Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

Diverse gammacoronaviruses detected in wild birds from Madagascar.

To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of gammacoronaviruses in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.

Does Avian Coronavirus Co-Circulate with Avian Paramyxovirus and Avian Influenza Virus in Wild Ducks in Siberia?

Avian coronaviruses (ACoV) have been shown to be highly prevalent in wild bird populations. More work on avian coronavirus detection and diversity estimation is needed for the breeding territories of migrating birds, where the high diversity and high prevalence of Orthomyxoviridae and Paramyxoviridae have already been shown in wild birds. In order to detect ACoV RNA, we conducted PCR diagnostics of cloacal swab samples from birds, which we monitored during avian influenza A virus surveillance activities. Samples from two distant Asian regions of Russia (Sakhalin region and Novosibirsk region) were tested. Amplified fragments of the RNA-dependent RNA-polymerase (RdRp) of positive samples were partially sequenced to determine the species of Coronaviridae represented. The study revealed a high presence of ACoV among wild birds in Russia. Moreover, there was a high presence of birds co-infected with avian coronavirus, avian influenza virus, and avian paramyxovirus. We found one case of triple co-infection in a Northern Pintail (Anas acuta). Phylogenetic analysis revealed the circulation of a Gammacoronavirus species. A Deltacoronavirus species was not detected, which supports the data regarding the low prevalence of deltacoronaviruses among surveyed bird species.

Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.

Physiological Changes in Chicken Embryos Inoculated with Drugs and Viruses Highlight the Need for More Standardization of this Animal Model.

Several studies have been developed using the Gallus gallus embryo as an experimental model to study the toxicity of drugs and infections. Studies that seek to standardize the evaluated parameters are needed to better understand and identify the viability of CEs as an experimental model. Therefore, we sought to verify whether macroscopic, histopathological, blood count, metabolites and/or enzymes changes and oxidative stress in CE of different ages are specific to the model. To achieve this goal, in ovo assays were performed by injecting a virus (Gammacoronavirus) and two drugs (filgrastim and dexamethasone) that cause known changes in adult animals. Although congestion and inflammatory infiltrate were visible in the case of viral infections, the white blood cell count and inflammation biomarkers did not change. Filgrastim (FG) testing did not increase granulocytes as we expected. On the other hand, CE weight and red blood cell count were lower with dexamethasone (DX), whereas white blood cell count and biomarkers varied depended on the stage of CE development. Our work reinforces the importance of standardization and correct use of the model so that the results of infection, toxicity and pharmacokinetics are reproducible.

New Insights into Avian Infectious Bronchitis Virus in Colombia from Whole-Genome Analysis.

Infectious Bronchitis (IB) is a respiratory disease caused by a highly variable Gammacoronavirus, which generates a negative impact on poultry health worldwide. GI-11 and GI-16 lineages have been identified in South America based on Infectious Bronchitis virus (IBV) partial S1 sequences. However, full genome sequence information is limited. In this study we report, for the first time, the whole-genome sequence of IBV from Colombia. Seven IBV isolates obtained during 2012 and 2013 from farms with respiratory disease compatible with IB were selected and the complete genome sequence was obtained by NGS. According to S1 sequence phylogenetic analysis, six isolates belong to lineage GI-1 and one to lineage GVI-1. When whole genome was analyzed, five isolates were related to the vaccine strain Ma5 2016 and two showed mosaic genomes. Results from complete S1 sequence analysis provides further support for the hypothesis that GVI-1, considered a geographically confined lineage in Asia, could have originated in Colombia. Complete genome information reported in this research allow a deeper understanding of the phylogenetic evolution of variants and the recombination events between strains that are circulating worldwide, contributing to the knowledge of coronavirus in Latin America and the world.

Coronaviruses in wild birds - A potential and suitable vector for global distribution.

The recurrent appearance of novel coronaviruses (CoVs) and the mortality and morbidity caused by their outbreaks aroused a widespread response among the global science community. Wild birds' high biodiversity, perching and migratory activity, ability to travel long distances and possession of a special adaptive immune system may make them alarming sources of zoonotic CoV-spreading vectors. This review gathers the available evidence on the global spread of CoVs in wild birds to date. The major wild birds associated with different types of CoVs are Anseriformes, Charadriiformes, Columbiformes, Pelecaniformes, Galliformes, Passeriformes, Psittaciformes, Accipitriformes, Ciconiiformes, Gruiformes and so on. However, the main type of CoVs found in wild birds is gammacoronavirus, followed by deltacoronavirus. Consequently, it is imperative to enable thorough research and continuous monitoring to fill the study gap in terms of understanding their role as zoonotic vectors and the frequent appearance of novel CoVs.

Host immune response to infectious bronchitis virus Q1 in two commercial broiler chicken lines.

This study investigated the pathogenesis of infectious bronchitis virus (Gammacoronavirus) strain Q1 in two commercial broiler chicken lines, and the host immune response to infection. Chicks from each line were grouped into either infected or control. Following Q1 infection at day-old, fast (Line-A) and slow (Line-B) growing chicks were monitored for clinical signs and body weights. At 3, 7, 9, 14, 21 and 28 days post infection (dpi), five birds were humanely euthanised, and trachea, kidney and proventriculus tissues were collected for quantitative RT-PCR and histopathology. Blood was collected weekly to determine IBV-specific ELISA antibody titres. Q1 infection significantly reduced the body weights of Line-A chicks at 14 and 21 dpi, but there were no significant differences in Line-B. Through qRT-PCR, significantly higher viral loads were found in the trachea, proventriculus and kidney tissues of Line-A chicks at 7-9 dpi. At day-old and at 28 dpi, the mean antibody titre in Line-B was notably higher than Line-A. Significant IFN-α mRNA expression was noted in the trachea and kidneys of Line-A, whereas no change occurred in Line-B. Chicks in Line-B, compared to those in Line-A, demonstrated a tissue-dependent increase of IFN-ß, TLR3, IL-1ß and IL-6 and LITAF gene transcription responses to IBV Q1. It appears that the level of maternal antibodies, growth rates, and other inherent host genetic factors could have influenced the differences in viral loads and immune responses.

A bio-safe multiple antigenic peptide (MAP) enzyme-linked immunoassay for the detection of antibodies to infectious bronchitis virus in chickens.

The objective of the study was to develop a bio-safe synthetic peptide ELISA for the detection of antibodies against the infectious bronchitis virus (IBV) using a novel multiple antigenic peptide approach (MAP). After initial ELISA optimization, diagnostic sensitivity (DSn) and specificity (DSp) for the linear peptides were determined using receiver operator curve (ROC) analysis. The peptide IBVP1 showed 90.44% DSn and 88.64% DSp at ROC cut off 22.8% while IBVP2 showed 88.24% DSn and 85.23% DSp at ROC cut off 23.05%. The multimerization of linear peptides to MAP design resulted in the improvement of the diagnostic efficiency up to 94.85% DSn and 92.05% DSp for IBVM1 with 19.95% cut off. A similar improvement in the performance was also observed with 92.65% DSn and 90.91% DSp for IBVM2 at 20.72% cut off. All the peptides were tested for diagnostic specificity and did not show the cross-reactivity with Newcastle disease virus and infectious bursal disease virus positive serum samples. In addition, repeatability testing for all linear and multimeric peptide showed that the coefficient of variation for intra-assay was within the expected limits, ranging from 2.4 to 10.4% and inter-assay coefficient of variation was ranging from 5.56 to 14.3%. In a nutshell, the present study used predicted B cell epitope, the synthetic peptide in linear and multimeric design for IBV antibody detection. The study also highlights peptide antigen with modified scaffold design could be a safe alternative to whole virion-based ELISA for IBV antibody detection.

Wild birds as reservoirs for diverse and abundant gamma- and deltacoronaviruses.

Wild birds interconnect all parts of the globe through annual cycles of migration with little respect for country or continental borders. Although wild birds are reservoir hosts for a high diversity of gamma- and deltacoronaviruses, we have little understanding of the ecology or evolution of any of these viruses. In this review, we use genome sequence and ecological data to disentangle the evolution of coronaviruses in wild birds. Specifically, we explore host range at the levels of viral genus and species, and reveal the multi-host nature of many viral species, albeit with biases to certain types of avian host. We conclude that it is currently challenging to infer viral ecology due to major sampling and technical limitations, and suggest that improved assay performance across the breadth of gamma- and deltacoronaviruses, assay standardization, as well as better sequencing approaches, will improve both the repeatability and interpretation of results. Finally, we discuss cross-species virus transmission across both the wild bird - poultry interface as well as from birds to mammals. Clarifying the ecology and diversity in the wild bird reservoir has important ramifications for our ability to respond to the likely future emergence of coronaviruses in socioeconomically important animal species or human populations.

Detection and Discovery of Coronaviruses in Wild Bird Populations.

Wild birds are natural hosts of multiple microbial agents, including a wide diversity of coronaviruses. Here we describe a pan-Coronavirus detection RT-PCR method to identify those viruses regardless of the coronavirus genus or nature of the specimen. We also describe a protocol using high-throughput sequencing technologies to obtain their entire genome, which overcomes the inherent difficulties of wild bird coronavirus sequencing, that is, their genetic diversity and the lack of virus isolation methods.

Divergent coronaviruses detected in wild birds in Brazil, including a central park in São Paulo.

Coronaviruses are single-stranded positive-sense RNA viruses associated with important avian diseases. Their relatively high rates of mutation and recombination frequencies allow them to adapt to new hosts and ecological niches. Although Brazil has 18% of global avian species diversity, studies regarding the presence of avian viral diseases in wild birds in South America are scarce. In this study, we performed a retrospective analysis of the presence of CoVs in 746 wild birds. Oropharyngeal and cloacal swabs were obtained and placed together in vials containing VTM transport medium collected in different regions of Brazil between 2006 and 2013. Screening for viral nucleic acid was performed using conventional RT-PCR and pancoronavirus nested PCR. Positive samples were characterized by partial sequencing of the RNA-dependent RNA polymerase (RdRp) gene, and ensuing phylogenetic analysis was performed to investigate the association between virus epidemiology and bird migration routes. Coronavirus RNA were detected and sequenced from six samples, in which three were related to gammacoronaviruses group and the other three to deltacoronavirus group. Our study documents the presence of CoVs related to avian gamma- and deltacoronaviruses circulating in both urban- and poultry-farm regions of Brazil, implicating wild birds as potential carriers of CoVs which may represent a risk to poultry farms and public health in Brazil.

Prevalence and genetic diversity of coronaviruses in wild birds, Finland.

Introduction: Migratory birds act as hosts for a number of zoonotic viruses, and have the ability to disperse these viruses to distant geographic locations. Coronaviruses (CoVs) represent a family of zoonotic viruses with wide variety of animal hosts, including birds and humans. The infections caused by coronaviruses vary from mild to severe, depending on the viral species and the host. Since the coronaviruses exhibit extraordinary large RNA genome, also the rate of homologous recombination is high, which in turn contributes to the genetic diversity and interspecies host-switches of CoVs. The emergence of novel CoVs has been rich during the last decades, and wild birds seem to serve as reservoirs for a variety of CoV strains. We examined the CoVs circulating among wild birds in Finland. Materials and methods: Samples (cloacal swab, tracheal swab, oropharyngeal swab, or tissue) representing 61 bird species were collected during 2010-2013, and examined by RT-PCR targeting the RdRp gene for the presence of CoV RNA. Results: Altogether 51/939 (5.4%) of the examined birds were found positive by RT-PCR. Diverse gamma- and deltacoronavirus sequences were detected. Discussion: Gamma- and deltacoronaviruses circulate among wild birds in Finland. The number of CoV-positive birds detected each year varies greatly.

Gamma and Deltacoronaviruses in quail and pheasants from Northern Italy1.

In view of the restricted knowledge on the diversity of coronaviruses in poultry other than chicken, this study aimed to investigate the genetic diversity of coronaviruses in quail, pheasant, and partridge from two regions of Northern Italy. To this end, pools of tracheal and cloacal swabs from European quail (Coturnix Coturnix) and intestinal tract from pheasants (Phasianus Colchicus) and partridge (Perdix Perdix) flocks, with or without enteric signs, were collected during 2015. Avian coronavirus (Gammacoronavirus) was detected in quail not vaccinated against Infectious Bronchitis Virus (IBV) and in pheasants vaccinated with an IBV Massachusetts serotype. Based on DNA sequences for the gene encoding the S protein, the avian coronaviruses detected in the quail and pheasant are related to the IBV 793B and Massachusetts types, respectively. However, RNA-dependent RNA polymerase (RdRp) analyses showed the susceptibility of quail also to Deltacoronaviruses, suggesting that quail and pheasant avian coronaviruses share spike genes identical to chicken IBV spike genes and quail might host Deltacoronavirus.

Gammacoronavirus and Deltacoronavirus in Quail.

This paper expands on a previous report about coronaviruses in quail. After surveillance carried out in 2009 and 2010, some farmers started vaccinating quail with the Massachusetts avian infectious bronchitis virus serotype. The samples for this study were collected in 2013 from São Paulo state in southeastern Brazil. Pools of trachea, lungs, reproductive tract, kidneys, and enteric contents from quail and laying hens kept in the same farms and from quail-only farms as well as from both healthy birds and those showing infectious bronchitis-like symptoms were sampled in this study. The samples were screened using nested RT-PCR targeting the 3'-untranslated region of the Gammacoronavirus genus. Based on the DNA sequence for the RNA-dependent RNA polymerase (RdRp) gene, the strains isolated from quail clustered within either the Gammacoronavirus or Deltacoronavirus genus, and sequences from both genera were found in one quail sample. The phylogeny based on the partial S1 subunit sequence showed that the gammacoronaviruses detected in quail and layers belonged to the Brazil type. These results suggest that quail are susceptible to Gammacoronavirus and Deltacoronavirus viruses and indicate that the Massachusetts vaccination was not controlling IBV in quail or chickens.

Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco.

BACKGROUND: A rapid, sensitive, and specific molecular method for the diagnosis of infectious bronchitis virus (IBV) infection is important in curbing infectious bronchitis outbreaks in Morocco and other countries. METHODS: In this study, an easy-to-perform SYBR green I real-time reverse transcriptase polymerase chain reaction (RT-PCR) targeting the nucleocapsid gene of IBV was developed and compared with conventional agarose gel-based RT-PCR for the detection of IBV infection. RESULTS: We found that the SYBR green I real-time RT-PCR was at least 10 times more sensitive than the agarose gel electrophoresis detection method. The assay exhibited high specificity for IBV infection. All negative controls, such as Newcastle disease virus, infectious bursal disease virus, and avian influenza virus, were not detected. CONCLUSION: The SYBR green I real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for diagnosis and control of infectious bronchitis outbreaks in Morocco. The test is a valuable and useful method as a routine assay for diagnosis of clinical IBV infection in commercial chickens.

The avian coronavirus spike protein.

Avian coronaviruses of the genus Gammacoronavirus are represented by infectious bronchitis virus (IBV), the coronavirus of chicken. IBV causes a highly contagious disease affecting the respiratory tract and, depending on the strain, other tissues including the reproductive and urogenital tract. The control of IBV in the field is hampered by the many different strains circulating worldwide and the limited protection across strains due to serotype diversity. This diversity is believed to be due to the amino acid variation in the S1 domain of the major viral attachment protein spike. In the last years, much effort has been undertaken to address the role of the avian coronavirus spike protein in the various steps of the virus' live cycle. Various models have successfully been developed to elucidate the contribution of the spike in binding of the virus to cells, entry of cell culture cells and organ explants, and the in vivo tropism and pathogenesis. This review will give an overview of the literature on avian coronavirus spike proteins with particular focus on our recent studies on binding of recombinant soluble spike protein to chicken tissues. With this, we aim to summarize the current understanding on the avian coronavirus spike's contribution to host and tissue predilections, pathogenesis, as well as its role in therapeutic and protective interventions.