A Temperature-Sensitive Recombinant of Avian Coronavirus Infectious Bronchitis Virus Provides Complete Protection against Homologous Challenge.

Avian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens' eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development. IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines.

Role of Stress Granules in Suppressing Viral Replication by the Infectious Bronchitis Virus Endoribonuclease.

Infectious bronchitis virus (IBV), a γ-coronavirus, causes the economically important poultry disease infectious bronchitis. Cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation. Previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. Here, we aimed to delineate the molecular mechanisms regulating the SG response to pathogenic IBV strain infection. We found that most chicken embryo kidney (CEK) cells formed no SGs during IBV infection and IBV replication inhibited arsenite-induced SG formation. This inhibition was not caused by changes in the integrity or abundance of SG proteins during infection. IBV nonstructural protein 15 (Nsp15) endoribonuclease activity suppressed SG formation. Regardless of whether Nsp15 was expressed alone, with recombinant viral infection with Newcastle disease virus as a vector, or with EndoU-deficient IBV, the Nsp15 endoribonuclease activity was the main factor inhibiting SG formation. Importantly, uridine-specific endoribonuclease (EndoU)-deficient IBV infection induced colocalization of IBV N protein/dsRNA and SG-associated protein TIA1 in infected cells. Additionally, overexpressing TIA1 in CEK cells suppressed IBV replication and may be a potential antiviral factor for impairing viral replication. These data provide a novel foundation for future investigations of the mechanisms by which coronavirus endoribonuclease activity affects viral replication. IMPORTANCE Endoribonuclease is conserved in coronaviruses and affects viral replication and pathogenicity. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal, and reproductive systems, causing millions of dollars in lost revenue to the poultry industry worldwide annually. Mutating the viral endoribonuclease poly(U) resulted in SG formation, and TIA1 protein colocalized with the viral N protein and dsRNA, thus damaging IBV replication. These results suggest a new antiviral target design strategy for coronaviruses.

Immunopathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus (IBV): Impact on the reproductive tract in layers.

IBV infection may lead to reduced egg production and poor egg quality in layer flocks. The DMV/1639 strain was recently identified as one of the most dominant IBV variants isolated from Canadian layer flocks with egg production problems. The current study aimed to investigate the immunopathogenesis of the Canadian DMV/1639 strain in laying chickens. Specific-pathogen-free (SPF) layers were infected at the peak of lay (29 weeks; n = 10) with an uninfected control group (n = 10). Egg production in the infected group dropped to 40% by the fifth day post-infection (dpi). Five birds from the infected and the control groups were euthanized at 5 and 10 dpi. Ovarian regression and shortened oviduct with marked histopathological changes were observed in the infected group at 10 dpi. An increase in the IBV viral load in reproductive tissues was accompanied by a significant recruitment (p < 0.05) of KUL01+ macrophages and CD4+ and CD8+ T cell subsets at 10 dpi. Additionally, anti-IBV antibody response was detected in serum and locally in the reproductive tract washes of the infected group. Overall, our findings contribute to the understanding of the pathogenicity of the Canadian DMV/1639 strain and the subsequent host responses in the reproductive tract of chickens.

Pathological effect of different avian infectious bronchitis virus strains on the bursa of Fabricius of chickens.

Infectious bronchitis is an acute and highly contagious disease caused by avian infectious bronchitis virus (IBV). As well as the typical clinical respiratory signs, such as dyspnoea and tracheal rales, QX genotype strains can also cause damage to the urinary system and reproductive system. Our previous studies found that chickens infected with QX-type IBV also displayed damage to the bursa of Fabricius. To investigate the effects of different genotypes of IBV on the bursa of Fabricius, we challenged one-week-old SPF chickens with Mass, QX and TW genotype IBV strains and compared the clinical signs, gross lesions, histopathological damage, viral loads, and expression levels of inflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-α,ß, γ and TNF-α). The results showed that all three strains caused tissue damage, while significant temporal variations in the viral loads of the different infected groups were detected. IBV infection seriously interfered with the natural immune response mediated by inflammatory cytokines (IFN-α, IFN-ß, IL-6 and IFN-γ) in chickens. Our results suggested that IBV has potential immunological implications for chickens that may lead to poor production efficiency. RESEARCH HIGHLIGHTSAvian coronavirus IBV is an important pathogen of chickens.IBV has potential immunological implications in chickens.The bursal viral load of different IBV strains varies significantly.

Infection with IBV DMV/1639 at a Young Age Leads to Increased Incidence of Cystic Oviduct Formation Associated with False Layer Syndrome.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes respiratory disease but can affect the reproductive tract of laying-type chickens. If infection occurs in pullets, false layer syndrome, which is characterized by the development of large, fluid-filled cystic oviducts, can occur. Recently, IBV strain DMV/1639 has been detected in parts of Canada and the U.S., where false layer syndrome has occurred, though it is not clear if IBV is the sole cause or if age at infection is an influencing variable. Our study investigates the role and timing of IBV infection on the development of false layer syndrome, using the IBV types DMV/1639 and Massachusetts (Mass). Six groups of 120 SPF chickens were challenged at either three, seven, or fourteen days of age, using either DMV/1639 or Mass IBV. Cystic oviducts were seen in all the challenged groups, and the pullets challenged at 14 days of age had fewer cystic oviducts than pullets challenged at 3 or 7 days of age. The highest percentage of severe histology lesion scores were seen in the 3-day challenge groups. The data collected in this experiment confirm that IBV DMV/1639 causes cystic oviducts and indicate that age at infection plays a role in the pathogenesis of false layer syndrome.

Identification of Amino Acids within Nonstructural Proteins 10 and 14 of the Avian Coronavirus Infectious Bronchitis Virus That Result in Attenuation In Vivo and In Ovo.

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.

Peering into Avian Influenza A(H5N8) for a Framework towards Pandemic Preparedness.

2014 marked the first emergence of avian influenza A(H5N8) in Jeonbuk Province, South Korea, which then quickly spread worldwide. In the midst of the 2020-2021 H5N8 outbreak, it spread to domestic poultry and wild waterfowl shorebirds, leading to the first human infection in Astrakhan Oblast, Russia. Despite being clinically asymptomatic and without direct human-to-human transmission, the World Health Organization stressed the need for continued risk assessment given the nature of Influenza to reassort and generate novel strains. Given its promiscuity and easy cross to humans, the urgency to understand the mechanisms of possible species jumping to avert disastrous pandemics is increasing. Addressing the epidemiology of H5N8, its mechanisms of species jumping and its implications, mutational and reassortment libraries can potentially be built, allowing them to be tested on various models complemented with deep-sequencing and automation. With knowledge on mutational patterns, cellular pathways, drug resistance mechanisms and effects of host proteins, we can be better prepared against H5N8 and other influenza A viruses.

Traceable surveillance and genetic diversity analysis of coronaviruses in poultry from China in 2019.

Coronavirus disease 2019 (COVID-19), caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China, and rapidly spread throughout the world. This newly emerging pathogen is highly transmittable and can cause fatal disease. More than 35 million cases have been confirmed, with a fatality rate of about 2.9% to October 9, 2020. However, the original and intermediate hosts of SARS-CoV-2 remain unknown. Here, 3160 poultry samples collected from 14 provinces of China between September and December 2019 were tested for SARS-CoV-2 infection. All the samples were SARS-CoV-2 negative, but 593 avian coronaviruses were detected, including 485 avian infectious bronchitis viruses, 72 duck coronaviruses, and 36 pigeon coronaviruses, with positivity rates of 15.35%, 2.28%, and 1.14%, respectively. Our surveillance demonstrates the diversity of avian coronaviruses in China, with higher prevalence rates in some regions. Furthermore, the possibility that SARS-CoV-2 originated from a known avian-origin coronavirus can be preliminarily ruled out. More surveillance of and research into avian coronaviruses are required to better understand the diversity, distribution, cross-species transmission, and clinical significance of these viruses.

Mutations in the Methyltransferase Motifs of L Protein Attenuate Newcastle Disease Virus by Regulating Viral Translation and Cell-to-Cell Spread.

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present.

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

Molecular Characterization and Demographic Study on Infectious Bursal Disease Virus in Faisalabad District.

The re-emergence of virulent strains of the Infectious Bursal Disease Virus (IBDV) leads to significant economic losses of poultry industry in Pakistan during last few years. This disease causes the infection of bursa, which leads to major immune losses. A total number of 30 samples from five IBD outbreaks during the period of 2019-20 were collected from different areas of Faisalabad district, Pakistan and assayed by targeting the IBD virus VP2 region through RT-PCR. Among all the outbreaks, almost 80% of poultry birds were found positive for the IBDV. The bursa tissues were collected from the infected birds and histopathological examination of samples revealed severe lymphocytic depletion, infiltration of inflammatory cells, and necrosis of the bursa of Fabricius (BF). Positive samples were subjected to re-isolation and molecular characterization of IBDV. The Pakistan IBDV genes were subjected to DNA sequencing to determine the virus nucleotide sequences. The sequences of 100 Serotype-I IBDVs showing nearest homology were compared and identified with the study sequence. The construction of the phylogenetic tree for nucleotide sequences was accomplished by the neighbor-joining method in MEGA-6 with reference strains. The VP2 segment reassortment of IBDVs carrying segment A were identified as one important type of circulating strains in Pakistan. The findings indicated the molecular features of the Pakistan IBDV strains playing a role in the evolution of new strains of the virus, which will contribute to the vaccine selection and effective prevention of the disease.

Two newly isolated GVI lineage infectious bronchitis viruses in China show unique molecular and pathogenicity characteristics.

During 2016 to 2020, GVI-1 type infectious bronchitis virus (IBV) strains were sporadically reported across China, indicating a new epidemic trend of the virus. Here we investigated the molecular characteristics and pathogenicity of two newly isolated GVI-1 type IBV virus strains (CK/CH/TJ1904 and CK/CH/NP2011) from infected chicken farms in China. Genetic evolution analysis of the S1 gene showed the highest homology with the GVI-1 representative strain, TC07-2. Phylogenetic analysis and recombination analysis of the virus genomes indicated that newly isolated strains in China may be independently derived from recombination events that occurred between GI-19 and GI-22 strains and early GVI-1 viruses. Interestingly, unlike the deduced parental GI-19 or GI-22 strains, CK/CH/TJ1904 and CK/CH/NP2011 showed affinity for the trachea rather than the kidney and were less pathogenic. This difference may be because of recombination events that occurred during the long co-existence of the GVI-1 viruses with prevalent GI-19 and GI-22 strains. Considering the new trend, it is very important to permanently monitor circulating strains and to develop new vaccines to counteract emerging new-type IBVs.

Genomic Analysis of Avian Infectious Bronchitis Viruses Recently Isolated in South Korea Reveals Multiple Introductions of GI-19 Lineage (QX Genotype).

Infectious bronchitis virus (IBV) was first identified in the 1930s and it imposes a major economic burden on the poultry industry. In particular, GI-19 lineage has spread globally and has evolved constantly since it was first detected in China. In this study, we analyzed S1 gene sequences from 60 IBVs isolated in South Korea. Two IBV lineages, GI-15 and GI-19, were identified in South Korea. Phylogenetic analysis suggested that there were six distinct subgroups (KM91-like, K40/09-like, and QX-like I to IV) of the South Korean GI-19 IBVs. Among them, QX-type III and IV subgroups, which are phylogenetically different from those reported in South Korea in the past, accounted for more than half of the total. Moreover, the phylogeographic analysis of the QX-like subgroups indicated at least four distinct introductions of GI-19 IBVs into South Korea during 2001-2020. The efficacy of commercialized vaccines against the recently introduced QX-like subgroups should be verified, and continuous international surveillance efforts and quarantine procedures should be enhanced to prevent the incursion of viruses.

Genetic and Pathogenic Characterization of QX(GI-19)-Recombinant Infectious Bronchitis Viruses in South Korea.

Infectious bronchitis viruses (IBVs) are evolving continuously via genetic drift and genetic recombination, making disease prevention and control difficult. In this study, we undertook genetic and pathogenic characterization of recombinant IBVs isolated from chickens in South Korea between 2003 and 2019. Phylogenetic analysis showed that 46 IBV isolates belonged to GI-19, which includes nephropathogenic IBVs. Ten isolates formed a new cluster, the genomic sequences of which were different from those of reference sequences. Recombination events in the S1 gene were identified, with putative parental strains identified as QX-like, KM91-like, and GI-15. Recombination detection methods identified three patterns (rGI-19-I, rGI-19-II, and rGI-19-III). To better understand the pathogenicity of recombinant IBVs, we compared the pathogenicity of GI-19 with that of the rGI-19s. The results suggest that rGI-19s may be more likely to cause trachea infections than GI-19, whereas rGI-19s were less pathogenic in the kidney. Additionally, the pathogenicity of rGI-19s varied according to the genotype of the major parent. These results indicate that genetic recombination between heterologous strains belonging to different genotypes has occurred, resulting in the emergence of new recombinant IBVs in South Korea.

Pathogenic evaluation of a turkey coronavirus isolate (TCoV NC1743) in turkey poults for establishing a TCoV disease model.

Turkey coronavirus (TCoV) can cause a highly contagious enteric disease in turkeys with severe economic losses in the global turkey industry. To date, no commercial vaccines are available for control of the disease. In the present study, we isolated a field strain (NC1743) of TCoV and evaluated its pathogenicity in specific-pathogen-free (SPF) turkey poults to establish a TCoV disease model. The results showed that the TCoV NC1743 isolate was pathogenic to turkey poults with a minimal infectious dose at 106 EID50/bird. About 50 % of one-day-old SPF turkeys infected with the virus's minimal infectious dose exhibited typical enteric disease signs and lesions from 6 days post-infection (dpi) to the end of the experiment (21 dpi). In contrast, fewer than 20 % of older turkeys (1- or 2-week-old) infected with the same amount of TCoV displayed enteric disease signs, which disappeared after 15-18 dpi. Although all infected turkeys, regardless of age, shed TCoV, the older turkeys shed less virus than the younger birds, and 50 % of the 2-week-old birds even cleared the virus at 21 dpi. Furthermore, the viral infection caused day-old turkeys more body-weight-gain reduction than older birds. The overall data demonstrated that the TCoV NC1743 isolate is a highly pathogenic strain and younger turkeys are more susceptible to TCoV infection than older birds. Thus, one-day-old turkeys infected with the minimal infectious dose of TCoV NC1743 could be used as a TCoV disease model to study the disease pathogenesis, and the TCoV NC1743 strain could be used as a challenge virus to evaluate a vaccine protective efficacy.

An attenuated TW-like infectious bronchitis virus strain has potential to become a candidate vaccine and S gene is responsible for its attenuation.

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.

Infectious bronchitis virus in Australia: a model of coronavirus evolution - a review.

Infectious bronchitis virus (IBV) was first isolated in Australia in 1962. Ongoing surveillance and characterization of Australian IBVs have shown that they have evolved separately from strains found throughout the rest of the world, resulting in the evolution of a range of unique strains and changes in the dominant wild-type strains, affecting tissue tropism, pathogenicity, antigenicity, and gene arrangement. Between 1961 and 1976 highly nephropathogenic genotype GI-5 and GI-6 strains, causing mortalities of 40% to 100%, predominated, while strains causing mainly respiratory disease, with lower mortality rates, have predominated since then. Since 1988, viruses belonging to two distinct and novel genotypes, GIII and GV, have been detected. The genome organization of the GIII strains has not been seen in any other gammacoronavirus. Mutations that emerged soon after the introduction of vaccination, incursion of strains with a novel lineage from unknown sources, recombination between IBVs from different genetic lineages, and gene translocations and deletions have contributed to an increasingly complex IBV population. These processes and the consequences of this variation for the biology of these viruses provide an insight into the evolution of endemic coronaviruses during their control by vaccination and may provide a better understanding of the potential for evolution of other coronaviruses, including SARS-CoV-2. Furthermore, the continuing capacity of attenuated IBV vaccines developed over 40 years ago to provide protection against viruses in the same genetic lineage provides some assurance that coronavirus vaccines developed to control other coronaviruses may continue to be effective for an extended period.

Genetic and antigenic heterogeneity of GI-1/Massachusetts lineage infectious bronchitis virus variants recently isolated in China.

Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns, and characteristics of the Mass-type IBV strains.

Lack of Susceptibility to SARS-CoV-2 and MERS-CoV in Poultry.

We challenged chickens, turkeys, ducks, quail, and geese with severe acute respiratory syndrome coronavirus 2 or Middle East respiratory syndrome coronavirus. We observed no disease and detected no virus replication and no serum antibodies. We concluded that poultry are unlikely to serve a role in maintenance of either virus.

Genetic, Antigenic, and Pathogenic Characteristics of Infectious Bronchitis Virus GI-7/TW-II in China.

Nine infectious bronchitis virus (IBV) strains belonging to the GI-7 lineage were isolated between 2009 and 2017 in China. Phylogenetic analysis and comparisons of full-length sequences of the S1 gene suggested that the GI-7 lineage should be further classified as Taiwan (TW)-I and TW-II sublineages, which correspond to the previous TW-I and TW-II genotypes. The nine IBV strains were clustered in the TW-II sublineage. Further investigation revealed that viruses in the TW-I and TW-II were not only genetically but also antigenically different. Moreover, the TW-II sublineage contained various clades and recombinants. A recombinant was found to originate from recombination events between field strains (TW-II ck/CH/LJL/090608- and GI-19 ck/ CH/LDL/091022-like viruses) in which the recombination in the S1 subunit coding sequences had led to changes in antigenicity of the viruses. A more in-depth investigation demonstrated that TW-II viruses appear to have undergone a significant evolution following introduction in mainland China, which resulted in the viruses diverging into different clades. The viruses between the different clades in TW-II sublineage exhibited a significant change in genetic and antigenic characteristics. In addition, the five TW-II viruses selected on the basis of the results of S1 nucleotide sequence phylogenetic trees showed different pathogenicity to specific-pathogen-free chickens, although they could induce nephritis in the infected chickens and thus were identified as nephropathogenic strains.

Características genéticas, antigénicas y patógenas del virus de la bronquitis infecciosa GI-7/TW-II en China. Nueve cepas del virus de la bronquitis infecciosa (IBV) que pertenecen al linaje GI-7 se aislaron entre 2009 y 2017 en China. El análisis filogenético y las comparaciones de las secuencias completas del gene S1 sugirieron que el linaje GI-7 debería ser clasificado además como sublinajes TW-I y TW-II, que corresponden a los anteriores genotipos TW-T y TW-II. Las nueve cepas del virus de la bronquitis infecciosa se agruparon en el sublinaje TW-II. La investigación adicional reveló que los virus en TW-I y TW-II no solo eran tanto genéticamente como antigénicamente diferentes. Además, el sublinaje TW-II contenía varios clados y recombinantes. Se descubrió que un recombinante se originaba a partir de eventos de recombinación entre cepas de campo (virus similares a las cepas TW-II ck/CH/LJL/090608 y GI-19 ck/CH/LDL/091022) en los que la recombinación en las secuencias de codificación de la subunidad de S1 condujo a cambios en la antigenicidad de los virus. Una investigación más profunda demostró que los virus TW-II parecen haber experimentado una evolución significativa después de su introducción en China continental, lo que resultó en la divergencia de los virus en diferentes clados. Los virus entre los diferentes clados en el sublinaje TW-II exhibieron un cambio significativo en las características genéticas y antigénicas. Además, los cinco virus TW-II seleccionados con base en los resultados de los árboles filogenéticos de las secuencias de nucleótidos de S1 mostraron patogenicidad diferente en los pollos libres de patógenos específicos, aunque pudieron inducir nefritis en los pollos infectados y, por lo tanto, se identificaron como cepas nefropatógenas.