key: cord-0954187-9405zwjp
authors: Li, Yao‐Tsun; Chen, Ting‐Chih; Lin, Shu‐Yi; Mase, Masaji; Murakami, Shin; Horimoto, Taisuke; Chen, Hui‐Wen
title: Emerging lethal infectious bronchitis coronavirus variants with multiorgan tropism
date: 2019-11-20
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13412
sha: e5ec08eed8d1ba75d074bbdae0ec5c1e15eb2724
doc_id: 954187
cord_uid: 9405zwjp

Infectious bronchitis virus (IBV) causes respiratory diseases in chickens and poses an economic threat to the poultry industry worldwide. Despite vaccine use, there have been field outbreaks of IBV in Taiwan. This study aimed to characterize the emerging IBV variants circulating in Taiwan. The analysis of the structural protein genes showed that these variants emerged through frequent recombination events among Taiwan strains, China strains, Japan strains and vaccine strains. Cross‐neutralization tests revealed that two of the variants exhibited novel serotypes. Clinicopathological assessment showed that two of the variants caused high fatality rates of 67% and 20% in one‐day‐old SPF chicks, and all the variants possessed multiorgan tropisms, including trachea, proventriculus and urogenital tissues. Furthermore, the commercial live‐attenuated Mass‐type vaccine conferred poor protection against these variants. This study identified novel genotypes, serotypes and pathotypes of emerging IBV variants circulating in Taiwan. There is an urgent need for effective countermeasures against these variant strains.

Avian infectious bronchitis is an acute, highly contagious upper respiratory tract disease of chickens. Chickens of all ages and types are susceptible to the disease, which usually causes clinical symptoms, including rales, sneezing, shakes and diarrhoea. In addition, the reproductive tract of chickens can be affected, resulting in decreased egg quality and production (Cook, Jackwood, & Jones, 2012; Jackwood et al., 2005) . When young chicks are affected, damage to the reproductive tract can lead to reduced production. The virus transmits very rapidly in naive chickens and poses an economic threat to the poultry industry worldwide (Jackwood, 2012) .

possessing a single-stranded positive-sense RNA genome enclosed by an envelope. The virus is made up primarily of four structural proteins: spike glycoprotein (S), envelope protein (E), membrane glycoprotein (M) and nucleocapsid protein (N). The S protein is the most important viral protein for virus subtyping because it contains epitopes for neutralizing antibodies and thus evolves very quickly by random mutation or recombination (Lin & Chen, 2017) . The protein also mediates cell attachment and virus-host membrane fusion, playing a critical role in tissue specificity. Therefore, changes in the IBV S protein can easily influence the virus phenotype (Cavanagh, 2007;  Sjaak de Wit, Cook, & Heijden, 2011) . In recent years, IBV variants presenting novel genotypes, serotypes or pathogenicity have been identified in China Gao et al., 2016; Zhong et al., 2016; Zhou et al., 2017) , Korea (Hong, Kwon, Kim, Mo, & Kim, 2012) , Egypt (Zanaty et al., 2016) and Australia (Hewson et al., 2014) . These variants caused different degrees of mortality in chickens in experimental inoculations.

In Taiwan, IBV was first isolated in the early 1960s; since then, the live-attenuated Massachusetts-type (H120) IBV vaccine has been used Summary Infectious bronchitis virus (IBV) causes respiratory diseases in chickens and poses an economic threat to the poultry industry worldwide. Despite vaccine use, there have been field outbreaks of IBV in Taiwan. This study aimed to characterize the emerging IBV variants circulating in Taiwan. The analysis of the structural protein genes showed that these variants emerged through frequent recombination events among Taiwan strains, China strains, Japan strains and vaccine strains. Cross-neutralization tests revealed that two of the variants exhibited novel serotypes. Clinicopathological assessment showed that two of the variants caused high fatality rates of 67% and 20% in one-day-old SPF chicks, and all the variants possessed multiorgan tropisms, including trachea, proventriculus and urogenital tissues. Furthermore, the commercial live-attenuated Mass-type vaccine conferred poor protection against these variants. This study identified novel genotypes, serotypes and pathotypes of emerging IBV variants circulating in Taiwan. There is an urgent need for effective countermeasures against these variant strains.

infectious bronchitis virus, multiorgan tropism, pathotype, recombinant variants, serotype to prevent and control the disease (Lin, Wang, & Wang, 2005; Wang, Hsieh, & Chang, 1996) . However, IBVs locally circulating in Taiwan   have been found to be genetically different from all other genotypes in   the world and can be divided into two groups, namely, Taiwan Group   I (TW-I) and Taiwan Group II (TW-II) (Wang & Tsai, 1996) . Because of the lack of specific vaccines against endemic strains of IBV in Taiwan, IBV infections remain a problem in the poultry industry. Since 2002, IBVs causing severe outbreaks have been isolated from the field and subsequently identified as viral variants that emerged through frequent recombination events, including strains 2992/02 (Chen, Huang, & Wang, 2009) , 3374/05 and 3382/06 (Chen, Huang, & Wang, 2010) , and TC3/13 and S78/14 (Tsai, Tsai, & Wang, 2016) . These variants have been circulating in domestic chickens. In this study, we retrospectively characterized the genotype, serotype, pathogenicity and vaccine protection of emerging IBV variants.

Virus propagation was performed using 10-day-old specific-pathogen free (SPF) embryonated eggs (Animal Health Research Institute, Tamsui, Taiwan) via an allantoic route as previously described (Chen et al., 2009 ). Viral samples were inoculated in the allantoic cavity of embryos and incubated at 37°C for 72 hr. Allantoic fluid was subsequently collected and stored at −80°C until use. For virus titration, samples were diluted tenfold with sterile PBS, and each 10-day-old SPF egg received 0.1 ml of the diluted sample. Infection was determined by the presence of dwarfing or malformation in embryos 7 days post-infection (dpi). Viral titres were expressed as a 50% egg infectious dose (EID 50 ). At least five eggs were used for each dilution, and the EID 50 values were calculated by the method of Reed and Muench (1938) .

Viral RNA was extracted from the harvested allantoic fluid by a commercial RNA extraction kit (Geneaid Biotech Ltd.) following the manufacturer's guidelines. Full S and N genes were RT-PCR amplified and sequenced using the primers and protocols described in Lin et al. (2016) . DNA sequencing was conducted by a commercial service (Tri-I Biotech). Each nucleotide was determined from at least three identical results generated from separate PCR products.

Sequences of the reference IBV strains were GenBank with the accession numbers listed in Table S1 . Sequence data were compiled using the Lasergene (DNASTAR), and sequence alignments were conducted with the Clustal W method available in BioEdit software.

Phylogenetic trees were constructed with the neighbour-joining method using MEGA software (Tamura, Dudley, Nei, & Kumar, 2007) , and the bootstrap values were determined from 1,000 replicates of the original data. Phylogenetic trees of the S1, S2 and N genes of IBVs were constructed, where the classification of S1 gene is according to Valastro et al. (2016) . The recombinant analysis was performed using the Recombination Detection Program 4 (RDP4) software (Martin, Murrell, Golden, Khoosal, & Muhire, 2015) .

Anti-sera against IBV strains were prepared in SPF chickens. Groups of three 3-week-old chickens were intranasally inoculated with virus at a titre of 10 5 EID 50 and further boosted after 2 and 4 weeks with the same strain at a titre of 10 6 EID 50 intravenously. Blood was obtained by cardiac puncture two weeks after the last inoculation. Serum was heat-inactivated and stored at −20°C. The anti-serum to Mass-type H120 was acquired from Charles River Laboratories (North Franklin, CT). A cross-neutralization test was conducted as previously published (Wang & Huang, 2000) . Fourfold diluted sera were mixed with the same volume of 100 EID 50 virus at room temperature for one hour.

The mixtures were inoculated into 10-day-old SPF eggs, and the eggs were observed for survival on a daily basis. Seven days after inoculation, the eggs were opened and examined for typical lesions caused by IBV infection (dwarfing or malformation). The neutralizing titre of each serum against the homologous or heterologous virus was determined by the last serum dilution that protected 50% of the embryo. In addition, r-values were calculated by the method described by Archetti and Horfall (1950) . The antigenic (serotype) difference between two given strains was denoted as follows: r: 70%-100%, same serotype; r: 33%-70%, different subtype (minor); r: 11%-32%, different subtype (major); and r: 0%-10%, different serotype.

One-day-old chicks were inoculated intranasally with 10 6 EID 50 virus;

for each strain, chicks were observed daily for clinical signs and survival for 21 days (n = 5 or 6 per group). The clinical scores of IBV were interpreted according to the methods described by Avellaneda, Villegas, Jackwood, and King (1994) . The clinical signs were evaluated as follows: 0 = no clinical signs; 1 = lacrimation, slight shaking, watery faeces or tracheal rales; 2 = lacrimation, presence of nasal exudate, depression, watery faeces, apparent sneezing or cough; 3 = same as 2 but stronger with severe watery faeces; and 4 = death. Mean scores of each group during the 21-day observation period were calculated. For the tissue tropism and pathological evaluations, following infection, chicks were killed at 4, 7, 11 and 14 dpi (n = 3 or 4 each time point); the blood was collected for ELISA and tissues, including trachea, proventriculus, kidney and oviduct were collected for viral detection. Half of the harvested tissues were homogenized in tryptose phosphate broth and clarified by centrifugation. Viral detection by N gene-based RT-PCR was performed as described above. Another half of the tissues were further processed for immunohistochemical staining.

Tissues stored in formalin were trimmed, embedded in paraffin, and cut into sections. Sections were first processed to remove the paraffin by xylene and rehydrate by ethanol. Citric buffer (10 mM, pH 6.0) was used to retrieve the viral antigens at 95°C followed by treatment with 3% hydrogen peroxide. After blocking with 1% bovine serum albumin solution, slides with sections were incubated with anti-S1 monoclonal antibody (mAb) 2296-B1 (prepared from S1 recombinant protein-immunized mice) as the primary antibody (1:500) at 37°C for 40 min and an anti-mouse IgG HRP conjugate (Jackson ImmunoResearch) as the secondary antibody. The antigens were visualized by applying a substrate of peroxidase (DAB). Finally, the slides were counterstained with haematoxylin and fixed by mounting buffer.

Indirect ELISA against IBV was performed. Briefly, the IBV antigen was prepared as previously described (Chen, Wang, & Cheng, 2011) .

The serum antibody response was evaluated. The virus-specific antibody response induced by the IBV strains was evaluated as previously described (Lin et al., 2016) .

To assess the protection conferred by a commercially available vaccine, groups of 10 one-day-old chicks were intranasally inoculated with 10 4 EID 50 live-attenuated H120 vaccine (Merial), the most common vaccine used in Taiwan. After 14 days, chickens were challenged with the above-studied IBV variant strains or the Mass-type strain as a control. Another mock group of chickens received PBS inoculation.

Chickens were euthanized at 7-and 21-days post-challenge (dpc), and their pathological manifestations were evaluated. Viral shedding was also examined by collecting throat and cloacal swabs from each chicken at 7 and 21 dpc. Lesions in the trachea were evaluated as follows: 0 = no lesion; 1 = slight increase of mucin; 2 = large increase of mucin; and 3 = large increase of mucin and mucosal congestion.

Lesions in the proventriculus were evaluated as follows: 0 = no lesion; 1 = slight increase in the thickness of the mucosa; 2 = large increase in the thickness of the mucosa; and 3 = large increase in the thickness of the mucosa and mucosal congestion. Lesions in the kidney were evaluated as follows: 0 = no lesions; 1 = swelling, urate visible only under stereomicroscopy; 2 = swelling with urate; and 3 = same as 2 with a large amount of urate deposition in the kidney.

The lesion scores from three organs were averaged.

Data were analysed by unpaired t tests or ANOVA followed by Dunnett's multiple comparisons using GraphPad Prism (GraphPad Software). The p values smaller than .05 were considered significant.

As listed in Table 1 , five viral recombinants were previously isolated in Taiwan during 2002-2014 (Chen et al., 2009 (Chen et al., , 2010 Huang, Lee, Cheng, & Wang, 2004; Tsai et al., 2016) . Among the recombinants, IBV TC3/13 and S78/14 strains were isolated from chickens and broilers native to Taiwan in 2013 and 2014, respectively. These two strains have similar genetic sequences and are grouped as Taiwan-Japan (TW-JP) recombinants. IBV 2992/02 and its homologous strain 3374/05 were previously reported to have arisen from multiple recombination events from strains from Taiwan and China; therefore, they have been defined as Taiwan-China (TW-CN) recombinants.

In contrast, IBV 3382/06 was identified from broiler chickens in a poultry slaughterhouse, and this virus displays a Taiwan-Mass-type (TW-Mass) recombination. In this study, the 3′ structural protein genomes of these IBV variants along with the putative parental strain, JP/Akita/92, were fully sequenced. The sequences were submitted to GenBank, and the accession numbers are shown in Table S1 .

The phylogenetic analyses were compared among Taiwan variants and other reference strains. The S1 comparison ( Figure 1a) 

high nucleotide homology in structural proteins genes. In addition, the S1 gene sequences of these two strains were found to be clus- strains. These results suggest that TC3/13 and S78/14 were generated from the recombination of Taiwan and Japan IBV strains. We, therefore, performed the recombination analysis. RDP4 was used to display the consecutive nucleotide identity and illustrate the crossover events among the queried strain (S78/14) and the parental strains (2575/98 and Akita/92). As shown in Figure 1a ,d crossover event in the 3′ of the S1 gene (nt 1,416) was suggested by the similarity plot.

There was a significant difference (p < .01) between the resultant divisions of informative sites. IBV isolate S78/14 was therefore identified as an inter-typic recombinant among the two putative parental strains. Three other variants, 2992/02, 3374/05 and 3382/06, have previously been reported to be derived from multiple recombination events with Chinese or Mass strains, and the recombination sites were located in the S1, S2, M and 5a genes (Chen et al., 2009 (Chen et al., , 2010 .

To examine the antigenicity of the three viral variants, anti-serum was cross neutralized using two well-characterized local strains, 2575/98 (TW-I) and 2296/95 (TW-II), along with a vaccine strain, H120. The neutralization capacity of each anti-serum was evaluated based on the viral infectivity in embryonated chicken eggs inoculated with a virusserum mixture, and the r-value was calculated to assess the differences in the antigenicity between viruses. The r-value of homologous virus-serum was set as 100. The results shown in Table 2 and 

To evaluate the pathogenicity of the three viral variants in chickens, SPF chickens were infected with the three viruses, and their clinical signs were recorded daily (Figure 2a and Figure 2b ). Among all tested viruses, S78/14 induced chick death beginning at 7 dpi and caused the highest mortality rate of 67% at 21 dpi. IBV 2992/02 and 3382/06 caused fewer mortalities, with rates of 20% and 0%, respectively. Clinical signs such as tracheal rales and lacrimation occurred since the day following infection in all study groups of chicks except for the 3382/06 group, which showed only mild symptoms beginning at 3 dpi. More apparent respiratory signs, including sneezing and coughing, began at 5-7 dpi. Severe clinical signs, such as depression, were only present before animal death. As representative pictures shown in Figure (Figure 4) . The results showed that antibody titres against IBV of 2992/02-and 3382/06-infected chicks increased significantly from 11 to 14 dpi, whereas the titres of the S78/14-infected chicks increased moderately. Notably, the antibody titres of chicks infected with S78/14 at 11 dpi were higher than those of chicks infected with the other two selected viral strains.

To further study the tissue tropism of these strains, tissues from the trachea, proventriculus, kidney and oviduct were collected at 4, 7, 11 and 14 dpi, and viral RNA was examined by RT-PCR (Table 3) 

Based on the results above, the three novel IBV strains exhibited substantial antigenic distances to the Mass vaccine strain, and two of the strains exhibited virulence to chickens. Therefore, we inoculated chicks with live H120 vaccine prior to the challenge with different strains of virus to assess the protective effect of the vaccine against each studied strain. From the throat and cloacal swabs, viruses in all three groups were detected, but the positive rates declined in a temporal manner (Table 4 ). Among the three groups of IBV variants-infected chickens, chickens inoculated with S78/14 had the highest levels of virus detected from two swabbed sites across two time points, with positive rates of 78% and 89% at 7 dpc in throat swabs and cloacal swabs, respectively. For the 2992/02 group, which began with slightly lower positive rates at 7 dpc in the cloacal swabs, a similar shedding pattern was observed compared to the S78/14 group. At 21 dpc, 40% and 60% of viral shedding from the throat swabs and cloacal swabs, respectively, were still observed from these two variants-challenged groups. On the contrary, vaccination conferred better protection to 3382/06-infected chickens, with 50 and 20% of infected animals presenting positive viral detection at 7 dpc in the throat and cloacal swabs, respectively, and viruses were undetectable at 21 dpc. The group of chickens received homologous viral challenge or mock challenge demonstrated a low or zero detection rate for virus shedding. In addition to viral shedding, the challenged chickens were killed at 7 and 21 dpc to examine the gross lesions made by viruses. As shown in Figure one-day-old chicks, with mortality rates of 67% and 20%, respectively. These two strains also caused more severe clinical signs than 3382/06, which did not kill chicks. Furthermore, an extensive nephropathic effect was observed in S78/14-and 2992/02-infected chicks, while viral RNA was detected systemically in infected hosts.

Finally, the results from the challenge experiments showed that the current vaccine based on the Mass strain could not stop viral transmission from host to host, and lesions made by viruses were not prevented in vaccinated chickens inoculated with the studied viruses.

Recombination in S78/14 and TC3/13 strains was first recognized through nucleotide identity searching, wherein the S1 gene sequences were found to be similar with the IBVs clustered in the JP-I group (Mase et al., 2004) , but the S2 and N gene sequences were found to be highly homologous with local viruses isolated in Taiwan.

Using the RDP4 analysis, S78/14 was confirmed to emerge through recombination events between Taiwan and Japan strains; however, in the one-day-old chicken experimental infection, S78/14 exhibited increased lethality (67%) as compared to the 33.3% lethality caused by the TW-I strain 2575/98 (Lin et al., 2016) . Furthermore, a previous study has described the abnormal egg production of chickens caused by the infection of the Japan IBV strains JP8127 (Shieh et al., 2004) , which is closely related to S78/14 in term of the S1 gene, supporting our finding on the oviduct tropism of S78/14. However, F I G U R E 2 Pathogenicity of three Taiwan IBV variants in chickens. (a) One-day-old chicks were intranasally inoculated with 10 6 EID 50 viruses, and their survival rates were recorded daily. (b) Following infection, the daily clinical scores were recorded.

The clinical signs were evaluated as follows: 0 = no clinical signs; 1 = lacrimation, slight shaking, watery faeces or tracheal rales; 2 = lacrimation, presence of nasal exudate, depression, watery faeces, apparent sneezing or cough; 3 = same as 2 but stronger with severe watery faeces; and 4 = death. Mean scores of each group during the 21-day observation period are indicated (n = 5 or 6 chickens per group). *p < .05 the number of examined female chickens in this study was relatively limited, and the experimental period was not long enough to observe pathological lesions. Further investigation using a defined group of female chickens is warranted to confirm this observation. In conclusions, this study reported recombinant IBVs with novel serotypes, multiorgan tropism and lethality (Table S3 ). These strains caused disease outbreaks in the field, and the use of commercial

Mass-type vaccine is not effective in preventing these infections.

Our study indicates that more intense surveillance efforts and a review of the current IBV vaccine strategy are required.

The authors thank Dr. Ching-Ho Wang for providing the viruses. 

University under an approved Institutional Animal Care and Use Committee (IACUC) protocol (no. NTU-103-EL-3). All animal experiments were carried out in accordance with the approved guidelines.

Authors declare that none of conflict interests exist.

HWC conceived and designed the experiments; YTL TCC SYL and HWC performed the experiments; YTL TCC SYL and HWC analysed the data; MM SM and TH contributed essential materials; YTL and HWC wrote the paper. All authors read the final manuscript and approved it for submission.

Vaccination against novel IBV isolates using the Mass vaccine. One-day-old chicks were intranasally inoculated with H120 live-attenuated vaccine (10 4 EID 50 ), and after 14 days, chicks were infected with S78/14, 2992/02 or 3382/06 viruses intranasally. At 7-and 21-days post-challenge (dpc), animals were killed to examine their gross lesions made by viruses. Lesions in the trachea were evaluated as follows: 0 = no lesion; 1 = slight increase of mucin; 2 = large increase of mucin; and 3 = large increase of mucin and mucosal congestion. Lesions in the proventriculus were evaluated as follows: 0 = no lesion; 1 = slight increase in the thickness of the mucosa; 2 = large increase the thickness of the mucosa; and 3 = large increase in the thickness of the mucosa and mucosal congestion. Lesions in the kidney were evaluated as follows: 0 = no lesions; 1 = swelling, urate visible only under stereomicroscopy; 2 = swelling with urate; and 3 = same as 2 with a large amount of urate deposition in the kidney. The lesion scores from trachea, proventriculus and kidney were averaged

Hui-Wen Chen https://orcid.org/0000-0002-0595-3420

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Additional supporting information may be found online in the Supporting Information section