key: cord-0707783-5gjib3aa
authors: IWAMOTO, Jiro; OKADA, Daisuke; KITAHARA, Syouei; CHITOSE, Kenichi
title: Change of pathotype and phylogenetic analysis of infectious bronchitis virus detected in Kagoshima prefecture, Japan
date: 2020-02-10
journal: J Vet Med Sci
DOI: 10.1292/jvms.19-0491
sha: 52d3a9673702325151d33c3a7ebac37dd94ec63d
doc_id: 707783
cord_uid: 5gjib3aa

Infectious bronchitis (IB) is a highly contagious disease in chickens, induced by IB virus (IBV) infection. The pathotype and S1 genotype of IBV field strain that was detected from 2008 to 2018 were investigated in Kagoshima prefecture, Japan. The frequency of cases that the renal lesion characteristic of IBV infection was histopathologically confirmed was significantly higher from 2014 to 2018 than from 2008 to 2009, suggesting the altered pathotype of IBV. Of 7 genotypes (JP-I, JP-II, JP-III, JP-IV, Mass, Gray, and 4/91) that have been detected in Japan, 6 genotypes except for JP-II were detected since 2008 and it appeared that the JP-III and JP-I have been predominant. The JP-IV with different antigenicity from other genotypes was detected since 2009.

nephritis, tubular degeneration, and infiltration by heterophils in the kidney [3] . The frequency of cases that the respiratory and renal lesions as described above were histopathologically observed in the trachea and kidney was compared between 2008-2009 and 2014-2018 by Fisher's exact test.

Eighteen of RNA samples (6 samples from 2008-2009 and 12 samples from 2014-2018) utilized for diagnosis in the 17 cases were subjected to the phylogenetic analysis (Table 1 and Fig. 1 ). The reverse transcription-polymerase chain reaction was performed by One-Step RT-PCR kit (Qiagen, Hilden, Germany) with the primer set which amplifies the S1 gene of IBV [17] . After 1.5% agarose gel electrophoresis, the amplification products were purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The commercialized outsourcing sequence service (Fasmac DNA sequence service, http://fasmac.co.jp/gene_loupe) was utilized for the sequence of amplification products. Based on the sequences of amplification products and several S1 gene sequences of IBV retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank), the phylogenetic tree was constructed by the neighbor-joining method (500 bootstrap replicates) using MEGA 6 Software (https://www.megasoftware.net). The S1 genotypes of each sample were defined according to the classification that Mase et al. identified [17] [18] [19] . The detection rate of each genotype was calculated though the investigation period and in two groups (2008-2009 and 2014-2018) , respectively. The detection rate between the two groups was compared by Fisher's exact test.

All statistical analyses were performed using free statistical software R (version 3.5.2), distributed by The R Project (https:// www.r-project.org). The significant level was P<0.05 in all tests.

There was no significant difference in the frequency of histopathological observation of respiratory lesion between two groups, a) The identical alphabets indicate identical farms. b) In Case 8, the two genotypes of infectious bronchitis virus were detected from the trachea and kidney respectively, which is a pooled sample of three chickens. c) +: The case that the lesions were histopathologically observed, -: not observed. d) The "suspected" indicates the case that it was difficult to make a definitive diagnosis because the histopathological lesions were not observed or were very mild, despite the pathogen detection such as isolation or specific gene detection of the virus. IB: Infectious bronchitis. (Table 3) .

In Japan, the most of reports regarding IB were with nephritis until around 1990 [7, 9, 10, 16, 24] since the first report of IB nephritis in 1971 [11] , although there were a few reports of IB which developed the respiratory sign alone in affected chickens [23] . Also, the major clinical sign caused by IBV isolates submitted from the various regions in Japan for phylogenetic analysis was mostly nephritis since 1989, whereas that was respiratory from 1951 to 1980 [17] . Hence, it appeared that the pathotype of IBV field strain chronologically changed from respiratory to nephropathogenic in Japan. In Kagoshima prefecture, there was no report regarding the pathotype of IBV field strain except for the report in 1981 [20] and the predominant pathotype of IBV has not been known for a long period. The result of the present study showed that the detection of IBV field strain with nephropathogenicity has significantly increased in Kagoshima prefecture since 2014, suggesting that the nephropathogenic strain of IBV became predominant in Kagoshima prefecture. Conversely, the change of pathogenicity from highly nephropathogenic to respiratory has been reported in the field strain in Australia [8] , and thus it appeared that IBV field strains have constantly altered the pathogenic features.

Cavanagh et al. [6] described that the relationship between the pathogenicity of IBV and S protein is "open question", although it has been known that the S protein of IBV is the determinant factor of cell tropism [4] . In this study, there was no significant difference in the detection rate of the S1 genotype between the two groups, despite the change of pathotype in field strain, and the relationship between the pathogenicity of IBV and S1 gene remained unclear. Recently, some studies suggested that non-structural protein of IBV such as replicase or accessory proteins involved the pathogenicity of IBV [2, 14] . Although the S gene might not be significant to determine the pathogenicity of IBV, further studies will be needed to elucidate the relationship.

It is assumed that the elicitation of the histopathological lesion by IBV infection and the determination of IBV pathotype would be affected by the various factors (genetic factors, host factors such as breeds, days of age, and vaccination history, and environmental factors such as season, hygiene management of the farm, and the pathogens other than IBV) in the IB field case. However, there were no significant relationships between the observation of histopathological lesion and days of age, vaccination, and month of occurrence in the 17 cases of the present study (data not shown). Also, the histopathological lesions characteristic of IBV infection were not observed in 4 cases (Case 2, 3, 6, and 7) despite the complication with other diseases, as shown in Table 1 . It would be hard to identify and explain the determination factors of IBV pathotype only by the data in the present study.

The detection rate of each genotype, shown in Table 3 , might not necessarily reflect the prevalent status of IBV genotype in Kagoshima prefecture since 2008 because of the small sample size and bias regarding the region and farm of samples subjected to the present study. However, the JP-III and JP-I have been constantly detected for a long period at the wide area in the prefecture, as shown in Fig. 1 . Therefore, it appeared that the two genotypes have been predominant in Kagoshima prefecture since 2008.

The JP-I is indigenous to Japan [17] , and it was speculated that the JP-I has originally evolved for a long time also in Kagoshima prefecture. On the other hand, the origin of JP-III is unclear. The phylogenetic analysis of present and previous studies suggests that the JP-III is closely related to the isolate in China [1, 17] . It has been believed that the IBV has a wide host range and infects not only poultry but also wild birds such as a duck [6, 15] . Mase et al. inferred the possibility of involvement of wild birds regarding the dissemination of IBV into Japan [17] . In Kagoshima prefecture, there is a large plain (called "Izumi plain") as an overwintering site for wild migrant birds including a duck that infects with a virus such as avian influenza virus and that migrates among neighboring countries including China [21] . The predominance of JP-III in Kagoshima prefecture might not be irrelevant to the existence of a "portal site", such as Izumi plain, for viruses derived from neighboring countries. It is essential for the prevention and control of IB to estimate the S1 genotype of IBV field strain because the S1 genotype of field strain is associated with the vaccine selection in a chicken farm. All of the S1 genotypes, except for JP-II, were detected in Kagoshima prefecture since 2008. It implied the diversity of IBV field strain in Kagoshima prefecture. A flexible vaccination strategy would be needed for the prevention and control of IB.

The JP-IV was detected in 3 of 18 samples subjected to the phylogenetic analysis in the present study. The 3 samples were collected from 3 different cases (Case 2, 8 and 11 in Table 1 ) and the earliest detection case was in January 2009 in a broiler farm (Case 2 in Table 1 ). On the other hand, the JP-IV was initially reported as a novel genotype of IBV in Japan, which was identified from the samples collected in September 2009 in a layer farm located at Ibaraki prefecture [19] , more than 1,000 km far away from Kagoshima prefecture. Although the JP-IV was found in the farms located at Ibaraki and Kagoshima prefecture almost during the corresponding period, there was no geographic and epidemiological relationship between the two farms. Hence, it appeared that JP-IV has widely disseminated into Japan since at least 2009. In addition, the virulence of JP-IV was believed to be low in the previous study [19] . However, the both of respiratory and renal lesions were histopathologically confirmed in 2 of 3 cases (Case 8 and 11, Table 1 ) that the JP-IV was detected, and the JP-IV alone was detected in Case 11 ( Table 1 ). The 2 cases suggest the altered pathogenicity of JP-IV strain in Japan although the virulence to affected chickens in the cases might not necessarily be due to the IBV alone because of the complications such as Coccidiosis and Colibacillosis. Moreover, the antigenicity of JP-IV differs from others detected in Japan [19] . Therefore, the JP-IV strain could be a great threat to all of the chicken farms in Japan. Further studies regarding the pathogenicity and the prevalent status of JP-IV strain in Japan would be needed for the prevention of IB caused by JP-IV strain. 

Classification of IBV S1 genotypes by direct reverse transcriptase-polymerase chain reaction (RT-PCR) and relationship between serotypes and genotypes of strains isolated between 1998 and 2008 in Japan

The replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity

Pathogenesis and diagnostic approaches of avian infectious bronchitis

Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism

Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes

Coronavirus avian infectious bronchitis virus

Concurrence of nephrosis-nephritis due to infectious bronchitis virus and infectious bursal disease in broiler chickens

Pathogenicity of Australian strains of avian infectious bronchitis virus

Comparative characteristics among infectious bronchitis viruses isolated from broiler chickens with nephritis

Diagnosis of avian infectious bronchitis by indirect fluorescent antibody technique using monoclonal antibody

Pathology of spontaneous cases of avian transmissible tubulonephrosis

Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide

Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization

Identification of an infectious bronchitis coronavirus strain exhibiting a classical genotype but altered antigenicity, pathogenicity, and innate immunity profile

Isolation of avian infectious bronchitis coronavirus from domestic peafowl (Pavo cristatus) and teal (Anas)

Typing of recent infectious bronchitis virus isolates causing nephritis in chicken

Phylogenetic analysis of avian infectious bronchitis virus strains isolated in Japan

Existence of avian infectious bronchitis virus with a European-prevalent 4/91 genotype in Japan

A novel genotype of avian infectious bronchitis virus isolated in Japan in 2009

Comparison of two strains of avian infectious bronchitis virus for their interferon induction, viral growth and development of virus-neutralizing antibody in experimentally-infected chickens

Genetic diversity of highly pathogenic H5N8 avian influenza viruses at a single overwintering site of migratory birds in Japan

Serological studies of infectious bronchitis vaccines against Japanese field isolates of homologous and heterologous genotypes

Typing of avian infectious bronchitis virus isolates in Hiroshima prefecture by RT-PCR and restriction enzyme analysis

Some characteristics of infectious bronchitis virus strains isolated from chickens with nephritis