A high-titer, multi-tissue-tropic strain of avian infectious bronchitis virus

Abstract

The present invention relates to the field of virology technology, and specifically to a high titer, multi-tissue tropic chicken infectious bronchitis virus CK / CH / GX / 202109, whose deposit number is: CCTCC NO: V202257. The strain has been isolated, cultured and purified for multiple times, and has been identified by EID. 50 The measurement can reach 10 ‑7.85 / mL, with high culture titer. The strain showed similar clinical symptoms when used for eye drop and oral challenge, especially in 1-day-old SPF chickens, a mortality rate of 30%-40% was observed. The main clinical symptoms of surviving and dead chickens were respiratory symptoms and nephritis. The virus was detected by immunohistochemistry in the kidney, glandular stomach, gizzard, jejunum, ileum and rectum. The strain showed multi-tissue tropism and artificially reproduced gizzarditis. The strain can be used to study the pathogenic mechanism of the virus and the mechanism of changes in tissue tropism, and provide candidate strains for the future preparation of oral models of infectious bronchitis in chickens and the development and preparation of oral vaccines. It is of great significance in the prevention and control of chicken epidemics.

Description

Technical Field

[0001] The present invention belongs to the technical field of virology, and in particular relates to a high-titer, multi-tissue-tropic chicken infectious bronchitis virus CK / CH / GX / 202109, and also relates to the strain characteristics of the virus. Background Art

[0002] Infectious bronchitis virus (IBV) is a single-stranded, positive-sense RNA virus belonging to the genus Gammacoronavirus in the family Coronaviridae. IBV can infect chickens of all ages, with clinical manifestations primarily including upper respiratory symptoms, nephritis, and decreased egg production. It can also co-infect with other viruses, causing significant losses to the poultry industry. Discontinuous RNA replication and incomplete proofreading by RNA polymerase, as well as clinical immune pressure and natural selection, can cause IBV to mutate. The virus is prone to mutation and homologous recombination between genomes of different strains, leading to the continuous emergence of new IBV genotypes or mutants. Clinically, different IBV strains exhibit distinct tissue tropisms and pathogenicity, with different strains being found in diverse organs and tissues, including the proventriculus, gizzard, duodenum, jejunum, cecum, kidney, bursa of Fabricius, and cloaca. This may be related to the S protein.

[0003] IBV strains can be isolated from diseased materials, but it is not easy to obtain a strain with high titer, strong pathogenicity, multi-tissue tropism, and the ability to cause disease by eye drops or oral administration. Traditional IBV vaccine immunization methods mainly use injection or nasal drops. Screening a strong strain that can cause disease by spray or drinking water can provide candidate strains for the future preparation of oral disease models of chicken infectious bronchitis and the development and preparation of oral vaccines. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain with high titer through multiple rounds of blind transmission and purification, which meets the actual clinical production needs. At the same time, it can cause disease through oral or eye drop artificial infection, has strong pathogenicity, and the virus is detected in multiple tissues. It can be used as a candidate oral vaccine for attacking the virus and preparing an oral infectious bronchitis model for chickens.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] By collecting 40 glandular stomach and gizzard samples from 28-day-old yellow-feathered broiler chickens with decreased feed intake and depression in a chicken farm in Guangxi, IBV was isolated, purified and identified, and a high-titer, multi-tissue-tropic avian infectious bronchitis virus strain was obtained and named CK / CH / GX / 202109. Through S1 gene amplification and sequencing, the strain was found to belong to the "tl / CH / LDT3 / 03" type IBV strain.

[0007] Avian infectious bronchitis virus CK / CH / GX / 202109 was deposited in the China Center for Type Culture Collection on July 8, 2022, with the deposit number CCTCC NO: V202257, and the deposit address is Wuhan University, Wuhan, China.

[0008] The present invention artificially infects one-day-old SPF chickens with pure culture of avian infectious bronchitis virus CK / CH / GX / 202109 orally or by eye drop, and conducts a pathogenicity test. The results are as follows: the pure culture of the virus has a strong pathogenicity to one-day-old chickens, which is manifested in respiratory symptoms in one-day-old SPF chickens after infection, and death begins on the 6th day. The total mortality rate of the oral group 14 days after infection is 30%, and the total mortality rate of the eye drop group is 40%. Nephritis, swelling of the junction of the glandular stomach and gizzard, disappearance of glandular stomach papillae, shedding of gizzard lining and atrophy of the bursa of Fabricius were observed during autopsy. Immunohistochemistry showed that the virus was detected in the kidney, glandular stomach, gizzard, jejunum, ileum and rectum, showing multi-tissue tropism. The results of the pathogenicity test show that the pure culture of the virus can cause disease in one-day-old chickens by oral or eye drop infection. The virus content of each dose of IBV vaccine strain should be ≥10 3.5 EID 50 , the virus content of 2000 samples per bottle should be ≥10 7.2 EID 50 The strain of the present invention has the characteristics of strong pathogenicity and high titer, EID 50 is 10 -7.85 / mL, which has reached the culture titer for vaccine preparation. At the same time, the strain has a wide range of tissue tropism. Therefore, the strain has the potential to be developed into a vaccine strain that can be immunized through drinking water or spray and to prepare an oral disease model of chicken infectious bronchitis.

[0009] The advantages of the present invention are:

[0010] The present invention has obtained a strain of avian infectious bronchitis virus through separation, purification and identification, which is classified and named as avian infectious bronchitis virus CK / CH / GX / 202109. The strain was deposited in the China Center for Type Culture Collection on July 8, 2022, with a deposit number of CCTCC NO: V202257, and the deposit address is Wuhan University, Wuhan, China. The strain has a high titer, and oral and eye infection can cause death in SPF chickens, and the virus has been detected in multiple tissues. The strain of the present invention has a high titer, and both oral and eye infection of the strain are pathogenic to one-day-old chickens, making it more suitable to develop the strain into an oral vaccine strain for the prevention of clinical IBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Clinical examination of one-day-old SPF chickens challenged with the virus. A: Depression (day 7); B: Air sac inflammation (day 7); C: Enlargement of the proventriculus and the junction of the proventriculus and gizzard (day 14); D: Loss of proventriculus papillae (day 14); E: Loss of gizzard lining (day 7); F: Enlarged kidney with urate crystals (day 7).

[0012] Figure 2 Immunohistochemical results of 1-day-old SPF chickens challenged with the virus. A: Glandular stomach (day 14); B: Gizzard stomach (day 7); C: Jejunum (day 7); D: Ileum (day 7); E: Rectum (day 7); F: Kidney (day 14). DETAILED DESCRIPTION

[0013] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0014] Example 1 Isolation, purification and identification of virus strains

[0015] The isolation, purification and identification of an avian infectious bronchitis virus strain comprises the following steps:

[0016] 1) Sample Handling: From March to May 2020, several flocks of Sanhuang broiler chickens at a large poultry farm in Guangxi Province presented with clinical symptoms of depression, decreased feed intake, and slow growth. Necropsy revealed proventriculus inflammation. The birds had been vaccinated with H120 and 4 / 91 vaccines. Disease occurred in multiple day-old flocks, and young flocks died. Infectious bronchitis virus infection was suspected. The proventriculus or gizzard diseased material was separated, and 5 proventriculus samples were pooled as one sample, and 5 gizzard samples were pooled as one sample, for a total of 20 samples. The tissues were homogenized according to conventional methods, frozen and thawed once, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was sterilized with a 0.45 μm filter. 140 μL of the supernatant was then tested for IBV by RT-PCR and inoculated into embryos. Ten samples were positive for IBV. The primer sequences for IBV detection refer to the national standard "Diagnostic Technology for Infectious Bronchitis in Chickens" GB / T 23197-2008. The primer sequences are:

[0017] 5'-GGAAGATAGGCATGTAGCTT-3'(F);

[0018] 5'-CTAACTCTATACTAGCCTAT-3'(R).

[0019] 2) Virus isolation: 0.2 mL of the sample supernatant was inoculated into 10-day-old SPF chicken embryos. A total of 10 embryos were inoculated and incubated for 7 days. The embryos were observed for pathological changes and embryos that died within 24 hours were discarded. The allantoic fluid of 2 chicken embryos was collected on days 4, 5, 6, and 7, and the mixed fluid was recorded as the first generation. On the same day, 0.2 mL of the first generation allantoic fluid was collected and inoculated into 10-day-old SPF chicken embryos. This was passaged four times in succession. Only 3 samples were blindly passaged into chicken embryos. After incubation to day 17, the typical "dwarf embryo" phenomenon, with small individual development, head and claws clasped together, and embryonic body curled up, was observed. These embryos were identified as IBV positive by RT-PCR.

[0020] 3) Virus purification: The third generation allantoic fluid was diluted to 10 by limiting dilution method. -8 , from 10 -4 to 10 -8 Five embryos were inoculated at each dilution, and dead chicken embryos within 24 hours were discarded. After inoculation, the incubation was continued for 5 days to collect a single allantoic fluid sample with high dilution and obvious lesions. The sample was diluted five times in succession, and the second limiting dilution of one sample was 10 -7 When the 3rd, 4th and 5th limiting dilutions were used, the dilution degree was 10 -7 One to two chicken embryo allantoic fluids were identified as IBV positive. The chicken embryos were smaller in size and had slight bleeding on the skin compared with other chicken embryos of the same dilution. The highly diluted allantoic fluids were collected within 12 hours and stored at -70°C.

[0021] 4) Propagation: The inventors inoculated 10 10-day-old SPF chicken embryos with the highest dilution of the above-mentioned virus solution, inoculating each embryo with 100 μL. Chicken embryos that died within 24 hours were discarded. Fresh chicken embryo allantoic fluid was harvested 36 hours later and stored in a -70°C refrigerator in 1 mL aliquots. The aliquots were labeled as the p0 strain.

[0022] 5) Exogenous virus test: The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The results showed that the isolated and purified virus strains were not contaminated by exogenous viruses.

[0023] 6) The inventors have made a biological deposit of avian infectious bronchitis virus CK / CH / GX / 202109, with the deposit number CCTCC NO: V202257, the deposit unit is the China Center for Type Culture Collection, the deposit date is July 8, 2022, and the NCBI accession number is OM970248 (not yet published).

[0024] The above-mentioned PBS for dilution was prepared as follows: 7.9 g NaCl, 0.2 g KCl, 0.24 g KH2PO4 and 1.8 g K2HPO4 were weighed and dissolved in 800 mL of double-distilled water. The pH value of the solution was adjusted to 7.4 with HCl, and finally the volume was adjusted to 1 L with distilled water.

[0025] Example 2 Titer determination of avian infectious bronchitis virus CK / CH / GX / 202109 p0 strain

[0026] Take 1 tube of p0 virus solution from -70℃ refrigerator, dissolve it, take 100 μL and dilute it with sterile PBS, dilute it 8 times in total, take 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Five 10-day-old chicken embryos were inoculated with each dilution, and the inoculation volume was 0.1 mL / egg. Chicken embryos that died within 24 hours were discarded. The embryos were observed and recorded daily and those that died after 24 hours were collected. After 6 days, all the chicken embryos were placed in a 4°C refrigerator overnight. The chicken embryo allantoic fluid was collected the next day. If dwarf embryos appeared in the surviving chickens, it was recorded as positive. RNA was extracted from the remaining collected allantoic fluid using an RNA extraction kit and subjected to RT-PCR detection. The IBV detection primer sequence referred to the national standard "Diagnostic Technology for Avian Infectious Bronchitis" GB / T 23197-2008, and the positive and negative were determined based on the test results.

[0027] The first and second determinations were performed using the Reed-Muench method to calculate the EID of the infectious strain. 50 Both are 10 -7.78 / mL; the third determination was calculated using the Reed-Muench method, and the EID50 of the infectious strain was 10 -8.0 / mL. The average value of the three determinations was obtained, and the EID of the strain was 50 is 10 -7.85 / mL (see Table 1a, Table 1b and Table 1c).

[0028] Table 1a EID calculated by Reed-Muench method 50

[0029]

[0030] Table 1b EID calculated by Reed-Muench method 50

[0031]

[0032] Table 1c EID calculated by Reed-Muench method 50

[0033]

[0034] Example 3 S1 gene amplification

[0035] RNA from the p0 passage of avian infectious bronchitis virus (CK / CH / GX / 202109) strain was extracted using an RNA extraction kit, and the full-length sequence was amplified using the S1 primer. The S1 primer sequences are as follows: 5'-TGAAAACTGAACAAAAGAC-3' (F); 5'-GTATGTACTCATCTGTAAC-3' (R). The PCR reaction procedure was as follows: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 1 minute, and extension at 68°C for 2 minutes; and a final extension at 68°C for 10 minutes. Twenty μL of the PCR product was electrophoresed on 1 wt% agarose gel, then excised under UV irradiation. The PCR product was recovered using a DNA recovery kit and sent to a biotechnology company for sequencing. The sequencing results showed that the full length of the S1 genome nucleotide sequence of the chicken infectious bronchitis virus CK / CH / GX / 202109 strain was 1807 bp (SEQ ID NO.5), belonging to the "tl / CH / LDT3 / 03" type strain.

[0036] Example 4 Pathogenicity test of avian infectious bronchitis virus CK / CH / GX / 202109 strain on one-day-old SPF chickens

[0037] 1) One-day-old SPF chickens were randomly divided into three groups: a negative control group, an eye-drop group, and an oral administration group, with 20 chickens in each group. The chickens were weighed. The eye-drop and oral administration groups were infected with the p0 generation of avian infectious bronchitis virus CK / CH / GX / 202109 strain, diluted with PBS. The infection dose for the eye-drop group was 50 μL / bird (containing 10 6.0 EID 50 ), the oral infection dose was 500 μL / bird (containing 10 6.0 EID 50 ), and were observed for 14 consecutive days.

[0038] 2) On days 7 and 14 post-infection, chickens were weighed and sacrificed, and clinical symptoms were observed. The proventriculus, gizzard, and bursa of Fabricius were weighed, and the proventriculus index, gizzard index, and bursa of Fabricius index were calculated and statistically analyzed. Samples of the proventriculus, gizzard, bursa of Fabricius, duodenum, jejunum, ileum, and cecum were also collected and fixed with 10% paraformaldehyde and sent to a biotechnology company for immunohistochemistry.

[0039] Results showed that the CK / CH / GX / 202109 strain of avian infectious bronchitis virus (IBDV) was pathogenic to one-day-old SPF chickens, administered either by eye drop or oral route. Clinical manifestations included depression, decreased feed intake, and diarrhea starting on the fourth day. Health began to recover by the 12th day, but diarrhea developed in nine birds in the oral group and six in the eye drop group. Mortality began on the sixth day after infection, with a total mortality rate of 30% in the oral group and 40% in the eye drop group throughout the trial (Table 2). On the 7th day after infection, the body weight, weight gain, and proventriculus weight of the eye-dropping and oral groups were significantly different from those of the control group, and the gizzard index, bursa weight, and bursal index of the oral group were significantly different from those of the control group. On the 14th day after infection, the body weight and weight gain of the oral group were significantly different from those of the control group, the body weight of the eye-dropping group was significantly different from that of the control group, the proventriculus index and gizzard index of the oral and eye-dropping groups were significantly different from those of the control group, and the bursa weight and bursal index of the oral group were significantly different from those of the control group (see Table 3a and Table 3b).

[0040] Table 2 Mortality of 1-day-old SPF chickens

[0041]

[0042] Table 3a Body weight, weight gain, proventriculus index, gizzard index and bursa index of 1-day-old SPF chickens on day 7 after infection

[0043]

[0044] Note: Means with different superscripts (lowercase letters) differed significantly (p<0.05), and means with the same superscript did not differ significantly (p>0.05), excluding dead chickens.

[0045] Table 3b Body weight, weight gain, proventriculus index, gizzard index and bursa index of 1-day-old SPF chickens on day 14 after infection

[0046]

[0047] Note: Means with different superscripts (lowercase letters) differed significantly (p<0.05), and means with the same superscript did not differ significantly (p>0.05), excluding dead chickens.

[0048] 3) Autopsy symptoms

[0049] Surviving chickens were autopsied 7 and 14 days after infection, and clinical symptoms such as swelling of the glandular stomach and gizzard junction, partial disappearance of glandular stomach papillae, thickening of the glandular stomach wall, and shedding of gizzard gizzard were observed in a small number of chickens. Both dead and surviving chickens showed enlarged kidneys and urate crystals (see Figure 1 ).

[0050] 4) Immunohistochemistry

[0051] In the negative control group of one-day-old SPF chickens, no virus was detected on the 7th and 14th days after infection. Virus was detected in the glandular stomach, gizzard, kidney and rectum of the oral group, and in the gizzard, jejunum, ileum and rectum of the eye-dot group (see Figure 2 ).

[0052] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An avian infectious bronchitis virus strain characterized by: The strain is classified and named as chicken infectious bronchitis virus CK / CH / GX / 202109, and was deposited in the China Center for Type Culture Collection on July 8, 2022. The preservation number is CCTCC NO: V202257, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the avian infectious bronchitis virus strain according to claim 1 in preparing an avian infectious bronchitis model.

3. Use of the avian infectious bronchitis virus strain according to claim 1 as a candidate strain for developing an oral vaccine for avian infectious bronchitis.

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