Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia.

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections. The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

The H9N2 avian influenza virus increases APEC adhesion to oviduct epithelia by viral NS1 protein-mediated activation of the TGF-ß pathway.

H9N2 avian influenza is a low-pathogenic avian influenza circulating in poultry and wild birds worldwide and frequently contributes to chicken salpingitis that is caused by avian pathogenic Escherichia coli (APEC), leading to huge economic losses and risks for food safety. Currently, how the H9N2 virus contributes to APEC infection and facilitates salpingitis remains elusive. In this study, in vitro chicken oviduct epithelial cell (COEC) model and in vivo studies were performed to investigate the role of H9N2 viruses on secondary APEC infection, and we identified that H9N2 virus enhances APEC infection both in vitro and in vivo. To understand the mechanisms behind this phenomenon, adhesive molecules on the cell surface facilitating APEC adhesion were checked, and we found that H9N2 virus could upregulate the expression of fibronectin, which promotes APEC adhesion onto COECs. We further investigated how fibronectin expression is regulated by H9N2 virus infection and revealed that transforming growth factor beta (TGF-ß) signaling pathway is activated by the NS1 protein of the virus, thus regulating the expression of adhesive molecules. These new findings revealed the role of H9N2 virus in salpingitis co-infected with APEC and discovered the molecular mechanisms by which the H9N2 virus facilitates APEC infection, offering new insights to the etiology of salpingitis with viral-bacterial co-infections.IMPORTANCEH9N2 avian influenza virus (AIV) widely infects poultry and is sporadically reported in human infections. The infection in birds frequently causes secondary bacterial infections, resulting in severe symptoms like pneumonia and salpingitis. Currently, the mechanism that influenza A virus contributes to secondary bacterial infection remains elusive. Here we discovered that H9N2 virus infection promotes APEC infection and further explored the underlying molecular mechanisms. We found that fibronectin protein on the cell surface is vital for APEC adhesion and also showed that H9N2 viral protein NS1 increased the expression of fibronectin by activating the TGF-ß signaling pathway. Our findings offer new information on how AIV infection promotes APEC secondary infection, providing potential targets for mitigating severe APEC infections induced by H9N2 avian influenza, and also give new insights on the mechanisms on how viruses promote secondary bacterial infections in animal and human diseases.

Avian coronavirus infectious bronchitis virus Beaudette strain NSP9 interacts with STAT1 and inhibits its phosphorylation to facilitate viral replication.

Avian coronavirus, known as infectious bronchitis virus (IBV), is the causative agent of infectious bronchitis (IB). Viral nonstructural proteins play important roles in viral replication and immune modulation. IBV NSP9 is a component of the RNA replication complex for viral replication. In this study, we uncovered a function of NSP9 in immune regulation. First, the host proteins that interacted with NSP9 were screened. The immune-related protein signal transducer and activator of transcription 1 (STAT1) was identified and the interaction between NSP9 and STAT1 was further confirmed. Furthermore, IBV replication was inhibited in STAT1-overexpressing cells but inversely affected in STAT1 knock-down cells. Importantly, NSP9 inhibited STAT1 phosphorylation. Finally, the expression of JAK/STAT pathway downstream genes IRF7 and ISG20 was significantly decreased in NSP9-overexpressing cells. These results showed the important role of IBV NSP9 in immunosuppression.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

IMPORTANCE: Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.

Phylogenotyping of infectious bursal disease virus in Vietnam according to the newly unified genotypic classification scheme.

Since 1987, infectious bursal disease virus (IBDV) has circulated and evolved in Vietnam, but little is known about the genotypes present. IBDV samples were collected in 1987, 2001-2006, 2008, 2011, 2015-2019, and 2021 in 18 provinces. We conducted phylogenotyping analysis based on an alignment of 143 VP2-HVR (hypervariable region) sequences from 64 Vietnamese isolates (26 previous and 38 additional sequences and two vaccines, and alignment of 82 VP1 B-marker sequences, including one vaccine and four Vietnamese field strains. The analysis identified three A-genotypes, A1, A3, and A7, and two B-genotypes, B1 and B3, among the Vietnamese IBDV isolates. The lowest average evolutionary distance (8.6%) was seen between the A1 and A3 genotypes, and the highest (21.7%) was between A5 and A7, while there was a distance of 14% between B1 and B3 and 17% between B3 and B2. Unique signature residues were observed for genotypes A2, A3, A5, A6, and A8, which could be used for genotypic discrimination. A timeline statistical summary revealed that the A3-genotype predominated (79.8% presence) in Vietnam from 1987 to 2021 and that it remained the dominant IBDV genotype over the last five years (2016-2021). The current study contributes to a better understanding of the circulating genotypes and evolution of IBDV in Vietnam and worldwide.

A Recombinant Duck Plague Virus Containing the ICP27 Deletion Marker Provides Robust Protection in Ducks.

Duck plague virus (DPV) is a member of Alphaherpesvirus genus and poses a major threat to waterfowl breeding. Genetic engineered vaccines that are capable of distinguishing naturally infected from vaccine-immunized animals are useful for eradicating duck plague. In this study, reverse genetics was used to develop an ICP27-deficient strain (CHv-ΔICP27), and its potential as a marker vaccination candidate was evaluated. The results showed that the CHv-ΔICP27 generated in this study exhibited good genetic stability in vitro and was highly attenuated both in vivo and in vitro. The level of neutralizing antibody generated by CHv-ΔICP27 was comparable to that induced by a commercial DPV vaccine, suggesting that it could protect ducks from virulent DPV attack. By using molecular identification techniques such as PCR, restriction fragment length polymorphism, immunofluorescence, Western blotting, and others, it is possible to differentiate the CHv-ΔICP27 from wild-type strains. Moreover, ICP27 can also be a potential target for the genetic engineering vaccine development of alphavirus or perhaps the entire herpesvirus family members due to the highly conservative of ICP27 protein in all herpesvirus family members. IMPORTANCE The development of distinguishable marker vaccines from natural infection is a key step toward eradicating duck plague. Here, we generated a recombinant DPV that carries an ICP27 deletion marker that could be easily distinguished from wild-type strain by molecular biological methods. It was highly attenuated in vitro and in vivo and could provide comparable protection to ducks after a single dose of immunizations, as commercial vaccines did. Our findings support the use of the ICP27-deficient virus as a marker vaccine for DPV control and future eradication.

Assessing avian leukosis virus proviral load and lesion correlates in fowl glioma-inducing virus-infected Japanese bantam chickens.

The fowl glioma-inducing virus prototype (FGVp) and its variants, which belong to avian leukosis virus subgroup A (ALV-A), induce cardiomyocyte abnormalities and gliomas in chickens. However, the molecular mechanisms underlying these myocardial changes remain unclear, and ALV-induced tumorigenesis, which is caused by proviral insertional mutagenesis, does not explain the early development of cardiac changes in infected chickens. We established a quantitative PCR (qPCR) assay to measure ALV-A proviral loads in the brains and hearts of FGV-infected Japanese bantam chickens and compared these results with morphologic lesions. Four of 22 bantams had both gliomas and cardiac lesions. Hearts with cardiac lesions had a higher proviral load (10.3 ± 2.7 proviral copies/nucleus) than those without cardiac lesions (0.4 ± 0.4), suggesting that the proviral load in hearts is correlated with the frequency of myocardial changes. Our qPCR method may be useful in the study of ALV-induced cardiomyocyte abnormalities.

Critical Role of Viral Protein Hexon in Hypervirulent Fowl Adenovirus Serotype-4-Induced Autophagy by Interaction with BAG3 and Promotion of Viral Replication in LMH Cells.

Hepatitis-pericardial syndrome (HHS) is an acute highly infectious avian disease caused by fowl adenovirus serotype 4 (FAdV-4), characterized by fulminant hepatitis and hydropericardium in broilers. Since 2015, a widespread epidemic has occurred in China due to the emergence of hypervirulent FAdV-4 (HPFAdV-4), causing huge losses to the stakeholders. However, the pathogenesis of HPFAdV-4 and the host responses to its infection remain elusive. Here, we show that infection of leghorn male hepatocellular (LMH) cells by HPFAdV-4 induced complete autophagy in cells and that the autophagy induced by recombinant HPFAdV-4-ON1 (rHPFAdV-4-ON1), a viral strain generated by replacing the hexon gene of wild-type HPFAdV-4 (HPFAdV-4-WT) with the one of nonpathogenic strain FAdV-4-ON1, was remarkably mitigated compared to that of the rHPFAdV-4-WT control, suggesting that HPFAdV-4 hexon is responsible for virus-induced autophagy. Importantly, we found that hexon interacted with a cellular protein, BAG3, a host protein that initiates autophagy, and that BAG3 expression increased in cells infected with HPFAdV-4. Furthermore, knockdown of BAG3 by RNA interference (RNAi) significantly inhibited HPFAdV-4- or hexon-induced autophagy and suppressed viral replication. On the contrary, expression of hexon markedly upregulated the expression of BAG3 via activating the P38 signaling pathway, triggering autophagy. Thus, these findings reveal that HPFAdV-4 hexon interacts with the host protein BAG3 and promotes BAG3 expression by activating P38 signaling pathway, thereby inducing autophagy and enhancing viral proliferation, which immensely furthers our understanding of the pathogenesis of HPFAdV-4 infection. IMPORTANCE HHS, mainly caused by HPFAdV-4, has caused large economic losses to the stakeholders in recent years. Infection of leghorn male hepatocellular (LMH) cells by HPFAdV-4 induced complete autophagy that is essential for HPFAdV-4 replication. By a screening strategy, the viral protein hexon was found responsible for virus-induced autophagy in cells. Importantly, hexon was identified as a factor promoting viral replication by interaction with BAG3, an initiator of host cell autophagy. These findings will help us to better understand the host response to HPFAdV-4 infection, providing a novel insight into the pathogenesis of HPFAdV-4 infection.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Infectivity and transmissibility of an avian H3N1 influenza virus in pigs.

In 2019 a low pathogenic H3N1 avian influenza virus (AIV) caused an outbreak in Belgian poultry farms, characterized by an unusually high mortality in chickens. Influenza A viruses of the H1 and H3 subtype can infect pigs and become established in swine populations. Therefore, the H3N1 epizootic raised concern about AIV transmission to pigs and from pigs to humans. Here, we assessed the replication efficiency of this virus in explants of the porcine respiratory tract and in pigs, using virus titration and/or RT-qPCR. We also examined transmission from directly, intranasally inoculated pigs to contact pigs. The H3N1 AIV replicated to moderate titers in explants of the bronchioles and lungs, but not in the nasal mucosa or trachea. In the pig infection study, infectious virus was only detected in a few lung samples collected between 1 and 3 days post-inoculation. Virus titers were between 1.7 and 4.8 log10 TCID50. In line with the ex vivo experiment, no virus was isolated from the upper respiratory tract of pigs. In the transmission experiment, we could not detect virus transmission from directly inoculated to contact pigs. An increase in serum antibody titers was observed only in the inoculated pigs. We conclude that the porcine respiratory tract tissue explants can be a useful tool to assess the replication efficiency of AIVs in pigs. The H3N1 AIV examined here is unlikely to pose a risk to swine populations. However, continuous risk assessment studies of emerging AIVs in pigs are necessary, since different virus strains will have different genotypic and phenotypic traits.

Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection.

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses.

Duck Tembusu Virus Inhibits Type I Interferon Production through the JOSD1-SOCS1-IRF7 Negative-Feedback Regulation Pathway.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that mainly causes a decrease in egg production in infected waterfowl. Similar to other members of the Flaviviridae family, it can proliferate in most mammalian cells and may also pose a potential threat to nonavian animals. In previous studies, we found that DTMUV infection can upregulate suppressor of cytokine signaling 1 (SOCS1) to inhibit type I interferon (IFN) production and promote virus replication, but the specific mechanism is unclear. Furthermore, little is known about the regulatory role of ubiquitination during flavivirus infection. In this study, we found that activation of Toll-like receptor 3 (TLR3) signaling rather than type I IFN stimulation led to the upregulation of SOCS1 during DTMUV infection. Further studies revealed that JOSD1 stabilized SOCS1 expression by binding to the SH2 domain of SOCS1 and mediating its deubiquitination. In addition, JOSD1 also inhibited type I IFN production through SOCS1. Finally, SOCS1 acts as an E3 ubiquitin ligase that binds to IFN regulatory factor 7 (IRF7) through its SH2 domain and mediates K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting type I IFN production mediated by IRF7 and promoting viral proliferation. These results will enrich and deepen our understanding of the mechanism by which DTMUV antagonizes the host interferon system. IMPORTANCE DTMUV is a newly discovered flavivirus that seriously harms the poultry industry. In recent years, there have been numerous studies on the involvement of ubiquitination in the regulation of innate immunity. However, little is known about the involvement of ubiquitination in the regulation of flavivirus-induced type I IFN signaling. In this study, we found that SOCS1 was induced by TLR3 signaling during DTMUV infection. Furthermore, we found for the first time that duck SOCS1 protein was also modified by K48-linked polyubiquitination, whereas our previous study found that SOCS1 was upregulated during DTMUV infection. Further studies showed that JOSD1 stabilized SOCS1 expression by mediating the deubiquitination of SOCS1. While SOCS1 acts as a negative regulator of cytokines, we found that DTMUV utilized SOCS1 to mediate the ubiquitination and proteasomal degradation of IRF7 and ultimately inhibit type I IFN production, thereby promoting its proliferation.

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.

The Emergence, Diversification, and Transmission of Subgroup J Avian Leukosis Virus Reveals that the Live Chicken Trade Plays a Critical Role in the Adaption and Endemicity of Viruses to the Yellow-Chickens.

The geographical spread and inter-host transmission of the subgroup J avian leukosis virus (ALV-J) may be the most important issues for epidemiology. An integrated analysis, including phylogenetic trees, homology modeling, evolutionary dynamics, selection analysis and viral transmission, based on the gp85 gene sequences of the 665 worldwide ALV-J isolates during 1988-2020, was performed. A new Clade 3 has been emerging and was evolved from the dominating Clade 1.3 of the Chinese Yellow-chicken, and the loss of a α-helix or ß-sheet of the gp85 protein monomer was found by the homology modeling. The rapid evolution found in Clades 1.3 and 3 may be closely associated with the adaption and endemicity of viruses to the Yellow-chickens. The early U.S. strains from Clade 1.1 acted as an important source for the global spread of ALV-J and the earliest introduction into China was closely associated with the imported chicken breeders in the 1990s. The dominant outward migrations of Clades 1.1 and 1.2, respectively, from the Chinese northern White-chickens and layers to the Chinese southern Yellow-chickens, and the dominating migration of Clade 1.3 from the Chinese southern Yellow-chickens to other regions and hosts, indicated that the long-distance movement of these viruses between regions in China was associated with the live chicken trade. Furthermore, Yellow-chickens have been facing the risk of infections of the emerging Clades 2 and 3. Our findings provide new insights for the epidemiology and help to understand the critical factors involved in ALV-J dissemination. IMPORTANCE Although the general epidemiology of ALV-J is well studied, the ongoing evolutionary and transmission dynamics of the virus remain poorly investigated. The phylogenetic differences and relationship of the clades and subclades were characterized, and the epidemics and factors driving the geographical spread and inter-host transmission of different ALV-J clades were explored for the first time. The results indicated that the earliest ALV-J (Clade 1.1) from the United States, acted as the source for global spreads, and Clades 1.2, 1.3 and 3 were all subsequently evolved. Also the epidemiological investigation showed that the early imported breeders and the inter-region movements of live chickens facilitated the ALV-J dispersal throughout China and highlighted the needs to implement more effective containment measures.

Hsp70 Inhibits the Replication of Fowl Adenovirus Serotype 4 by Suppressing Viral Hexon with the Assistance of DnaJC7.

Fowl adenovirus serotype 4 (FAdV-4) infection results in serious hepatitis-hydropericardium syndrome (HHS) in broilers, which has caused great economic losses to the poultry industry; however, the specific host responses to FAdV-4 remain unknown. In this study, we identified 141 high-confidence protein-protein interactions (PPIs) between the main viral proteins (Hexon, Fiber 1, Fiber 2, and Penton bases) and host proteins via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. We found that heat shock protein 70 (Hsp70), the protein with the highest score, and its cofactor DnaJ heat shock protein 40 family member C7 (DnaJC7) could negatively regulate the replication of FAdV-4. Furthermore, the nucleotide binding domain (NBD) of Hsp70 and the J domain of DnaJC7 were necessary for inhibiting FAdV-4 replication. We verified that DnaJC7 as a bridge could bind to Hsp70 and Hexon, assisting the indirect interaction between Hsp70 and Hexon. In addition, we found that FAdV-4 infection strongly induced the expression of autophagy proteins and cellular Hsp70 in a dose-dependent manner. Blockage of Hexon by Hsp70 overexpression was significantly reduced when the autophagy pathway was blocked by the specific inhibitor chloroquine (CQ). Our results showed that Hsp70 was co-opted by DnaJC7 to interact with viral Hexon and inhibited Hexon through the autophagy pathway, leading to a considerable restriction of FAdV-4 replication. IMPORTANCE FAdV-4, as the main cause of HHS, has quickly spread all over the world in recent years, seriously threatening the poultry industry. The aim of this study was to identify the important host proteins that have the potential to regulate the life cycle of FAdV-4. We found that Hsp70 and DnaJC7 played crucial roles in regulating the amount of viral Hexon and extracellular viral titers. Moreover, we demonstrated that Hsp70 interacted with viral Hexon with the assistance of DnaJC7, followed by suppressing Hexon protein through the autophagy pathway. These results provide new insight into the role of the molecular chaperone complex Hsp70-DnaJC7 in FAdV-4 infection and suggest a novel strategy for anti-FAdV-4 drug development by targeting the specific interactions among Hsp70, DnaJC7 and Hexon.

Evolution of the North American Lineage H7 Avian Influenza Viruses in Association with H7 Virus's Introduction to Poultry.

The incursions of H7 subtype low-pathogenicity avian influenza virus (LPAIV) from wild birds into poultry and its mutations to highly pathogenic avian influenza virus (HPAIV) have been an ongoing concern in North America. Since 2000, 10 phylogenetically distinct H7 virus outbreaks from wild birds have been detected in poultry, six of which mutated to HPAIV. To study the molecular evolution of the H7 viruses that occurs when changing hosts from wild birds to poultry, we performed analyses of the North American H7 hemagglutinin (HA) genes to identify amino acid changes as the virus circulated in wild birds from 2000 to 2019. Then, we analyzed recurring HA amino acid changes and gene constellations of the viruses that spread from wild birds to poultry. We found six HA amino acid changes occurring during wild bird circulation and 10 recurring changes after the spread to poultry. Eight of the changes were in and around the HA antigenic sites, three of which were supported by positive selection. Viruses from each H7 outbreak had a unique genotype, with no specific genetic group associated with poultry outbreaks or mutation to HPAIV. However, the genotypes of the H7 viruses in poultry outbreaks tended to contain minor genetic groups less observed in wild bird H7 viruses, suggesting either a biased sampling of wild bird AIVs or a tendency of having reassortment with minor genetic groups prior to the virus's introduction to poultry. IMPORTANCE Wild bird-origin H7 subtype avian influenza viruses are a constant threat to commercial poultry, both directly by the disease they cause and indirectly through trade restrictions that can be imposed when the virus is detected in poultry. It is important to understand the genetic basis of why the North American lineage H7 viruses have repeatedly crossed the species barrier from wild birds to poultry. We examined the amino acid changes in the H7 viruses associated with poultry outbreaks and tried to determine gene reassortment related to poultry adaptation and mutations to HPAIV. The findings in this study increase the understanding of the evolutionary pathways of wild bird AIV before infecting poultry and the HA changes associated with adaptation of the virus in poultry.