Genetic characterization of duck hepatitis B viruses from Anhui Province, China.

Duck hepatitis B virus (DHBV) infection model was frequently used as the experimental model for human hepatitis B virus (HBV) research. In order to decipher the genetic characteristics of DHBVs from Anhui province of China, 120 duck liver tissue samples were collected and subjected to PCR screening, and 28 samples were detected as DHBV positive. Subsequently, five DHBV-positive samples were selected for genome-wide amplification and a comprehensive analysis. Comparative analysis of complete genome sequences using the MegAlign program showed that five strains of DHBVs shared 94.5-96.3% with each other and 93.2-98.7% with other reference strains in GenBank. The phylogenetic analysis showed that all five DHBV strains belonged to the evolutionary branch of "Chinese DHBV" isolates or DHBV-2. Importantly, three potential intra-genotypic recombination events, between strains AAU-6 and Guilin, strains AAU-1 and GD3, and strains AAU-6 and AAU-1, were respectively found using the RDP and SimPlot softwares and considered the first report in avihepadnaviruses. These results not only improve our understanding for molecular prevalence status of DHBV among ducks, but also provide a reference for recombination mechanism of HBV.

Pathogenicity and immunogenicity of gI/gE/TK-gene-deleted Felid herpesvirus 1 variants in cats.

BACKGROUND: Felid herpesvirus 1 (FHV-1) is a major pathogenic agent of upper respiratory tract infections and eye damage in felines worldwide. Current FHV-1 vaccines offer limited protection of short duration, and therefore, do not reduce the development of clinical signs or the latency of FHV-1. METHODS: To address these shortcomings, we constructed FHV ∆gIgE-eGFP, FHV ∆TK mCherry, and FHV ∆gIgE/TK eGFP-mCherry deletion mutants (ΔgI/gE, ΔTK, and ΔgIgE/TK, respectively) using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISP-associated protein 9 (Cas9) system (CRISPR/Cas9), which showed safety and immunogenicity in vitro. We evaluated the safety and efficacy of the deletion mutants administered with intranasal (IN) and IN + subcutaneous (SC) vaccination protocols. Cats in the vaccination group were vaccinated twice at a 4-week interval, and all cats were challenged with infection 3 weeks after the last vaccination. The cats were assessed for clinical signs, nasal shedding, and virus-neutralizing antibodies (VN), and with postmortem histological testing. RESULTS: Vaccination with the gI/gE-deleted and gI/gE/TK-deleted mutants was safe and resulted in significantly lower clinical disease scores, fewer pathological changes, and less nasal virus shedding after infection. All three mutants induced virus-neutralizing antibodies after immunization. CONCLUSIONS: In conclusion, this study demonstrates the advantages of FHV-1 deletion mutants in preventing FHV-1 infection in cats.

Simultaneous detection and identification of Peste des petits ruminants Virus Lineages II and IV by MCA-Based real-time quantitative RT-PCR assay within single reaction.

BACKGROUND: Peste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV. RESULTS: To achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV. CONCLUSION: Since the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.

A cDNA-based reverse genetics system for feline calicivirus identifies the 3' untranslated region as an essential element for viral replication.

Virulent systemic feline calicivirus (VS-FCV) is a newly emerging FCV variant that is associated with a severe acute multisystem disease in cats that is characterized by jaundice, oedema, and high mortality (approximately 70%). VS-FCV has spread throughout the world, but there are no effective vaccines or therapeutic options to combat infection. VS-FCV may therefore pose a serious threat to the health of felines. The genomic characteristics and functions of VS-FCV are still poorly understood, and the reason for its increased pathogenicity is unknown. Reverse genetics systems are powerful tools for studying the molecular biology of RNA viruses, but a reverse genetics system for VS-FCV has not yet been reported. In this study, we developed a plasmid-based reverse genetics system for VS-FCV in which infectious progeny virus is produced in plasmid-transfected CRFK cells. Using this system, we found that the 3' untranslated region (UTR) and poly(A) tail are important for maintaining the infection and replication capacity of VS-FCV and that shortening of the poly(A) tail to less than 28 bases eliminated the ability to rescue infectious progeny virus. Whether these observations are unique to VS-FCV or represent more-general features of FCV remains to be determined. In conclusion, we successfully established a rapid and efficient VS-FCV reverse genetics system, which provides a good platform for future research on the gene functions and pathogenesis of VS-FCV. The effects of the deletion of 3' UTR and poly(A) tail on VS-FCV infectivity and replication also provided new information about the pathogenesis of VS-FCV.

Simultaneous degradation and separation of antibiotics in sewage effluent by photocatalytic nanofiltration membrane in a continuous dynamic process.

Bifunctional photocatalytic nanofiltration (PNF) membrane is increasingly concerned in practical micro-polluted water purification, but there are still several bottlenecks that inhibit its practicality. In this context, the feasibility of a novel metal-free and visible light-responsive surface-anchored PNF membrane for simultaneously removing target antibiotics in real sewage effluent in a continuous dynamic process was explored. The results showed that the optimal PNF-4 membrane was expectedly consisted of an inside tight sub-nanopore structured separation layer and an outside thinner, smoother, super hydrophilic mesoporous degradation layer, respectively. Consequently, the activated PNF-4 membrane could synergistically reduce trimethoprim and sulfamethoxazole concentrations to below two orders of magnitude, accompanying with almost constant high water permeability, suggesting that the hydrophilic modification of the mesoporous degradation layer basically offsets its inherent hydraulic resistance. Also, after repeating the fouling-physical rinsing process three times lasted for 78 h, only sporadic adherent contaminants remained onto the top surface, together with the minimal total and irreversible fouling ratios (as low as 7.2% and 1.2%, respectively), strongly demonstrated that PNF-4 membrane displayed good self-cleaning performance. Undoubtedly, this will significantly reduce its potential cleaning frequency and maintenance cost in long-term operation. Meanwhile, the acute and chronic biotoxicities of its permeate to Virbrio qinghaiensis sp. -67 were also reduced sharply to 2.22% and 0.45%, respectively. All of these evidences suggest that the dual functions of PNF-4 membrane are synergetic in an uninterrupted permeating process. It will provide useful insights for continuously enhancing the practicality and effectiveness of PNF membrane in actual micro-polluted water purification scenarios.

SLC1A3 facilitates Newcastle disease virus replication by regulating glutamine catabolism.

As obligate intracellular parasites, viruses rely completely on host metabolic machinery and hijack host nutrients for viral replication. Newcastle disease virus (NDV) causes acute, highly contagious avian disease and functions as an oncolytic agent. NDV efficiently replicates in both chicken and tumour cells. However, how NDV reprograms host cellular metabolism for its efficient replication is still ill-defined. We previously identified a significantly upregulated glutamate transporter gene, solute carrier family 1 member 3 (SLC1A3), during NDV infection via transcriptome analysis. To investigate the potential role of SLC1A3 during NDV infection, we first confirmed the marked upregulation of SLC1A3 in NDV-infected DF-1 or A549 cells through p53 and NF-κB pathways. Knockdown of SLC1A3 inhibited NDV infection. Western blot analysis further confirmed that glutamine, but not glutamate, asparagine, or aspartate, was required for NDV replication. Metabolic flux data showed that NDV promotes the decomposition of glutamine into the tricarboxylic acid cycle. Importantly, the level of glutamate and glutaminolysis were reduced by SLC1A3 knockdown, indicating that SLC1A3 propelled glutaminolysis for glutamate utilization and NDV replication in host cells. Taken together, our data identify that SLC1A3 serves as an important regulator for glutamine metabolism and is hijacked by NDV for its efficient replication during NDV infection. These results improve our understanding of the interaction between NDV and host cellular metabolism and lay the foundation for further investigation of efficient vaccines.

Free ISG15 Inhibits the Replication of Peste des Petits Ruminants Virus by Breaking the Interaction of Nucleoprotein and Phosphoprotein.

Peste des petits ruminants virus (PPRV) causes a highly contagious disease in small ruminants and severe economic losses in developing countries. PPRV infection can stimulate high levels of interferon (IFN) and many IFN-stimulated genes (ISGs), such as ISG15, which may play a key role in the process of viral infection. However, the role of ISG15 in PPRV infection and replication has not yet been reported. In this study, we found ISG15 expression to be significantly upregulated after PPRV infection of caprine endometrial epithelial cells (EECs), and ISG15 inhibits the proliferation of PPRV. Further analysis showed that free ISG15 could inhibit PPRV proliferation. Moreover, ISG15 does not affect the binding, entry, and transcription but does suppress the replication of PPRV. A detailed analysis revealed that ISG15 interacts and colocalizes with both viral N and P proteins and that its interactive regions are all located in the N-terminal domain. Further studies showed that ISG15 can competitively interact with N and P proteins and significantly interfere with their binding. Finally, through the construction of the C-terminal mutants of ISG15 with different lengths, it was found that amino acids (aa) 77 to 101 play a key role in inhibiting the binding of N and P proteins and that interaction with the P protein disappears after the deletion of 77 to 101 aa. The present study revealed a novel mechanism of ISG15 in disrupting the activity of the N0-P complex to inhibit viral replication. IMPORTANCE PPRV, a widespread and fatal disease of small ruminants, is one of the most devastating animal diseases in Africa, the Middle East, and Asia, causing severe economic losses. IFNs play an important role as a component of natural immunity against pathogens, yet the role of ISG15, an IFN-stimulated gene, in protecting against PPRV infection is currently unknown. We demonstrated, for the first time, that free ISG15 inhibits PPRV proliferation by disrupting the activity of the N0-P complex, a finding that has not been reported in other viruses. Our results provide important insights that can further understand the pathogenesis and innate immune mechanisms of PPRV.

Peste des Petits Ruminants Virus Exhibits Cell-Dependent Interferon Active Response.

Peste des petits ruminants (PPR) is an acute and highly pathogenic infectious disease caused by peste des petits ruminants virus (PPRV), which can infect goats and sheep and poses a major threat to the small ruminants industry. The innate immune response plays an important role as a line of defense against the virus. The effect of PPRV on the active innate immune response has been described in several studies, with different conclusions. We infected three goat-derived cell lines with PPRV and tested their innate immune response. PPRV proliferated in caprine endometrial epithelial cells (EECs), caprine skin fibroblasts cells (GSFs), and goat fibroblast cells (GFs), and all cells expressed interferon (IFN) by poly (I: C) stimulation. PPRV infection stimulated expression of type I and type III IFN on EECs, and expression of the latter was significantly stronger, but IFN was not stimulated in fibroblasts (GSFs and GFs). Our results suggested that the effect of PPRV on IFN was cell-type specific. Nine IFN-stimulated genes (ISGs) were detected in EECs, but only ISG15 and RSAD2 were significantly upregulated. The effects of PPRV on IFN and IFN-induced ISGs were cell-type specific, which advances our understanding of the innate immune response induced by PPRV and creates new possibilities for the control of PPRV infection.

PseudoRHDV constructed with feline calicivirus genome as vector has the characteristics of well proliferation in vitro.

Rabbit hemorrhagic disease virus (RHDV) is a major member of the Caliciviridae. which is fatal to wild and domestic European rabbit. Because RHDV does not reproduce stably in vitro, molecular studies on this pathogen have been limited. Feline calicivirus (FCV), also a member of the Caliciviridae, reproduces well in vitro and is a good viral vector. As these viruses share similar genomic structures, we hypothesized that a chimeric infectious clone could be constructed by replacing the corresponding regions of the FCV genome with the structural proteins VP60 and VP10 and the 3' non-translated region of the RHDV genome. Transfection of the infectious clone into RK13 cells made it possible to rescue the chimeric virus, named pseudoRHDV, which reproduced in an RK13 cell line with high titer. An infectious pseudoRHDV was produced, which proliferated in RK13 cells to at least 15 generations. PseudoRHDV caused significant cytopathic changes in the RK13 cells, with a viral titer was 9.74 log10 TCID50 / mL. The pseudoRHDV constructed in this study will be helpful for investigating the molecular biology of RHDV, especially its interaction with the host. The model can also be used to explore some common laws between FCV and RHDV.

Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking.

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding, and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M protein nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were analyzed. Here, two types of combined NLSs and NESs were identified within the NDV-M protein. The Herts/33-type M protein was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M protein was retained mostly in the nuclei and showed retarded VLP production. Two critical residues, namely, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, of which its modification regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued LaSota (rLaSota) strains rLaSota-R247K, -S263R, and -double mutation (DM) showed about 2-fold higher hemagglutination (HA) titers and 10-fold higher 50% egg infective dose (EID50) titers than wild-type (wt) rLaSota. Furthermore, the mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of those recombinant viruses were slightly higher than those of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV and even those of all other paramyxoviruses. This information is beneficial for the development of vaccines and therapies for paramyxoviruses. IMPORTANCE Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked Newcastle disease (ND) as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and open up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach for improving paramyxovirus vaccines.

Genome-Wide Analysis of Alternative Splicing during Host-Virus Interactions in Chicken.

The chicken is a model animal for the study of evolution, immunity and development. In addition to their use as a model organism, chickens also represent an important agricultural product. Pathogen invasion has already been shown to modulate the expression of hundreds of genes, but the role of alternative splicing in avian virus infection remains unclear. We used RNA-seq data to analyze virus-induced changes in the alternative splicing of Gallus gallus, and found that a large number of alternative splicing events were induced by virus infection both in vivo and in vitro. Virus-responsive alternative splicing events preferentially occurred in genes involved in metabolism and transport. Many of the alternatively spliced transcripts were also expressed from genes with a function relating to splicing or immune response, suggesting a potential impact of virus infection on pre-mRNA splicing and immune gene regulation. Moreover, exon skipping was the most frequent AS event in chickens during virus infection. This is the first report describing a genome-wide analysis of alternative splicing in chicken and contributes to the genomic resources available for studying host-virus interaction in this species. Our analysis fills an important knowledge gap in understanding the extent of genome-wide alternative splicing dynamics occurring during avian virus infection and provides the impetus for the further exploration of AS in chicken defense signaling and homeostasis.

Comparison of the protective antigen variabilities of prevalent Newcastle disease viruses in response to homologous/heterologous genotype vaccines.

The genotype VII Newcastle disease virus (NDV) vaccine has begun to replace the traditional genotype II NDV vaccine and is widely used in the commercial poultry of China. However, the effect of homologous and heterogeneous anti-NDV serum on the evolution of prevalent NDV is unknown. To understand the effect of genotype II and VII anti-NDV serum on the evolution of genotype VII NDV strains, ZJ1 (waterfowl origin) and CH/SD/2008/128 (ND128; chicken origin) were used for serial passage of 30 generations in DF-1 cells without anti-NDV serum or with genotype II and VII anti-NDV serum independently. The F and HN genes of the 2 viruses were amplified for the 10th, 20th, and 30th generations of each serial passage group and compared with their respective original viruses. We found that there was only one mutation at position 248 in the F gene of ZJ1 due to the serum pressure of genotype VII anti-NDV. Similarly, mutations at residue 527 of the F gene, and position 9 and 319 of the HN gene of ND128 were noted in both anti-NDV serum groups. The results show that the nonsynonymous (NS)-to-synonymous (S) ratio of the F gene of ZJ1 virus was 1.6, and for the HN gene, it was 2.5 in the anti-II serum group. In the anti-VII serum group, the NS/S ratio for the F gene was 2.1, and for the HN gene, it was 2.5. The NS/S ratio of the F gene of the ND128 virus was 0.8, and for the HN gene, it was 3 in the anti-II serum group. Furthermore, the NS/S ratio of the F gene was 0.8, and the HN gene was 2.3 in the anti-VII group. Taken together, our findings highlight that there was no significant difference in the variation of protective antigens in genotype VII NDV under the selection pressure of homologous and heterogeneous genotype NDV inactivated vaccines.

Zinc finger antiviral protein (ZAP) inhibits small ruminant morbillivirus replication in vitro.

Small ruminant morbillivirus (SRMV) is a highly contagious and economically important viral disease of small domestic and wild ruminants. Difficulty with its stable proliferation in ovis aries-derived cells has led to a relative lag in the study of its natural immunity and pathogenesis. Here we report the antiviral properties of ZAP against SRMV, a single-stranded negative-stranded RNA virus of the genus Morbillivirus. ZAP expression was significantly induced in sheep endometrial epithelial cells following SRMV infection. ZAP inhibited SRMV replication in cells after infection, while its overexpression in Vero-SLAM cells significantly increased their resistance to SRMV replication. The ZAP protein co-localized with SRMV RNA in the cytoplasm and ZAP-responsive elements were mapped to the 5' untranslated region of SRMV nucleocapsid, phosphoprotein, matrix, and fusion. In summary, ZAP confers resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication. Our findings further extend the ranges of viral targets of ZAP and help elucidate the mechanism of SRMV replication.

Caspase-Dependent Cleavage of DDX21 Suppresses Host Innate Immunity.

DEAD (Glu-Asp-Ala-Glu) box RNA helicases have been proven to contribute to antiviral innate immunity. The DDX21 RNA helicase was identified as a nuclear protein involved in rRNA processing and RNA unwinding. DDX21 was also proven to be the scaffold protein in the complex of DDX1-DDX21-DHX36, which senses double-strand RNA and initiates downstream innate immunity. Here, we identified that DDX21 undergoes caspase-dependent cleavage after virus infection and treatment with RNA/DNA ligands, especially for RNA virus and ligands. Caspase-3/6 cleaves DDX21 at D126 and promotes its translocation from the nucleus to the cytoplasm in response to virus infection. The cytoplasmic cleaved DDX21 negatively regulates the interferon beta (IFN-ß) signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. Thus, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immune response to virus. IMPORTANCE Innate immunity serves as the first barrier against virus infection. DEAD (Glu-Asp-Ala-Glu) box RNA helicases, originally considered to be involved in RNA processing and RNA unwinding, have been shown to play an important role in antiviral innate immunity. The precise regulation of innate immunity is critical for the host because the aberrant production of cytokines leads to unexpected pathological consequences. Here, we identified that DDX21 was cleaved at D126 by virus infection and treatment with RNA/DNA ligands via the caspase-3/6-dependent pathway. The cytoplasmic cleaved DDX21 negatively regulates the IFN-ß signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. In sum, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immune response to virus.

Caprine MAVS Is a RIG-I Interacting Type I Interferon Inducer Downregulated by Peste des Petits Ruminants Virus Infection.

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-ß promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.

Newcastle Disease Virus Induced Pathologies Severely Affect the Exocrine and Endocrine Functions of the Pancreas in Chickens.

Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry. ND causes heavy economic losses to the global poultry industry by decreasing the growth rate, decrease in egg production high morbidity and mortality. Although significant advances have been made in the vaccine development, outbreaks are reported in vaccinated birds. In this study, we report the damage caused by NDV infection in the pancreatic tissues of vaccinated and specific-pathogen-free chickens. The histopathological examination of the pancreas showed severe damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of the pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone and somatostatin were observed in NDV-infected chicken at three- and five- days post infection (DPI). A slight decrease in the plasma concentrations of insulin was noticed at 5 DPI. Significant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection decreased the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Taken together, our findings highlight that NDV induces extensive tissue damage in the pancreas, decreases the activity and expression of pancreatic enzymes, and increases plasma corticosterone and somatostatin. These findings provide new insights that a defective pancreas may be one of the reasons for decreased growth performance after NDV infection in chickens.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

Upregulation of DUSP6 impairs infectious bronchitis virus replication by negatively regulating ERK pathway and promoting apoptosis.

Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.

Safety and Efficacy Evaluation of Recombinant Marek's Disease Virus with REV-LTR.

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek's disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.