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We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014–2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide., In this study, the authors present a genomic surveillance of avian influenza genomes sampled from live poultry markets in China. They report that a number of variants have emerged since 2016 that pose an increased risk to humans. They highlight the importance of continuous genome surveillance of circulating influenza strains.

With the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and growing fear, people have become very concerned about whether this novel coronavirus could be transmitted by mosquitoes. We evaluate the infectivity of SARS-CoV-2 in Aedes albopictus and Aedes aegypti derived cell lines. The results indicated that SARS-CoV-2 could not replicate in both C6/36, Sf9 and Aag2 cells. Further, no SARS-CoV-2 RNA was detected in the field collected Culex, and Anopheles mosquitoes during the months of April and May in Wuhan in 2020. Our findings highlight the restricted replication of SARS-CoV-2 in mosquito cells and field-caught mosquitoes.

Type I interferons (IFN-I) play a key role in the host defense against virus infection, but porcine reproductive and respiratory syndrome virus (PRRSV) infection does not effectively activate IFN-I response, and the underlying molecular mechanisms are poorly characterized. In this study, a novel transcription factor of the heme oxygenase-1 (HO-1) gene, homeobox A3 (HOXA3), was screened and identified. Here, we found that HOXA3 was significantly increased during PRRSV infection. We demonstrated that HOXA3 promotes PRRSV replication by negatively regulating the HO-1 gene transcription, which is achieved by regulating type I interferons (IFN-I) production. A detailed analysis showed that PRRSV exploits HOXA3 to suppress beta interferon (IFN-β) and IFN-stimulated gene (ISG) expression in host cells. We also provide direct evidence that the activation of IFN-I by HO-1 depends on its interaction with IRF3. Then we further proved that deficiency of HOXA3 promoted the HO-1-IRF3 interaction, and subsequently enhanced IRF3 phosphorylation and nuclear translocation in PRRSV-infected cells. These data suggest that PRRSV uses HOXA3 to negatively regulate the transcription of the HO-1 gene to suppress the IFN-I response for immune evasion. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, leads the pork industry worldwide to significant economic losses. HOXA3 is generally considered to be an important molecule in the process of body development and cell differentiation. Here, we found a novel transcription factor of the HO-1 gene, HOXA3, can negatively regulate the transcription of the HO-1 gene and play an important role in the suppression of IFN-I response by PRRSV. PRRSV induces the upregulation of HOXA3, which can negatively regulate HO-1 gene transcription, thereby weakening the interaction between HO-1 and IRF3 for inhibiting the type I IFN response. This study extends the function of HOXA3 to the virus field for the first time and provides new insights into PRRSV immune evasion mechanism.

MicroRNAs (miRNAs) play critical roles in the complex networks of virus–host interactions. Our previous research showed that porcine reproductive and respiratory syndrome virus (PRRSV) infection markedly upregulates miR-c89 expression, suggesting that miR-c89 may play an important role in PRRSV infection. The present study sought to determine the function of miR-c89 and its molecular mechanism during PRRSV infection. Using quantitative reverse transcription PCR (RT-qPCR) verification, we demonstrated that both highly pathogenic PRRSV and low-pathogenic PRRSV infection induced miR-c89 expression. The overexpression of miR-c89 significantly suppressed the replication of a variety of PRRSV strains, regardless of the timing of infection. Further, miR-c89 can directly target the 3′UTR of porcine retinoid X receptor β (RXRB) mRNA in a sequence-specific manner. Knockdown affected RXRB expression, as siRNA can suppress the replication of a variety of PRRSV strains. This work not only provides new insights into PRRSV–cell interactions, but also highlights the potential for the use of miR-c89 in the development of new antiviral strategies to combat PRRSV infection.

Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in West Africa have revealed the lack of investigation on this notorious virus., Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

Recombination is an important phenomenon that accelerates evolution and enriches the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant PRRSV isolates sometimes have different genetic backgrounds. In this study, we report a recombinant PRRSV (SD-YL1712) isolated from a pig farm. The genome of SD-YL1712 is 15,014 nucleotides in length, and its nucleotide and amino acid sequence conservation is higher than that of PRRSV strain JXA1 except within the NSP2 region. The NSP2 region of SDYL1712 shares the highest nucleotide (85.9%) and amino acid (84.1%) sequence identity with PRRSV strain NADC30. SD-YL1712 was found to contain a characteristic 131-amino-acid deletion in the NSP2 region. Two recombination breakpoints were detected at nt 2134 and nt 3958 within the NSP2 region, which revealed that SD-YL1712 originated from a recombination event between NADC30-like and HP-PRRSV-derived MLV-like strains. Interestingly, SD-YL1712 had an additional deletion at position 586, similar to that found in strain TJnh1501. Moreover, the pathogenicity of strain SD-YL1712 was found to be similar to that of HP-PRRSV JXA1, which was higher than that of the CH1a strain. Further analysis indicated that SD-YL1712 might be a transitional intermediate in the evolution of TJbd1401 to TJnh1501.

Background Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. Results Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. Conclusions Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.

Hepatitis E virus (HEV) has been detected in rabbits, a recently identified natural reservoir. In this study, anti-HEV antibodies and viral RNA were detected in rabbits sourced from a specific-pathogen-free (SPF) rabbit vendor in Shaanxi Province, China. BLAST results of partial HEV ORF2 genes cloned here indicated that two viral strains circulated in the rabbits. Sequence determination of the complete genome (7302bp) of one strain and a partial ORF1 gene (1537bp) of the other strain showed that they shared 90% identity with one another and 78%-94% identity with other known rabbit HEVs. In addition, inoculation with rabbit HEV from SPF rabbits studied here resulted in infection of SPF pigs; this cross-species transmission was evidenced by seroconversion, viremia and faecal virus shedding. These results suggest that to prevent spread of this zoonotic pathogen, rabbits should be tested routinely for HEV RNA in SPF vendor facilities.

Highlights • BVDV NS5B-specific nanobodies were identified. • NS5B-specific nanobody Nb1 suppresses BVDV infection and replication. • Nb1 interacts with NS5B protein during BVDV infection., Bovine viral diarrhea virus (BVDV) infection causes significant economic losses to the cattle industry worldwide and still represents a huge pressure on agricultural production. Thus, the development of novel anti-BVDV strategies are urgently needed. The nonstructural protein 5 (NS5B) of BVDV is essential for viral replication. Further, the camel single-domain antibody (nanobody) represents a promising antiviral approach with the advantages of small size, stable structure, high specificity and solubility, and the recognition of specific epitopes. However, no NS5B-specific nanobodies against BVDV have been reported. In this study, NS5B-specific nanobodies were isolated from a phage display library of variable domains of Camellidae heavy chain-only antibodies (VHHs). Further, an MDBK cell line stably expressing Nb1 was established to explore antiviral activity. Results showed that Nb1 could markedly suppress BVDV replication and interact with the BVDV NS5B protein. This suggests that nanobodies have potential for the development of novel antiviral drugs against BVDV infection.

// Na Li 1, 2 , Yunhuan Yan 1, 2 , Angke Zhang 1, 2 , Jiming Gao 1, 2 , Chong Zhang 1, 2 , Xue Wang 1, 2 , Gaopeng Hou 1, 2 , Gaiping Zhang 1, 3 , Jinbu Jia 4, 5 , En-Min Zhou 1, 2 , Shuqi Xiao 1, 2 1 College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China 2 Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi 712100, China 3 College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China 4 College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China 5 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China Correspondence to: Shuqi Xiao, email: xiaoshuqi@nwsuaf.edu.cn En-Min Zhou, email: zhouem@nwsuaf.edu.cn Keywords: microRNA, PRRSV, viral small RNAs (vsRNAs), viral replication, autoregulation Received: July 05, 2016 Accepted: October 03, 2016 Published: October 17, 2016 ABSTRACT Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. MicroRNAs (miRNAs) play vital roles in virus-host interactions by regulating the expression of viral or host gene at posttranscriptional level. Our previous research showed that PRRSV infection down-regulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, and overexpression of HO-1 inhibits PRRSV replication. In this study, we demonstrate that host miRNA miR-22 can downregulate HO-1 expression by directly targeting its 3′ untranslated region. Suppression of HO-1 expression by miR-22 facilitates PRRSV replication. This work suggests that PRRSV may utilize cellular miRNA to modify antiviral host factor expression, enabling viral replication, which not only provides new insights into virus-host interactions during PRRSV infection, but also suggests potential therapies for PRRSV infection.

Porcine reproductive and respiratory syndrome virus(PRRSV) is an important immunosuppressive virus which can suppresses infected cells proliferation. In this work, we examined PRRSV ability to manipulate cell cycle progression of MARC-145 cells and explored the potential molecular mechanisms. The results showed that PRRSV infection imposed a growth-inhibitory effect on MARC-145 cells by inducing cell cycle arrest at G2/M phase. This arrest was due to the significant decrease of Cdc2-cyclinB1 complex activity in PRRSV-infected cells and the activity reduction was a result of Cdc2 Tyr15 phosphorylation and the accumulation of Cdc2 and cyclinB1 in the nucleus. Not only elevated Wee1 and Myt1 expression and inactivated Cdc25C, but also increase of p21 and 14–3-3σ in a p53-dependent manner caused the inhibitory Tyr15 phosphorylation of Cdc2. PRRSV infection also activated Chk1. Our data suggest PRRSV infection induces G2/M arrest via various molecular regulatory mechanisms. These results provide a new insights for PRRSV pathogenesis.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread viral disease affecting the swine industry, with no cure or effective treatment. Current vaccines are inefficient mainly due to the high degree of genetic and antigenic variation within PRRS virus (PRRSV) strains. Thus, the development of novel anti-PRRSV strategies is an important area of research. The nonstructural protein 9 (Nsp9) of PRRSV is essential for viral replication, and its sequence is relatively conserved, making it a logical antiviral target for PRRSV. Camel single-domain antibodies (nanobodies) represent a promising antiviral approach because of their small size, high specificity, and solubility. However, no nanobodies against PRRSV have been reported to date. In this study, Nsp9-specific nanobodies were isolated from a phage display library of variable domains of Camellidaeheavy chain-only antibodies (VHH). One of the isolated nanobodies, Nb6, was chosen for further investigation. Co-immunoprecipitation experiments indicated that Nb6 can still maintain antigen binding capabilities when expressed in the cell cytoplasm. A MARC-145 cell line stably expressing Nb6 was established to investigate its potential antiviral activity. Our results showed that intracellularly expressed Nb6 could potently suppress PRRSV replication by inhibiting viral genome replication and transcription. More importantly, Nb6 could protect MARC-145 cells from virus-induced cytopathic effect (CPE) and fully block PRRSV replication at an MOI of 0.01 or lower. To our knowledge, this is the first report of a nanobody based antiviral strategy against PRRSV, and this finding has the potential to lead to future developments of novel antiviral treatments for PRRSV infection.

Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-κB signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

PRRSV (porcine reproductive and respiratory syndrome virus) is a major economically significant pathogen that has adversely impacted the global swine industry for almost 30 years. Currently PRRSV is estimated to cause losses of almost US$600 million per year in the USA. Except for new mutants that continually emerge during PRRSV outbreaks, our understanding of the virology, origin, and evolution of PRRSV and the host's immune response are largely inadequate. Such limited knowledge impedes development of effective methods to eradicate this virus. In this review, we systematically describe recent advances in anti-PRRSV research, especially focusing on those techniques with the potential to transform current anti-PRRSV strategies. Furthermore, a combination of these new techniques may provide creative insights to guide future PRRSV control and prevention.

Virus replication depends upon host-cell processes in infected cells, and this is true for porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS that is a worldwide threat to the swine industry. Heme oxygenase-1 (HO-1) is a ubiquitously expressed inducible isoform of the first and rate-limiting enzyme for heme degradation. Our previous research suggested that HO-1 may play an important role in PRRSV infection. However, the function of HO-1 in PRRSV infection is unclear. In the present study, Marc-145, PK-15(CD163) cell lines and porcine alveolar macrophages (PAMs) were used to evaluate the effects of HO-1 induction and over-expression on the replication of two different PRRSV strains. Induction of HO-1 markedly decreased the replication of PRRSV strains in the different cells. Similarly, adenoviral-mediated over-expression of HO-1 also greatly decreased the replication of PRRSV. In contrast, ablation of HO-1 using small interfering RNA concomitantly increased PRRSV replication. Therefore, the data were consistent with HO-1 acting as an antiviral factor and these findings suggested that over-expression or induction of HO-1 may provide a potential therapeutic strategy against PRRSV infection.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified into four major genotypes (1 to 4) and swine is the main natural reservoir for genotypes 3 and 4. In this study, a total of 106 bile samples from a slaughterhouse in the Shandong province of China were tested for the partial ORF2 gene of HEV by RT-nPCR to determine the virus genotypes, and two indirect ELISA were developed for the detection of swine HEV specific IgM and IgG antibodies in 980 serum samples from 24 farms, in order to investigate the seroprevalence. Thirty-two out of 106 (30.2%) bile samples were positive for HEV and a high degree of partial ORF2 sequence similarity (86.8-100%) was observed among 20 samples. The viral sequences belonged to genotype 4, subtypes 4a and 4d. One complete genome sequence of a subtype 4d HEV was further determined and characterized. The seroprevalence of HEV IgG and IgM antibodies was 100% (24/24) and 41.7% (10/24) for herds, and 66.4% (651/980) and 1.6% (16/980) for the individual pigs, respectively. These results suggested a high prevalence of genotype 4 of swine HEV infection both in swine farms and at the slaughterhouse in Shandong province, which further raise public-health concerns for zoonosis and pork safety.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine, which causes large economic losses to the swine industry worldwide. There is considerable strain variation in PRRSV and two examples of this are the highly virulent Chinese-type PRRSV (H-PRRSV) and the classical North American type PRRSV (N-PRRSV), both with different pathogenesis. These differences may be due in part to genetic and phenotypic differences in virus replication, but also interaction with the host cell. MicroRNAs (miRNAs) are crucial regulators of gene expression and play vital roles in virus and host interactions. However, the regulation role of miRNAs during PRRSV infection has not been systematically investigated. In order to better understand the differential regulation roles of cellular miRNAs in the host response to PRRSV, miRNA expression and a global mRNA transcriptome profile was determined in primary cells infected with either H-PRRSV or N-PRRSV as multiple time points during the viral lifecycle. miRNA-mRNA interactome networks were constructed by integrating the differentially expressed miRNAs and inversely correlated target mRNAs. Using gene ontology and pathway enrichment analyses, cellular pathways associated with deregulated miRNAs were identified, including immune response, phagosome, autophagy, lysosome, autolysis, apoptosis and cell cycle regulation. To our knowledge, this is the first global analysis of strain-specific host miRNA molecular signatures associated with H- and N-PRRSV infection by integrating miRNA and mRNA transcriptomes and provides a new perspective on the contribution of miRNAs to the pathogenesis of PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both a PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV; however, it did not affect PRRSV entry into host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cyclic GMP/protein kinase G (cGMP/PKG) signaling pathway. CO significantly inhibits PRRSV-induced NF-κB activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced proinflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks. IMPORTANCE PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Current antiviral strategies do not effectively prevent and control PRRSV. Recent reports show that microRNAs (miRNAs) play vital roles in viral infections by post transcriptionally regulating the expression of viral or host genes. Our previous research showed that non-muscle myosin heavy chain 9 (MYH9) is an essential factor for PRRSV infection. Using bioinformatic prediction and experimental verification, we demonstrate that MYH9 expression is regulated by the miRNA let-7f-5p, which binds to the MYH9 mRNA 3′UTR and may play an important role during PRRSV infection. To understand how let-7f-5p regulates PRRSV infection, we analyzed the expression pattern of both let-7f-5p and MYH9 in porcine alveolar macrophages (PAMs) after infection with either highly pathogenic PRRSV (HP-PRRSV) or classical type PRRSV (N-PRRSV) using a deep sequencing approach with quantitative real-time PCR validation. Our results showed that both HP-PRRSV and N-PRRSV infection reduced let-7f-5p expression while also inducing MYH9 expression. Furthermore, let-7f-5p significantly inhibited PRRSV replication through suppression of MYH9 expression. These findings not only provide new insights into the pathogenesis of PRRSV, but also suggest potential new antiviral strategies against PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is an important swine disease worldwide. PRRSV has a limited tropism for certain cells, which may at least in part be attributed to the expression of the necessary cellular molecules serving as the virus receptors or factors on host cells for virus binding or entry. However, these molecules conferring PRRSV infection have not been fully characterized. Here we show the identification of non-muscle myosin heavy chain 9 (MYH9) as an essential factor for PRRSV infection using the anti-idiotypic antibody specific to the PRRSV glycoprotein GP5. MYH9 physically interacts with the PRRSV GP5 protein via its C-terminal domain and confers susceptibility of cells to PRRSV infection. These findings indicate that MYH9 is an essential factor for PRRSV infection and provide new insights into PRRSV-host interactions and viral entry, potentially facilitating development of control strategies for this important swine disease.

To isolate specific nanobodies to porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 4 (Nsp4) and investigate their potential antiviral activities. Three PRRSV Nsp4-specific nanobodies were isolated from a phage display library of the variable domains of camelid heavy chain-only antibodies. Nanobody genes were introduced into MARC-145 cells using lentivirus vectors to establish cell lines stably expressing nanobodies. These intracellularly expressed nanobodies were tested for interaction with PRRSV-encoded Nsp4 within PRRSV-infected MARC-145 cells. Nb41 and Nb43 intrabodies each potently inhibited PRRSV replication, protected MARC-145 cells from PRRSV-induced cytopathic effect and fully blocked PRRSV replication at an MOI of 0.001 or lower. Intracellularly expressed Nb41 and Nb43 potently suppressed PRRSV replication in MARC-145 cells. Nanobodies hold great potential for development as novel antiviral treatments for PRRSV infection.

Viral cycle progression depends upon host-cell processes in infected cells and this is true for bovine viral diarrhoea virus (BVDV), the causative agent of BVD that is a worldwide threat to the bovine industry. Heme oxygenase-1 (HO-1) is a ubiquitously expressed inducible isoform of the first and rate-limiting enzyme for heme degradation. Recent studies have demonstrated that HO-1 has significant antiviral properties, inhibiting the replication of viruses such as ebola virus, human immunodeficiency virus, hepatitis C virus and porcine reproductive and respiratory syndrome virus. However, the function of HO-1 in BVDV infection is unclear. In the present study, the relationship between HO-1 and BVDV was investigated. In vitro analysis of HO-1 expression in BVDV-infected MDBK cells demonstrated that a decrease in HO-1 as BVDV replication increased. Increasing HO-1 expression through adenoviral-mediated overexpression or induction with cobalt protoporphyrin (CoPP, a potent HO-1 inducer), pre- and postinfection, effectively inhibited BVDV replication. In contrast, HO-1 siRNA knockdown in BVDV-infected cells increased BVDV replication. Therefore, the data were consistent with HO-1 acting as an anti-viral factor and these findings suggested that induction of HO-1 may be a useful prevention and treatment strategy against BVDV infection.

The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

The major neutralizing epitope of porcine reproductive and respiratory syndrome virus (PRRSV) is mainly located on virus glycoprotein 5 (GP5). Immunization with exogenous GP5 or exposure to native GP5 by means of DNA immunization can provide some degree of immune protection to PRRSV infection in pigs. However, during PRRSV infection in pigs, the production of neutralization antibodies induced by GP5 is delayed or suppressed. This suggests that the synthesis of GP5 is late than some PRRSV proteins or other PRRSV proteins interfering with the function of GP5 in inducing host responses during virus infection. Here, to exclude the impacts of the other PRRSV proteins and determine the role of GP5 in the replication of PRRSV in vitro, a Marc-145 cell line stably expressing GP5 (Marc-145-GP5(Flag)) was constructed. Cell proliferation and cell apoptosis measurements indicated that the expression of GP5 in Marc-145 cells did not disturb the cells' viability. Following infection with different PRRSV strains PRRSV replication in Marc-145-GP5(Flag) cells was inhibited significantly. Type I interferon assay results showed that beta interferon (IFN-β) in the Marc-145-GP5(Flag) cells were increased at mRNA and protein levels. When siRNA was introduced into the cells to knock down IFN-β mRNA, PRRSV infectivity of these cells was recovered. These data suggest that early GP5 expression is not favorable for further infection by PRRSV, because it not only stimulates production of neutralization antibodies in pigs, but also induces IFN-β production in host cells. Therefore, GP5 is an important protein in the induction of self-protection responses from the host.

Porcine reproductive and respiratory syndrome (PRRS) is a leading disease in pig industry worldwide and can result in serious economic losses each year. The PRRS epidemic situation in China has been very complicated since the unprecedented large-scale highly pathogenic PRRS (HP-PRRS) outbreaks in 2006. And now the HP-PRRS virus (HP-PRRSV) and classical North American type PRRSV strains have coexisted in China. Rapid differential detection of the two strains of PRRSV is very important for effective PRRS control. The real-time RT-PCR for simultaneous detection and differentiation of HP-PRRSV and PRRSV by using both SYBR Green and TaqMan probes was developed and validated. Both assays can be used for rapid detection and strain-specific identification of HP-PRRSV and PRRSV. However, the TaqMan probe method had the highest detection rate whereas the conventional RT-PCR was the lowest. The real-time RT-PCR developed based on SYBR Green and TaqMan probe could be used for simultaneous detection and differentiation of HP-PRRSV and PRRSV in China, which will benefit much the PRRS control and research.

Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection.

Background There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV) remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology. Results H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity. Conclusions The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified.

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.

Background Porcine reproductive and respiratory syndrome with PRRS virus (PRRSV) infection, which causes significant economic losses annually, is one of the most economically important diseases affecting swine industry worldwide. In 2006 and 2007, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) happened in China and Vietnam. However little data is available on global host response to PRRSV infection at the protein level, and similar approaches looking at mRNA is problematic since mRNA levels do not necessarily predict protein levels. In order to improve the knowledge of host response and viral pathogenesis of highly virulent Chinese-type PRRSV (H-PRRSV) and Non-high-pathogenic North American-type PRRSV strains (N-PRRSV), we analyzed the protein expression changes of H-PRRSV and N-PRRSV infected lungs compared with those of uninfected negative control, and identified a series of proteins related to host response and viral pathogenesis. Results According to differential proteomes of porcine lungs infected with H-PRRSV, N-PRRSV and uninfected negative control at different time points using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry identification, 45 differentially expressed proteins (DEPs) were identified. These proteins were mostly related to cytoskeleton, stress response and oxidation reduction or metabolism. In the protein interaction network constructed based on DEPs from lungs infected with H-PRRSV, HSPA8, ARHGAP29 and NDUFS1 belonged to the most central proteins, whereas DDAH2, HSPB1 and FLNA corresponded to the most central proteins in those of N-PRRSV infected. Conclusions Our study is the first attempt to provide the complex picture of pulmonary protein expression during H-PRRSV and N-PRRSV infection under the in vivo environment using 2D-DIGE technology and bioinformatics tools, provides large scale valuable information for better understanding host proteins-virus interactions of these two PRRSV strains.

Background The inability of current vaccines to provide effective protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection is not fully understood. One of the reasons might be the presence of anti-idiotypic antibodies (Ab2s) to the envelope glycoprotein GP5 induced by PRRSV infection since our previous studies demonstrated the presence of auto-Ab2s (aAb2s) in pigs infected with PRRSV. To test this hypothesis, PRRSV negative piglets were injected with a monoclonal Ab2 (Mab2-5G2) and aAb2s that are specific for anti-GP5 antibody, vaccinated with the attenuated PRRSV vaccine CH-1R and then challenged with the highly pathogenic PRRSV HuN4 strain. The animals were evaluated for clinical signs, pathological changes of the thymus and lungs, viremia, levels of serum antibodies and cytokines. Results The piglets injected with Mab2-5G2 or aAb2, and who received the attenuated PRRSV vaccine CH-1R before challenge, produced high levels of anti-N antibodies, IL-2 and IL-4, but low levels of neutralizing antibodies. After PRRSV HuN4 challenge, the animals showed obvious clinical signs, including lung lesions, severe thymus atrophy and decreased production of IL-4 and higher level of viremia. Conclusion When anti-GP5 Ab2s are present, the use of attenuated PRRSV vaccine CH-1R against HP-PRRSV infection is not recommended. It can result in poor health status with pneumonia and thymus atrophy.