Getavirus passage attenuated strain and application thereof

Abstract

The invention discloses a GETV (Getah virus) passage attenuated strain HuN1-230, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: V2024111. The GETV passage attenuated strain HuN1-230 is named as GETV HuN1-230. The GETV HuN1-230 is named as GETV HuN1-230. The GETV HuN1-230 is named as GETV HuN1-230. The attenuated virus strain can be used for immunizing mice and pigs to generate a high-level neutralizing antibody, and 100% immune protection can be provided for virulent virus attack. The getavirus passage attenuated strain provided by the invention has the advantages of high culture titer, low biological safety risk, strong immunogenicity, good immune protection efficacy and the like, and is a safe and effective getavirus vaccine candidate attenuated strain.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a Geta virus attenuated strain and application thereof. Background Art

[0002] Getah virus (GETV) is an arbovirus with a wide host range, including humans, monkeys, birds, pigs, horses and other mammals. GETV is an important pathogen of animal infectious diseases and is pathogenic to many mammals. It can cause death of piglets and reproductive disorders in sows, fever, urticaria and hind limb edema in horses, and fever, anorexia, neurological symptoms and even death in foxes. Although there have been no reports of human diseases caused by GETV infection, some studies have speculated that GETV may be related to certain febrile diseases in humans, and in the future the virus may become an important pathogen threatening animal and human health.

[0003] In recent years, there has been a significant increase in the number of GETV infection cases in my country, which is a "new onset of an old disease". Due to the long-term neglect of diseases caused by GETV infection, there is still a lack of commercial vaccines available in China to prevent and control GETV infection. Inactivated vaccines and live attenuated vaccines have been widely used in the prevention of animal diseases. Among them, inactivated vaccines are highly safe, but the protective effect lasts for a short time due to limited mucosal immunity and cellular immune responses. In contrast, live attenuated vaccines can induce strong mucosal immunity and cellular immunity, and the induced immune protection can last longer, but the safety of live attenuated vaccines needs to be carefully considered. Therefore, there is an urgent need to develop safe and effective vaccines to prevent and control GETV.

[0004] CN 118147089 A discloses a Geta virus attenuated vaccine strain GETV-LA11, in which the NSP3 protein of the virus mutates, resulting in weakened virulence. The strain has the characteristics of genetic stability, good safety, and high protection efficiency in the preparation of attenuated live vaccines. However, the strain still has the disadvantages of strong virulence and low immune efficacy. Summary of the invention

[0005] The purpose of the present invention is to provide a Geta virus attenuated strain and its application to solve the problems existing in the above-mentioned prior art. The attenuated strain can effectively prevent and control Geta virus disease and is a candidate attenuated strain for a vaccine with high safety.

[0006] To achieve the above object, the present invention provides the following solutions: The present invention conducts a plaque test on a strong strain of porcine Geta virus isolated from a clinic (NCBI's GenBank accession number: MF741771), and selects Geta virus with a smaller plaque morphology for continuous cell propagation in vitro; first, it is continuously propagated on PK-15 cells to 100 generations; then, it is propagated to 230 generations under a changed culture cell temperature, and a weakened Geta virus strain with base changes is obtained. The sequencing results show that the weakened strain mutates at multiple sites of the nsP1, nsP2, nsP3, nsP4, E2, and E1 genes, and the amino acid mutations are mainly concentrated in the E2 protein, accounting for 46.67% of the total mutant amino acids. The applicant named the strain as GETV HuN1-230 strain.

[0007] The results of growth characteristics and immune efficacy evaluation showed that the highest titer of GETV HuN1-230 strain on BHK-21 cells was 10 9.27 TCID 50 / mL, it can induce high levels of neutralizing antibodies after immunization of mice and pigs, and the measured neutralizing antibody titers are significantly higher than those of the known GETV-LA11 strain.

[0008] GETV-negative sows in early pregnancy were selected and inoculated with equal amounts of GETV HuN1 and GETVHuN1-230 strains through intravenous injection to evaluate the pathogenicity of GETV wild-type strains and passaged strains. The results showed that the virulence of GETV HuN1-230 strain was significantly weakened compared with that of GETV HuN1 strain, and there were no adverse reactions in infecting pregnant sows. GETV HuN1 strain and GETV HuN1-230 strain were inoculated into ICR mice in early pregnancy, respectively, and GETV HuN1-230 strain also showed significantly weakened virulence, and there were no adverse reactions in infecting pregnant female mice.

[0009] GETV HuN1-230 exhibits strong immunogenicity, and both mice and pigs can produce high levels of antibodies after immunization, providing 100% immune protection against virulent attacks. The above results show that the Geta virus attenuated strain (GETV HuN1-230) obtained by the present invention has high safety, strong immunogenicity, and good immune protection efficacy, and can be used as an attenuated vaccine.

[0010] The attenuated strain of Getah virus provided by the present invention is named as Getah virus HuN1-230 strain, and its Latin name is: Getah virus (GETV) HuN1-230; the preservation number is CCTCC NO: V2024111, the preservation time is November 22, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0011] The present invention also provides the use of the attenuated Geta virus strain in preparing a Geta virus vaccine.

[0012] Preferably, the vaccine includes attenuated live vaccines and inactivated vaccines.

[0013] The invention also provides a Geta virus vaccine, which contains the attenuated Geta virus strain.

[0014] Preferably, the vaccine further comprises a pharmaceutically acceptable adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the cytopathic effect diagram of GETV HuN1-230.

[0016] Figure 2 Immunofluorescence images for IFA identification of GETV HuN1-230 infected cells.

[0017] Figure 3 This is a plaque image of GETV HuN1-230.

[0018] Figure 4 is the amino acid mutation rate of GETV HuN1-230.

[0019] Figure 5 This is the phylogenetic tree of GETV HuN1-230.

[0020] Figure 6 The virus titer of GETV HuN1-230 was determined.

[0021] Figure 7 Neutralizing antibody detection after GETV HuN1-230 immunization of mice and pigs, A: neutralizing antibody after mouse immunization; B: neutralizing antibody after pig immunization. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are only used to explain the present invention and are not intended to limit the present invention. In addition, the experimental operations involved in the embodiments, the parts not particularly described in the text are all routine operations in the art, and those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature or related specifications, manuals, etc. before the application date of the present invention for implementation.

[0023] Example 1: Screening and identification of attenuated GETV strains 1. Screening of attenuated GETV strains The GETV-positive material was filtered and inoculated into PK-15 cells, and the cytopathic effect was observed by blind passage for 3 generations. On the 4th day after blind passage for 2 generations on the cells, obvious cytopathic effect appeared, manifested as cell shrinkage, rounding, and detachment. The virus was identified by RT-PCR detection and indirect immunofluorescence (IFA) and other methods. The results showed that a strain of GETV was successfully isolated and named GETV HuN1 strain. Subsequently, the strong HuN1 virus was attenuated by continuous passage on PK-15 cells.

[0024] The isolated HuN1 strain was subjected to TCID 50 The virus was then diluted tenfold according to the TCID 50 The results of the virus solution were taken from two dilutions before and after the endpoint dilution (a total of five concentrations), and 0.2 mL of the virus solution was added to the cell culture plate respectively. After adsorption at 37°C for 1 hour, the virus solution was discarded; a 2% agarose solution was prepared and placed in a 40-50°C water bath for use; the above agarose and 2× cell maintenance solution were mixed at a ratio of 1:1, and 2 mL per well was added to each culture well, and solidified into a covering layer after cooling; the culture plate was inverted and placed in a 37°C carbon dioxide incubator for culture; the cell pathological changes were observed every day, and a second covering was performed when obvious lesions appeared; the culture plate was inverted and placed in a 37°C carbon dioxide incubator for continued culture, and the results were observed within 48 hours; GETV with smaller plaque morphology was selected for the next step of proliferation and identification. GETV with smaller plaque morphology was continuously propagated in vitro on PK-15 cells for 100 generations, and then propagated to 230 generations at a temperature higher than the normal culture cell temperature (39°C), and the passage strain was named GETV HuN1-230 strain.

[0025] 2. Biological identification of GETV attenuated strains (1) IFA identification When GETV HuN1-230 was inoculated into BHK-21 cells at an MOI of 0.01, typical lesions (shrinkage and rounding) appeared in the cells 12 h after inoculation. However, after GETV HuN1 inoculation, BHK-21 cells showed visible lesions at 24 hpi, which was 12 h later than GETV HuN1-230 ( Figure 1 ). Mouse anti-GETV E2 protein monoclonal antibody was used to identify infected cells by IFA. Infected cells showed a large number of IF-stained cells, and no green fluorescence was observed in negative control cells ( Figure 2 ).

[0026] (2) Plaque identification BHK-21 cells were cultured in 6-well plates and GETV HuN1-230 was added at 200, 100, 50 and 10 TCID 50 / 0.1mL of the titer inoculated cells; after incubation at 37°C for 1 hour, the virus was removed and the cells were rinsed with PBS; then the cells were covered with 2 ml of fresh DMEM containing 2% NBCS, 1% P&S and 1% low-temperature agarose gel, and cultured in a 5% CO2 environment. After 4 dpi, the cells were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and the size of the plaques was measured with a sliding caliper. The results showed that the plaque size of the GETVHuN1-230 strain on the cells was 0.97±0.32 mm, while the plaque size of the GETV HuN1 strain was 5.33±0.75 mm, and the plaque size of the GETV HuN1-230 strain was significantly reduced ( Figure 3 ).

[0027] (3) Genetic variation of GETV HuN1-230 strain The whole genome of GETV HuN1-230 strain was sequenced, and the full-length genome sequence of the strain was compared using MEGA 6.06 software. The results showed that compared with the genome sequence of GETV HuN1 strain, the amino acid changes of GETV HuN1-230 strain were at positions 214, 220, and 450 of the nsP1 gene; at position 650 of the nsP2 gene; at positions 344 and 446 of the nsP3 gene; at position 175 of the nsP4 gene; at positions 4, 173, 178, 214, 222, 232, and 322 of the E2 gene; and at position 323 of the E1 gene (Table 1). The amino acid changes of GETV HuN1-230 strain were mainly concentrated in the E2 protein, accounting for 46.67% of the total mutated amino acids ( Figure 4 ). Phylogenetic analysis was performed using the full-length gene nucleotide sequences of the other 40 GETV strains obtained from NCBI. The results showed that these GETV strains can be divided into four different groups (GⅠ-GⅣ). The GETV HuN1-230 strain belongs to group Ⅲ, which includes strains from China, Japan, South Korea, and Mongolia. GETV HuN1-230 is closely related to many Chinese strains ( Figure 5 ).

[0028] Table 1 Amino acid differences in each gene between GETV HuN1 strain and GETV HuN1-230 strain

[0029] a The location of amino acid variation sites in each gene of GETV-HuN1 strain (GenBank # MF741771).

[0030] Example 2: Growth characteristics and immune efficacy of GETV HuN1-230 strain To evaluate the growth characteristics and immune efficacy of GETV HuN1-230 strain, this study used TCID 50 The virus titer was determined by the virus neutralization test method, and the antibody titer was determined by the virus neutralization test method. The results were compared with the GETV-LA11 strain reported in CN 118147089 A. The results are as follows: (1) Virus titer determination To determine the growth characteristics of GETV HuN1-230, BHK-21 cells were cultured in a 6-well plate as monolayers; GETV HuN1-230 was then inoculated at an MOI of 0.01, the virus was removed after incubation at 37°C for 1 hour, and the cells were rinsed with phosphate-buffered saline (PBS); fresh DMEM supplemented with 2% NBCS and 1% P&S was then added, and the culture was incubated in a 5% CO2 environment. Cell samples were collected at predetermined time points (12, 24, 36, 48, 60, 72, 84, and 96 hours after inoculation) for virus titration. TCID 50 The results showed that the virus titer of GETV HuN1-230 strain reached a peak at 36 hpi, with the highest titer of 10 9.27 TCID 50 / mL, and then the virus titer decreased ( Figure 6 GETV-LA11 strain reached the highest titer of 10 at 36 hpi. 7.5 TCID 50 / mL, the maximum virus titer of GETV HuN1-230 strain on BHK-21 cells was nearly 100 times that of GETV-LA11 strain, indicating that GETV HuN1-230 strain had stronger replication ability.

[0031] (2) Neutralizing antibody titer determination 1) Neutralizing antibody titer of GETV HuN1-230 strain in mice To evaluate the immune efficacy of GETV HuN1-230 strain in mice, adult female mice were intravenously immunized with 0.1 mL of 10 6 TCID 50 / mL of GETV HuN1-230 strain and GETV-LA11 strain virus suspension. The control group of mice was immunized by the same route with an equal amount of saline, 6 mice in each group. Blood samples were collected from each group 7 days, 14 days and 21 days after immunization, placed at 37°C, and serum was separated after 1 hour and placed in a 56°C water bath for 30 minutes to inactivate complement. The serum was diluted 2 times, and the serum diluted at different times was compared with the GETV HuN1 strain standard virus (200TCID 50 / mL) were mixed in equal volumes and incubated in a 37°C incubator for 1 h. They were inoculated into confluent PK-15 cells, with 2 replicates for each sample. The cell status was observed daily. After 5 days, the pathological changes in each well were counted, and the neutralizing antibody titer was calculated according to the Reed-Muench method. The results are shown in Figure 7 As shown in A, neutralizing antibodies appeared 7 days after immunization with GETV HuN1-230 and GETV-LA11 strains, with an antibody positive rate of 100%. The average antibody titer of GETV HuN1-230 strain was 1:80, and the average antibody titer of GETV-LA11 strain was 1:41.33. At 14 days, the average antibody titer of GETV HuN1-230 strain was 1:213.33, and the average antibody titer of GETV-LA11 strain was 1:112. At 21 days, the average antibody titer of GETV HuN1-230 strain was 1:192, and the average antibody titer of GETV-LA11 strain was 1:96.

[0032] 2) Neutralizing antibody titer of GETV HuN1-230 strain in pigs To evaluate the immune efficacy of GETV HuN1-230 strain in pigs, adult sows were immunized intravenously with 4 mL of 10 6 TCID 50 / mL of GETV HuN1-230 strain and GETV-LA11 strain virus suspension. The control group of sows was immunized by the same route with an equal amount of saline, 6 in each group. Blood samples were collected from each group 7 days, 14 days, and 21 days after immunization to determine the neutralizing antibody titer. The results are shown in Figure 7 As shown in B, neutralizing antibodies appeared 7 days after immunization with GETV HuN1-230 and GETV-LA11 strains, with an antibody positive rate of 100%. The average antibody titer of GETV HuN1-230 strain was 1:85.33, and the average antibody titer of GETV-LA11 strain was 1:42.67. At 14 days, the average antibody titer of GETV HuN1-230 strain was 1:362.67, and the average antibody titer of GETV-LA11 strain was 1:170.67. At 21 days, the average antibody titer of GETV HuN1-230 strain was 1:277.33, and the average antibody titer of GETV-LA11 strain was 1:122.67.

[0033] The above results show that after immunization with the attenuated strain GETV HuN1-230, the animal body can be induced to produce high levels of neutralizing antibodies, and the titer of neutralizing antibodies induced by the GETV HuN1-230 strain is significantly higher than that induced by the GETV-LA11 strain.

[0034] Example 3: Safety and immune protection test of GETV HuN1-230 strain (1) Pathogenicity test of GETV HuN1-230 strain on pregnant ICR female mice To evaluate the pathogenicity of GETV HuN1 and GETV HuN1-230, 0.1 mL of 10 6 TCID 50 / mL of virus suspension. The control group mice were injected with an equal amount of saline by the same route, with 6 mice in each group. The clinical manifestations of pregnant mice were monitored every day after vaccination until the estimated due date; the weight of newborn mice was recorded every 4 days within 13 days after birth. The results showed that pregnant mice inoculated with GETV HuN1 strain showed severe pathogenicity, all embryos were completely absorbed during pregnancy, and there was no live birth (Table 2). In contrast, pregnant mice inoculated with GETV HuN1-230 strain at the same gestational age all gave birth successfully, with a survival rate of 100% (Table 2). Within 13 days after birth, the weight of newborn mice in the GETV HuN1-230 strain inoculation group was not significantly different from that of the negative control group (Table 3). These results indicate that the virulence of GETV HuN1-230 strain is significantly attenuated compared with the wild-type GETV HuN1 strain.

[0035] Table 2 Pregnant female mice intravenously inoculated with GETV wild-type strain (HuN1) and passaged strain (HuN1-230)

[0036] a The embryo was absorbed during pregnancy and no offspring was born.

[0037] Table 3 Body weight of newborn mice in the GETV HuN1-230 challenge group and the control group

[0038] (2) Pathogenicity experiment of GETV HuN1-230 strain in pregnant sows To evaluate the pathogenicity of GETV HuN1 and GETV HuN1-230 to pigs, 11-day-old gestation sows were intravenously injected with 4 mL of 10 6 TCID 50 / mL of virus suspension. The control group sows were injected with an equal amount of saline by the same route, with 2 pigs in each group. The clinical manifestations of pregnant sows were monitored daily after vaccination until the estimated due date; the weight of newborn piglets was recorded every 4 days within 28 days after birth. The results showed that pregnant sows inoculated with GETV HuN1 strain showed severe pathogenicity, abnormal return to estrus during pregnancy, and no piglets were born (Table 4). In contrast, sows inoculated with GETV HuN1-230 strain at the same gestational age all gave birth successfully, with a survival rate of 100% (Table 4). Within 28 days after birth, the weight of newborn piglets in the GETV HuN1-230 strain inoculation group was not significantly different from that in the negative control group (Table 5). These results indicate that the virulence of GETVHuN1-230 strain to pigs is significantly weakened compared with the wild-type GETV HuN1 strain.

[0039] The above results indicate that GETV HuN1-230 strain is highly safe in pregnant mice and sows.

[0040] Table 4 Pregnant sows intravenously inoculated with wild-type GETV strain (HuN1) and passaged strain (HuN1-230)

[0041] a Abnormal return of estrus during pregnancy, no offspring.

[0042] Table 5 Body weight of newborn piglets in the GETV HuN1-230 challenge group and the control group

[0043] (3) Immunoprotective study of GETV HuN1-230 strain on pregnant ICR female mice To evaluate the immune protection of GETV HuN1-230 strain on pregnant ICR female mice, adult female mice were intravenously inoculated with 0.1 mL of 10 6 TCID 50 / mL of GETV HuN1-230 virus suspension. The control group of mice was injected with an equal amount of saline through the same route, with 6 mice in each group. Female mice were mated 13 days after immunization, and 14 days after immunization, 1-day-old female mice were intravenously injected with 0.1 mL of 10 6 TCID 50 / mL of GETV HuN1 strong strain was used for challenge. The clinical manifestations of pregnant mice were monitored every day after vaccination until the estimated due date, and the challenge protection rate was determined. The results showed that the pregnant mice in the challenge control group showed severe pathogenic effects, all embryos were completely absorbed during pregnancy, and there was no live birth (Table 6). In contrast, the pregnant mice in the immunized group showed no clinical symptoms, all gave birth successfully, and the survival rate reached 100% (Table 6).

[0044] Table 6 Immune protection of GETV HuN1-230 on pregnant female mice

[0045] a The embryo was absorbed during pregnancy and no offspring was born.

[0046] (4) Immunoprotective test of GETV HuN1-230 strain on pregnant sows To evaluate the immune protection of GETV HuN1-230 strain in pregnant sows, adult sows were intravenously inoculated with 4 mL of 10 6 TCID 50 / mL of GETV HuN1-230 virus suspension. The control group of sows was injected with an equal amount of saline through the same route, with 2 sows in each group. Sows were mated 3 days after immunization, and 14 days after immunization, 11-day-old sows were intravenously injected with 4 mL of 10 6 TCID 50 / mL of GETV HuN1 strong strain was used for challenge. The clinical manifestations of pregnant sows were monitored every day after vaccination until the estimated due date to determine the challenge protection rate. The results showed that the sows in the challenge control group showed severe pathogenic effects, abnormal return to estrus during pregnancy, and no piglets were born (Table 7). In contrast, the sows in the immunized group did not show clinical symptoms, all gave birth successfully, and the survival rate reached 100% (Table 7).

[0047] The above results show that the attenuated strain GETV HuN1-230 can provide 100% immune protection against virulent attacks. The vaccine developed with this attenuated strain can effectively resist virulent attacks in mice and pigs, and has good immune protection, making it a candidate attenuated strain of vaccine with high safety.

[0048] Table 7 Immune protection of GETV HuN1-230 in pregnant sows

[0049] a Abnormal return of estrus during pregnancy, no offspring.

Claims

1. A Getah virus (GETV) attenuated strain, named GETV HuN1-230, was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: V2024111.

2. Use of the attenuated Geta virus strain according to claim 1 in the preparation of a Geta virus vaccine.

3. The use according to claim 2, characterized in that The vaccines include attenuated live vaccines and inactivated vaccines.

4. A Geta virus vaccine, characterized in that: The vaccine contains the attenuated strain described in claim 1.

5. The vaccine according to claim 4, characterized in that: The vaccine also includes a pharmaceutically acceptable adjuvant.

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