentrations, and proprietary modifications to the Wes® protocol. Collectively, these modifications (i.e., optimizations) demonstrated consistent and reliable detection of PCMV anti-IgG antibodies.

[0247] The following examples validated serological Western blot assays for PCMV using known PCMV-positive, PCMV-negative, and latent infection samples.

[0248] method PCMV antigen preparation: Porcine CMV glycoprotein B (accession number: FJ595497.1;2309~2771) was expressed as a 10×His-tagged protein in a bacterial expression vector. Cloning of the antigen's DNA sequence, protein purification, and quality control were performed by a contract manufacturer (Origene Technologies Inc.). The predicted molecular weight of the PCMV antigen was determined using rabbit anti-His primary antibody (assay-positive control) (17kDa and 40kDa).

[0249] Study subjects: Nine pigs of the same age (60–116 days old) were included in the study. Five of these pigs were weaned early from sows and kept inside a biosecure barrier for their entire lives. These five animals were PCMV-negative by previous PCR analysis. The other four pigs were raised in a general herd and had been shown to be PCMV-positive by previous PCR analysis. Four older pigs (238, 240, 317, and 323 days old) that were PCMV-positive by previous PCR analysis were also included in the analysis. Whole blood was obtained from each of these pig animals for PCR and PCMV serological Western blot assays.

[0250] PCMV Serological Western Blot Setup: Following the manufacturer's (Simple Wes®) sample preparation protocol, PCMV antigen was diluted to 0.29 μg / μl in PBS buffer and loaded at 0.52 ug per lane. Porcine serum was diluted 1:75; 1:300; and 1:1200 in serum dilution buffer. Rabbit anti-porcine IgG HRP secondary antibody was diluted 1:250 in antibody dilution buffer. All reagents were loaded into Simple Wes® 22-250 kDa module plates. Serum incubation time with antigen was increased to 60 minutes, regardless of the manufacturer's recommendation. PCR analysis was performed in a diagnostic laboratory or in-house (data not presented).

[0251] result Samples from pigs reared in a biosecure high-health facility showed no detectable antibodies to PCMV. Serum samples (#1 - 5) from pigs reared in a biosecure high-health facility (i.e., a barrier) and previously negative for PCMV by PCR analysis, and a serum sample (#6) from a known PCMV-positive pig were subjected to serological Western blot analysis for PCMV. No signal was detected in serum samples from five pigs reared in the high-health facility, while the serum sample from the known PCMV PCR-positive pig showed strong signals at 17 kDa and 40 kDa up to a dilution of 1:1200. The serological Western blot analysis of PCMV was consistent with the PCR results (data not shown), indicating that the serological assay could distinguish PCMV-positive pigs from negative pigs.

[0252] Figures 2 - 4 show Western blot results using a serological Western blot assay for porcine cytomegalovirus (PCMV). The experiment was conducted using sera from six different pig animals reared under two different conditions, demonstrating the detection of anti-PCMV antibodies in pig animals reared outside a biosecure high-health facility (i.e., a barrier) facility compared to their littermates reared inside the biosecure high-health facility (i.e., a barrier) facility. The positive control contained a rabbit anti-His primary antibody used to determine the expected molecular weights (17 kDa and 40 kDa) of the PCMV antigen. Specifically, samples #1, #2, and #3 were pig serum samples obtained from three different pigs reared inside the biosecure high-health facility (i.e., a barrier) facility. As shown in Figures 2 - 4, samples from all three pigs showed no detectable antibodies to PCMV.

[0253] Samples from pigs reared outside a biosecure high-health facility showed detectable antibodies to PCMV. Serological Western blotting for PCMV was performed on serum samples from pigs (#6-9) and older pigs (#10-13), all of which were housed in a typical herd and had previously tested positive for PCMV by PCR DNA analysis (data not shown). A serum sample from a known PCMV PCR-positive pig (#6) was also included in the assay (data not shown). Like the positive control, all pigs (6-9, 10-13) showed a positive signal at the expected molecular weight, indicating positive PCMV infection. However, the PCR assay was negative for PCMV, indicating that the infection was in the incubation period (data not shown). These data demonstrate that this novel serological assay is sensitive enough to detect latent PCMV virus in all tested pigs, including older pigs.

[0254] Figures 2–4 show the results from experiments conducted using serum from six different pig animals raised under two different conditions. Samples #4, #5, and #6 were porcine serum samples from three different pigs raised outside the barrier facility and littermates of pigs #1, #2, and #3 mentioned above. As shown in Figures 2–4, all three samples showed detectable antibodies against PCMV at dilutions of 1:75 and 1:300. The negative control did not contain serum. The positive control contained rabbit anti-His primary antibody, which was used to determine the expected molecular weight (17 kDa and 40 kDa) of the PCMV antigen.

[0255] [Example 2] Sensitivity of PCMV serological Western blot assay compared to PCR-based assay To determine the sensitivity of the serological Western blot assay for PCMV, time-series experiments were conducted to detect anti-PCMV antibodies at different infection stages in pigs raised outside a biosecure barrier facility using PCR-based assays and the serological Western blot assay for PCMV. This example demonstrates that the serological Western blot assay for PCMV was as sensitive as or more sensitive than the PCR-based assay.

[0256] Serum samples were collected from pigs between 12 and 103 days of age and tested for PCMV in an independent veterinary diagnostic laboratory using PCR-based assays and serological Western blot assays for PCMV. Figure 5 shows the Western blot results for porcine cytomegalovirus (PCMV) serological Western blot assays, prepared using Compass software. In 12-day-old animals, two weak positive signals were detected in the correct Western blot (MW). A stronger signal was observed in 83-day-old animals, and the signal intensity was enhanced in 88-day-old and 103-day-old animals. Positive controls contained serum from pigs known to be PCMV-positive by PCR analysis from an independent veterinary diagnostic laboratory and were used to determine the expected molecular weight of the PCMV antigen. Negative controls did not contain serum (e.g., buffer only).

[0257] Table 1 summarizes the time series of results obtained from an independent veterinary diagnostic laboratory and serological Western blot assays of PCMV. On day 12, the PCR result from the independent veterinary diagnostic laboratory was negative. However, the serological Western blot assay of PCMV showed a weak positive signal. This weak signal could have been passively transmitted maternal antibodies. Therefore, the data demonstrate that the serological Western blot assay of PCMV can detect passively transmitted maternal antibodies.

[0258] On days 83 and 88, PCR results from an independent veterinary diagnostic laboratory were positive for PCMV. Both PCR analysis and serological Western blot assays for PCMV from an independent veterinary diagnostic laboratory on days 83 and 88 showed a positive PCMV signal. On day 103, serological Western blot assays for PCMV continued to show positive results. Collectively, Table 1 and Figure 5 demonstrate the ability to detect anti-PCMV antibodies during early PCMV viremia.

[0259] The table shows a timeline of tests, including relevant results for PCR analysis and serological Western blot assays for PCMV from an independent veterinary diagnostic laboratory, illustrating the importance of serological Western blot assays for PCMV in detecting maternal antibodies and antibodies produced during early-stage viremia.

[0260] [Example 3] "The PCMV serological Western blot assay can detect the presence of PCMV in latent-stage PCMV infections." To further evaluate the sensitivity of the serological Western blot assay for PCMV, its ability to detect latent-stage infections was determined using animals housed in a biosecure barrier facility.

[0261] Twenty-one pigs housed in a biosecure barrier facility were tested using a PCR-based assay. All 21 animals received negative PCMV PCR results from an independent veterinary diagnostic laboratory. These animals were then retested using a serological Western blot assay for PCMV. Figure 6 shows Western blot results generated by Compass software illustrating the efficacy and sensitivity of the serological Western blot assay for porcine cytomegalovirus (PCMV). It demonstrates the detection of anti-PCMV antibodies during the incubation period of PCMV infection in two of the 21 pigs housed in the biosecure barrier facility (samples #12 and #19).

[0262] As shown in Figure 6, the 19 re-examined animals were also negative for PCMV using a serological Western blot assay. Surprisingly, two of the 21 pig animals (samples #12 and #19) were positive for PCMV. Sample #12 was >3 months old, and sample #19 was 14 months old.

[0263] These data demonstrate that the serological Western blot assay for PCMV was able to reliably detect PCMV in its latent stage throughout the lifespan of animals. Furthermore, the serological Western blot assay for PCMV was able to reliably detect latent PCMV infections that were undetectable by PCR.

[0264] conclusion This is the first demonstration of a serological Western blot assay for PCMV using SimpleWes®. Thus, a serological Western blot assay for PCMV had never been performed before. The results described herein provide a novel serological assay that may potentially be performed in any diagnostic laboratory under GLP conditions.

[0265] Each assay can process at least approximately 24 samples in 4–5 hours, compared to at least 8–24 hours for traditional Western blotting, to produce final results. Furthermore, the novel serological assays disclosed herein can detect PCVM antibodies at various life stages of any subject (e.g., pigs). For example, the assays disclosed herein can detect anti-PCVM antibodies in colostrum, in newborn piglets, in potentially infected young piglets (e.g., approximately 2–3 months old), in donor pigs for xenotransplantation (e.g., >4 months old), in young sows (e.g., >7 months old), in boars (e.g., >12 months old), and / or in mature growing sows (e.g., >12 months old).

[0266] Furthermore, this novel PCMV serological Western blot assay is a qualified assay for detecting anti-PCMV antibodies in preclinical porcine serum samples intended for non-human primate (NHP) or human xenotransplantation.

[0267] Table 2 shows the number of porcine serum samples assayed for the presence of anti-PCMV antibodies using this novel PCMV serological Western blot assay.

[0268] [Table 2]

[0269] [Example 4] PLHV Serological Assay PCR-based methods have been developed by academic and diagnostic laboratories to detect gamma herpesvirus and PLHV infections. These methods are cumbersome and only capable of detecting the presence of the virus in the active viremia stage. After the virus enters the incubation period, the assays produce false-negative results. To date, no veterinary diagnostic laboratory has validated serological assays for PLHV-1, PLHV-2, and PLHV-3. Serological assays for PLHV-1, PLHV-2, and PLHV-3 published in peer-reviewed academic journals are cumbersome, insensitive, and unreliable. We designed novel serological Western blot assays for PLHV-1, PLHV-2, and PLHV-3 and validated their ability to detect the presence of PLHV-1, PLHV-2, and PLHV-3 IgG antibodies in porcine serum using known positive and negative PLHV-infected serum samples.

[0270] method PLHV1 antigen preparation Porcine lymphotropic herpesvirus 1 GD33 envelope glycoprotein B (accession number: KT844939.1;1~798) was expressed as a 10×His-tagged protein in a bacterial expression vector. The DNA sequence of the antigen was cloned, the protein purified, and quality control was performed by a contract manufacturer (Origene Technologies Inc.). The expected protein molecular weight was confirmed using a rabbit anti-His primary antibody (assay-positive control).

[0271] PLHV2 antigen preparation Porcine lymphotropic herpesvirus-2 glycoprotein B (accession number: NC_038265.1;1~897) was expressed as a 10×His-tagged protein in a bacterial expression vector. The DNA sequence of the antigen was cloned, the protein purified, and quality control was performed by a contract manufacturer (Origene Technologies Inc.). The expected protein molecular weight was confirmed using a rabbit anti-His primary antibody (assay-positive control).

[0272] PLHV3 antigen preparation Porcine lymphotropic herpesvirus-2 glycoprotein B (accession number: NC_0552341.1;1~951) was expressed as a 10×His-tagged protein in a bacterial expression vector. The DNA sequence of the antigen was cloned, the protein purified, and quality control was performed by a contract manufacturer (Origene Technologies Inc.). The expected protein molecular weight was confirmed using a rabbit anti-His primary antibody (assay-positive control).

[0273] PLHV Serological Western Blot Setup Dilute PLHV-1, PLHV-2, and PLHV-3 antigens to 0.29 μg / μl in PBS buffer and load 0.52 ug per lane according to the manufacturer's protocol for sample preparation. Dilute porcine serum to 1:37.5; 1:75; 11:300, or 1:1200 in serum dilution buffer. Dilute rabbit anti-porcine IgG HRP secondary antibody to 1:2400 in antibody dilution buffer. Load all reagents into Simple WES® 22-250 kDa module plates. Increase the serum incubation time with antigens to 60 or 90 minutes. This increased incubation time deviates from the manufacturer's recommendation.

[0274] Serological Western blot assays for PLHV-1, PLHV-2, and / or PLHV-3 using Simple WES® have never been performed before. The combination of Simple WES®, a novel antigen design, and increased incubation time may provide a novel serological system that can be performed in any veterinary diagnostic laboratory (VDL) under GLP conditions. Each assay can process at least approximately 24 samples in a time required of 4–5 hours, compared to at least 8–24 hours to produce final results. Furthermore, the novel serological assays can detect PLHV-1, PLHV-2, and PLHV-3 antibodies at various life stages in any subject (e.g., pigs). For example, the assays disclosed herein can detect maternal anti-PLHV-1, PLHV-2, and / or PLHV-3 antibodies in colostrum, in newborn piglets, in potentially infected young piglets (e.g., about 2-3 months old), in donor pigs for xenotransplantation (e.g., >4 months old), in young sows (e.g., >7 months old), in boars (e.g., >12 months old), and / or mature growing sows (e.g., >12 months old).

[0275] result "Samples from pigs raised outside biosecure, high-hard-health barns showed detectable antibodies against PLHV1." Figure 7 shows Western blot results created with Compass software illustrating the efficacy and sensitivity of a serological Western blot assay for porcine lymphotropic virus (PLHV)-1. The experiment was conducted using serum from three different pigs housed under two different conditions, demonstrating the detection of anti-PLHV-1 antibodies in pigs housed outside a biosecure, high-hard-health (i.e., barrier) facility compared to pigs housed inside a biosecure, high-hard-health (i.e., barrier) facility. Specifically, samples #1 and #2 are porcine serum samples obtained from two different pigs housed inside a biosecure, high-hard-health (i.e., barrier) facility, while sample #3 is a porcine serum sample obtained from a pig housed outside a biosecure, high-hard-health facility.

[0276] As shown in Figure 7, anti-PLHV-1 antibodies were detected in pigs raised outside the biosecure barrier facility (sample #3; infected), but not in pigs raised inside the biosecure barrier facility (samples #1 and #2). Samples from the two pigs showed no detectable antibodies against PLHV1, while the serum from pig sample #3 showed detectable PLHV1 antibodies.

[0277] Samples #1 and #2 also had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. Therefore, as expected, the samples from the two pigs showed no detectable antibodies against PLHV1, while the pig serum from sample #3 showed detectable PLHV1 antibodies.

[0278] The manipulated PLHV-1 antigen was detected using rabbit anti-histidine (His) primary antibody and anti-porcine IgG HRP conjugate. Positive controls, containing rabbit anti-His primary antibody, were used to determine the expected molecular weight of the PLHV-1 antigen. The tested PLHV-1 antigen had a molecular weight of approximately 31 kDa. Negative controls did not contain serum.

[0279] "Samples from pigs raised outside biosecure, high-hard-health barns showed detectable antibodies against PLHV2." Figure 8 shows Western blot results created with Compass software illustrating the effectiveness of a serological Western blot assay for porcine lymphotropic virus (PLHV)-2. The experiment was conducted using serum from three different pigs housed under two different conditions, demonstrating the detection of anti-PLHV-2 antibodies in pigs housed outside a biosecure, high-hard-health (i.e., barrier) facility compared to pigs housed inside a biosecure, high-hard-health (i.e., barrier) facility. Specifically, samples #1 and #2 are porcine serum samples obtained from two different pigs housed inside a biosecure, high-hard-health (i.e., barrier) facility, while sample #3 is a porcine serum sample obtained from a pig housed outside a biosecure, high-hard-health facility.

[0280] As shown in Figure 8, anti-PLHV-2 antibodies were detected in pigs raised outside the biosecure barrier facility (sample #3; infected), but not in pigs raised inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. As expected, the samples from the two pigs showed no detectable antibodies against PLHV2, while the serum from sample #3 showed detectable PLHV2 antibodies.

[0281] The manipulated PLHV-2 antigen was detected using rabbit anti-histidine (His) primary antibody and anti-porcine IgG HRP conjugate. Positive controls, containing rabbit anti-His primary antibody, were used to determine the expected molecular weight of the PLHV-2 antigen. The tested PLHV-2 antigen had a molecular weight of approximately 41 kDa. Negative controls did not contain serum.

[0282] "Samples from pigs raised outside biosecure, high-hard-health barns showed detectable antibodies against PLHV3." Figure 9 shows Western blot results created with Compass software illustrating the effectiveness of a serological Western blot assay for porcine lymphotropic virus (PLHV)-3. The experiment was conducted using serum from three different pigs housed under two different conditions, demonstrating the detection of anti-PLHV-3 antibodies in pigs housed outside a biosecure, high-hard-health (i.e., barrier) facility compared to pigs housed inside a biosecure, high-hard-health (i.e., barrier) facility. Samples #1 and #2 are porcine serum samples obtained from two different pigs housed inside a biosecure, high-hard-health (i.e., barrier) facility, while sample #3 is a porcine serum sample obtained from a pig housed outside a biosecure, high-hard-health facility.

[0283] As shown in Figure 9, anti-PLHV-3 antibodies were detected in pigs raised outside the biosecure barrier facility (sample #3; infected), but not in pigs raised inside the biosecure barrier facility (samples #1 and #2). Samples #1 and #2 had negative PLHV PCR results from an independent veterinary diagnostic laboratory, while sample #3 had a positive PLHV PCR result. As expected from the PCR results, the samples from the two pigs showed no detectable antibodies against PLHV3, while the serum from sample #3 showed detectable PLHV3 antibodies.

[0284] The manipulated PLHV-3 antigen was detected using rabbit anti-histidine (His) primary antibody and anti-porcine IgG HRP conjugate. A positive control containing rabbit anti-His primary antibody was used to determine the expected molecular weight of the PLHV-3 antigen. The tested PLHV-3 antigen had a molecular weight of approximately 40 kDa. The negative control did not contain serum.

[0285] "The PCR test results did not distinguish between PLHV-1, PLHV-2, and PLHV-3." Serological Western blot assays are more sensitive than PCR assays. This is because the assays distinguished between PLHV-1, PLHV-2, and PLHV-3 (see Figures 7, 8, and 9). For example, the tested PLHV-1 antigen had a molecular weight of approximately 31 kDa (Figure 7). The tested PLHV-2 antigen had a molecular weight of approximately 41 kDa (Figure 8). The tested PLHV-3 had a molecular weight of approximately 40 kDa (Figure 9). In contrast, PCR-based assays performed by independent veterinary diagnostic laboratories did not distinguish between PLHV-1, PLHV-2, and PLHV-3. These results further demonstrate the superior sensitivity of the novel serological Western blot assay compared to methods used in the art for the detection of PLHV-1, PLHV-2, and PLHV-3.

[0286] Equivalents This technology should not be limited in terms of the specific embodiments described in this application, which are intended as examples of individual aspects of this technology. Many modifications and variations of this technology can be made without departing from the spirit and scope of this technology, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of this technology will be apparent to those skilled in the art from the foregoing description, in addition to those enumerated herein. Such modifications and variations are intended to fall within the scope of this technology. It should be understood that this technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which are naturally subject to change. It should also be understood that the terminology used herein is intended only to describe and not to limit specific embodiments.

[0287] In addition, if any feature or aspect of the present disclosure is described in relation to a group of Markush members, a person skilled in the art will recognize that the present disclosure also describes any individual member or subgroup of any member of the group of Markush members.

Claims

1. A method for detecting porcine cytomegalovirus (PCMV) in a biological sample derived from a subject, comprising the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with a PCMV antigen for a time sufficient to enable binding between the corresponding antibody in the diluted serum sample and the PCMV antigen; and detecting the presence or absence of an antibody derived from the diluted serum sample that reacts with the PCMV antigen using Western blotting analysis.

2. A method for detecting anti-porcine cytomegalovirus (PCMV) antibodies in a biological sample derived from a target, wherein the method is (a) The step of introducing one or more operationalized PCMV antigens, a biological sample derived from the subject, one or more anti-immunoglobulin antibodies, and one or more chemiluminescent molecules into one or more capillaries of a microfluidic device; (b) The step of separating one or more of the operationalized PCMV antigens by electrophoresis; (c) Immobilizing one or more electrophoretically separated operationalized PCMV antigens onto the capillary wall; (d) The step of bringing one or more immobilized operationalized PCMV antigens into contact with the biological sample from the subject; (e) Incubating one or more immobilized operationalized PCMV antigens with the biological sample for about 10 minutes to about 120 minutes; and (f) A step of detecting the binding of one or more operationalized PCMV antigens to the biological sample by immunodetection and / or chemiluminescence detection. A method comprising the presence of an immunodetection signal and / or a chemiluminescent signal indicating the presence of one or more anti-PCMV antibodies in the biological sample.

3. An automated serological method for detecting anti-porcine cytomegalovirus (PCMV) antibodies in porcine animals, (a) The step of contacting one or more operationalized PCMV antigens with the biological sample derived from the pig; (b) Incubating one or more operationalized PCMV antigens with the biological sample for about 10 minutes to about 120 minutes; and (c) A step of detecting the binding of one or more operationalized PCMV antigens to the biological sample by immunodetection and / or chemiluminescence detection. Automated serological methods, including [specific method / method].

4. The automated serological method according to claim 3, wherein, prior to step (a), one or more operationalized PCMV antigens are separated electrophoretically by mass and immobilized on a solid support.

5. The automated serological method according to claim 4, wherein the solid support is a capillary wall of a microfluidic device.

6. The method according to any one of claims 1 to 5, performed in a closed-loop automated capillary immunoassay system.

7. The method according to any one of claims 1 to 6, wherein the one or more operationalized PCMV antigens are incubated with the biological sample for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

8. The method according to any one of claims 1 to 7, wherein the anti-PCMV antibody is an IgM antibody or an IgG antibody.

9. The method according to any one of claims 1 to 8, wherein the one or more operationalized PCMV antigens are selected from the group consisting of envelope glycoprotein B (gB), envelope glycoprotein H (gH), envelope glycoprotein L (gL), envelope glycoprotein M (gM), envelope glycoprotein N (gN), major tegument phosphorylated protein 1 (U54A), major tegument phosphorylated protein 2 (U54B), and U100p.

10. The method according to any one of claims 1 to 9, wherein the one or more operationalized PCMV antigens comprises glycoprotein B (gB).

11. The method according to claim 10, wherein the one or more operationalized PCMV antigens include an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, or 32.

12. The method according to claim 10 or 11, wherein the one or more operationalized PCMV antigens include the amino acid sequence of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, 32, or a combination thereof.

13. The method according to any one of claims 10 to 12, wherein the one or more operationalized PCMV antigens comprise the amino acid sequence of SEQ ID NO: 3 or 32.

14. The method according to any one of claims 10 to 13, wherein the one or more operationalized PCMV antigens comprises the amino acid sequences of SEQ ID NOs: 2, 3, 8, 9, 25, 26, 27, 28, 29, 30, 31, and 32.

15. The method according to any one of claims 9 to 14, wherein the one or more operationalized PCMV antigens are encoded by a polynucleotide sequence selected from SEQ ID NOs: 1, 5, 6, 7, 10, 11, or a combination thereof.

16. The method according to any one of claims 1 to 15, wherein the one or more operationalized PCMV antigens comprise glycoprotein gH and / or glycoprotein gL.

17. The method according to claim 16, wherein the one or more operationalized PCMV antigens comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 18 or 22.

18. The method according to claim 15 or 16, wherein one or more operationalized PCMV antigens comprise the amino acid sequence of SEQ ID NO: 18 and / or 22.

19. The method according to any one of claims 15 to 17, wherein the one or more operationalized PCMV antigens are encoded by a polynucleotide sequence selected from SEQ ID NOs: 17, 21, or a combination thereof.

20. The method according to any one of claims 1 to 18, wherein the one or more operationalized PCMV antigens comprises U100p(gQ).

21. The method according to any one of claims 1, 2, and 6 to 20, wherein the subject is a mammal selected from primates, non-human primates, humans, or pigs.

22. A method for detecting porcine lymphotropic herpesvirus (PLHV) in a biological sample derived from a subject, comprising the steps of: obtaining a serum sample from the subject; diluting the serum sample with a diluent; contacting the diluted serum sample with a PLHV antigen for a time sufficient to enable binding between the corresponding antibody in the diluted serum sample and the PLHV antigen; and detecting the presence or absence of an antibody derived from the diluted serum sample that reacts with the PLHV antigen using Western blotting analysis.

23. A method for detecting anti-porcine lymphotropic herpesvirus antibodies in a biological sample derived from a target, wherein the method is (a) The step of introducing one or more operationalized PLHV antigens, a biological sample derived from the subject, one or more anti-immunoglobulin antibodies, and one or more chemiluminescent molecules into one or more capillaries of a microfluidic device; (b) Separating one or more of the operationalized PLHV antigens by electrophoresis; (c) Immobilizing one or more electrophoretically separated operationalized PLHV antigens onto the capillary wall; (d) The step of bringing one or more immobilized operational PLHV antigens into contact with the biological sample from the subject; (e) Incubating one or more immobilized operationalized PLHV antigens with the biological sample for about 10 minutes to about 120 minutes; and (f) A step of detecting the binding of one or more operationalized PLHV antigens to the biological sample by immunodetection and / or chemiluminescence detection. A method comprising the presence of an immunodetection signal and / or a chemiluminescent signal indicating the presence of one or more anti-PLHV-1 antibodies in the biological sample.

24. An automated serological method for detecting anti-porcine lymphotropic herpesvirus antibodies in porcine animals, (a) The step of contacting one or more operationalized PLHV antigens with a biological sample derived from a pig; (b) Incubating one or more operationalized PLHV antigens with the biological sample for about 10 minutes to about 120 minutes; and (c) A step of detecting the binding of one or more operationalized PLHV antigens to the biological sample by immunodetection and / or chemiluminescence detection. Automated serological methods including [specific method / method].

25. The automated serological method according to claim 24, wherein, prior to step (a), one or more operationalized PLHV antigens are separated electrophoretically by mass and immobilized on a solid support.

26. The automated serological method according to claim 25, wherein the solid support is a capillary wall of a microfluidic device.

27. The method according to any one of claims 22 to 26, wherein the porcine lymphotropic herpesvirus (PLHV) is selected from PLHV-1, PLHV-2, PLHV-3, or a combination thereof.

28. The method according to any one of claims 22 to 27, performed in a closed-loop automated capillary immunoassay system.

29. The method according to any one of claims 22 to 28, wherein the one or more operationalized PLHV antigens are incubated with the biological sample for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

30. The method according to any one of claims 22 to 29, wherein the anti-PLHV antibody is an IgM antibody or an IgG antibody.

31. The method according to claim 22, wherein the one or more operationalized PLHV antigens are selected from the group consisting of envelope glycoprotein B (gB), envelope glycoprotein H (gH), envelope glycoprotein L (gL), envelope glycoprotein M (gM), envelope glycoprotein N (gN), major tegument phosphorylated protein 1 (U54A), major tegument phosphorylated protein 2 (U54B), and U100p.

32. The method according to any one of claims 22 to 31, wherein the one or more operationalized PLHV antigens comprises glycoprotein B (gB).

33. The method according to claim 32, wherein the one or more operationalized PLHV antigens comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs. 34, 35, 36, 37, 38, 39, 40, 41, 42, or a combination thereof.

34. The method according to claim 32 or 33, wherein the one or more operationalized PLHV antigens comprises the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, or a combination thereof.

35. The one or more of the processed PLHV antigens described above (a) PLHV-1 antigen containing the amino acid sequence of SEQ ID NOs: 34, 35, 36, or a combination thereof; (b) PLHV-2 containing the amino acid sequence of SEQ ID NOs: 37, 38, 39, or any combination thereof; and / or (c) PLHV-3 containing the amino acid sequences of SEQ ID NOs. 40, 41, 42, or combinations thereof. The method according to any one of claims 32 to 34, wherein one or more of the above.

36. The method according to any one of claims 22 to 35, wherein the one or more operationalized PLHV antigens comprises the amino acid sequences of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, and 42.

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