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During 2018–2020, we isolated 32 Eurasian avian-like swine influenza A(H1N1) viruses and their reassortant viruses from pigs in China. Genomic testing identified a novel reassortant H3N1 virus, which emerged in late 2020. Derived from G4 Eurasian H1N1 and H3N2 swine influenza viruses. This virus poses a risk for zoonotic infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes great economic losses globally to the swine industry. Innate immune RNA receptors mainly sense it during infection. As a DNA sensor, cyclic GMP-AMP synthase (cGAS) plays an important role in sensing cytosolic DNA and activating innate immunity to induce IFN-I and establish an antiviral cellular state. In contrast, the role of innate immune DNA sensors during PRRSV infection has not been elucidated. In this study, we found that cGAS facilitates the production of IFN-β during PRRSV infection. Western blot and virus titer assays suggested that cGAS overexpression suppressed the replication of multiple PRRSV strains, while knockout of cGAS increased viral titer and nucleocapsid protein expression. Besides, our results indicated that the mitochondria were damaged during PRRSV infection and leaked mitochondrial DNA (mtDNA) into the cytoplasm. The mtDNA in the cytoplasm co-localizes with the cGAS, and the cGAMP activity was increased when the cGAS was overexpressed during PRRSV infection. Furthermore, the cGAMP also possesses an anti-PRRSV effect. These results indicate for the first time that cGAS restricts PRRSV replication by sensing the mtDNA in the cytoplasm to increase cGAMP activity, which not only explains the molecular mechanism by which cGAS inhibits PRRSV replication but also provides research ideas for studying the role of the cGAS-STING signaling pathway in the process of RNA virus infection.

Novel porcine circovirus type 3 (PCV3), first identified in the United States, has been detected in many other countries. Porcine circovirus is associated with postweaning multisystemic wasting syndrome, reproductive failure, congenital tremors, and other clinical symptoms. In this study, we established a double polymerase chain reaction assay for detecting both porcine circovirus type 2 (PCV2) and PCV3. This is the first study to detect and characterize the PCV3 genome in the Tianjin region of North China. We collected a total of 169 tissue samples from seven farms between 2016 and 2018. The PCV3-positive rate of all tissue samples was 37.3% (63/169) and the rate of PCV2 and PCV3 coinfection was 14.8% (25/169). PCV2 and PCV3 coinfections with more serious clinical symptoms were found in only three farms. We sequenced three PCV3 strains selected from tissue samples that were positively identified. The complete genome sequences of the three strains shared 97.6–99.4% nucleotide identities with the PCV3 strains in GenBank. Our results showed the extent of PCV3’s spread in Tianjin, and the need to further study PCV3’s pathobiology, epidemiology, isolation, and coinfection.

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109–174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.

Background: Inflammation is supposed to play a key role in the pathophysiological processes of intestinal ischemia-reperfusion injury (IIRI), and Candida albicans in human gut commonly elevates inflammatory cytokines in intestinal mucosa. This study aimed to explore the effect of C. albicans on IIRI. Methods: Fifty female Wistar rats were divided into five groups according to the status of C. albicans infection and IIRI operation: group blank and sham; group blank and IIRI; group cefoperazone plus IIRI; group C. albicans plus cefoperazone and IIRI (CCI); and group C. albicans plus cefoperazone and sham. The levels of inflammatory factors tumor necrosis factor (TNF)-μ, interleukin (IL)-6, IL-1β, and diamine oxidase (DAO) measured by enzyme-linked immunosorbent assay were used to evaluate the inflammation reactivity as well as the integrity of small intestine. Histological scores were used to assess the mucosal damage, and the C. albicans blood translocation was detected to judge the permeability of intestinal mucosal barrier. Results: The levels of inflammatory factors TNF-μ, IL-6, and IL-1β in serum and intestine were higher in rats undergone both C. albicans infection and IIRI operation compared with rats in other groups. The levels of DAO (serum: 44.13 ± 4.30 pg/ml, intestine: 346.21 ± 37.03 pg/g) and Chiu scores (3.41 ± 1.09) which reflected intestinal mucosal disruption were highest in group CCI after the operation. The number of C. albicans translocated into blood was most in group CCI ([33.80 ± 6.60] ×102 colony forming unit (CFU)/ml). Conclusion: Intestinal C. albicans infection worsened the IIRI-induced disruption of intestinal mucosal barrier and facilitated the subsequent C. albicans translocation and dissemination.

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61–vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.

Pseudorabies (PR) is one of the most devastating diseases in the pig industry. To identify changes in microRNA (miRNA) expression and post-transcriptional regulatory responses to PRV infection in porcine kidney epithelial (PK15) cells, we sequenced a small RNA (sRNA) library prepared from infected PK15 cells and compared it to a library prepared from uninfected cells using Illumina deep sequencing. Here we found 25 novel viral miRNAs by high-throughput sequencing and 20 of these miRNAs were confirmed through stem-loop RT-qPCR. Intriguingly, unlike the usual miRNAs encoded by the α-herpesviruses, which are found clustered in the large latency transcript (LLT), these novel viral miRNAs are throughout the PRV genome like β-herpesviruses. Viral miRNAs are predicted to target multiple genes and form a complex regulatory network. GO analysis on host targets of viral miRNAs were involved in complex cellular processes, including the metabolic pathway, biological regulation, stimulus response, signaling process and immune response. Moreover, 13 host miRNAs were expressed with significant difference after infection with PRV: 8 miRNAs were up-regulated and 5 miRNAs were down-regulated, which may affect viral replication in host cell. Our results provided new insight into the characteristic of miRNAs in response to PRV infection, which is significant for further study of these miRNAs function.

In the original publication of this article [1], the author found the brand of vimentin antibody was wrong in Fig. 3. The legend of Fig. 3, ‘mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight’ should be ‘mouse anti-vimentin mAb (Sigma-Aldrich) at 4 °C overnight’.

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is still a serious threat to the swine industry. However, the pathogenic mechanism of HP-PRRSV remains unclear. We infected host porcine alveolar macrophages (PAMs) with the virulent HuN4 strain and the attenuated HuN4-F112 strain and then utilized fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) to screen for intracellular proteins that were differentially expressed in host cells infected with the two strains. There were 153 proteins with significant different expression (P

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development. [ABSTRACT FROM AUTHOR]

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China. [ABSTRACT FROM AUTHOR]

Background: Pigs have been implicated as mixing reservoir for the generation of new pandemic influenza strains, control of swine influenza has both veterinary and public health significance. Unlike human influenza vaccines, strains used for commercially available swine influenza vaccines are not regularly replaced, making the vaccines provide limited protection against antigenically diverse viruses. It is therefore necessary to develop broadly protective swine influenza vaccines that are efficacious to both homologous and heterologous virus infections. In this study, two forms of DNA vaccines were constructed, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2 swine influenza viruses. Another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). Their protective efficacies against homologous and heterologous challenges were tested. Results: BALB/c mice were immunized twice by particle-mediated epidermal delivery (gene gun) with the two DNA vaccines. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2 strains representative of different evolutional and antigenic clusters were used to challenge the vaccine-immunized mice (homosubtypic challenge). Results indicated that both of the DNA vaccines prevented homosubtypic virus infections completely. The vaccines' heterologous protective efficacies were further tested by challenging with a H1N1 swine influenza virus and a reassortant 2009 pandemic strain. It was found that MNHa reduced the lung viral titers significantly in both challenge groups, histopathological observation showed obvious reduction of lung pathogenesis as compared to MHa and control groups. Conclusions: The combined utility of the consensus HA and the conserved M2e and CTL epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse model. This may provide a basis for the development of universal swine influenza vaccines. [ABSTRACT FROM AUTHOR]

Background: The regions in the middle of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) have been shown to be nonessential for PRRSV replication, and these nonessential regions are different in various viral strains. Finding: In this study, the nonessential regions of the nsp2 of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus were identified based on an infectious cDNA clone of HuN4-F112. The results demonstrated that the segments of nsp2 [amino acids (aa) 480 to 667] tolerated deletions. Characterization of the mutants demonstrated that those with small deletions did not affect the viral growth on Marc-145 cells, but deletion of these regions led to earlier PRRSV replication increased (before 36 h after infectious in vitro). Conclusion: The functional roles of nsp2 variable middle region for PRRSV HuN4-F112 replication have been identified. Our results also suggested that none-essential region might be an ideal insertion region to express foreign gene in PRRSV genome. [ABSTRACT FROM AUTHOR]

Purpose of work: The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies. The 3C-like proteinase (3CLpro) of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and characterized. The optimal temperature and pH for its proteolytic activity were 8°C and 7.5, respectively. Na (1000 mM) and K (500 mM) were not inhibitory to its activity but Cu, Zn, PMSF and EDTA were significantly inhibitory. His, Asp and Ser residues were identified to form the catalytic triad of PRRSV 3CLpro by a series of site-directed mutagenesis analysis. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510–672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539–559), S1C4 (548–567), S1C7/8 (583–606), and S1C10/11 (607–630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome. [ABSTRACT FROM AUTHOR]

Abstract A phage display peptide library targeting the nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a was generated and used for epitope mapping. After 3 rounds of biopanning with the monoclonal antibody (MAb) N3H2 directed against the N protein, 3 positive phages were screened and sequenced. These phages share a consensus sequence, IQTAFNQGA, which corresponds to the amino acid (AA) 7987 segment of the CH-1a N protein. A small DNA fragment coding for IQTAFNQGA was expressed as a fusion product, and reacted to N3H2 in Western blots and indirect ELISA. Four truncated peptides (IQTAFNQG, IQTAFNQ, QTAFNQGA, and TAFNQGA) expressed as GST fusion products failed to react with N3H2. The sequences around the N3H2-binding site among the N proteins of 57 PRRSV strains were compared. Our results indicate that the IQTAFNQGA motif is highly conserved among North American and European isolates. We concluded that the precisely defined nona-peptide epitope is a novel conserved Linear B cell epitope on the N protein of PRRSV. [ABSTRACT FROM AUTHOR]

Interferon, an important cytokine, is an immunomodulator and possesses antiviral and anti-tumour activity. In vitro, it can be administrated in the treatment of diseases alone or with genetically engineered vaccine to enhance the immune effect of the latter. The recombinant transferring vector pSY681–ChIFN-γ was obtained in this study by inserting the chicken type II interferon (ChIFN-γ) gene into the Fowlpox virus (FPV) transferring vector pSY681. The resulting plasmid was then transfected into chicken embryo fibroblast (CEF) cell cultures pre-infected with the parental FPV S-FPV-017. Finally, the recombinant Fowlpox virus (rFPV) expressing ChIFN-γ (rFPV–ChIFN-γ) was produced by homologous recombination with the FPV gene in CEF. rFPV-positive plaques were verified by polymerase chain reaction (PCR), restriction analysis and indirect immunofluorescence assays. The rFPV–ChIFN-γ supernatants, cultured in CEF for 72 h and inoculated into rat fibroblasts (L929), had an inhibitory effect on the replication of Rous sarcoma virus (RSV) with an antiviral titre of 2048 U/ml. [ABSTRACT FROM PUBLISHER]

The article describes the development of a single-tube, one-step, real-time accelerated reverse transcription (RT)-loop-mediated isothermal amplification (LAMP) for the detection of Polvine teschovirus (PTV). It also notes that LAMP is a sensitive method for dinucleic acid (DNA) amplification. The results demonstrated that the test is simple, fast, accurate, and sensitive for the detection of PTV when 43 clinical samples were tested by the RT-LAM P method.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen that affects the pig industry, is a highly genetically diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus have not been completely elucidated. In this study, comparative analyses of all available genomic sequences of North American (NA)-type PRRSVs (n = 355, including 138 PRRSV genomes sequenced in this study) in China and the United States during 2014–2018 revealed a high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and the GP2 to GP3 regions. Lineage 1 (L1) PRRSV was found to be susceptible to recombination among PRRSVs both in China and the United States. The recombinant major parent between the 1991–2013 data and the 2014–2018 data showed a trend from complex to simple. The major recombination pattern changed from an L8 to L1 backbone during 2014–2018 for Chinese PRRSVs, whereas L1 was always the major backbone for US PRRSVs. Intralineage recombination hot spots were not as concentrated as interlineage recombination hot spots. In the two main clades with differential diversity in L1, NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, NADC34-like PRRSVs have been relatively stable in population genetic diversity for years. Systematic analyses of insertion and deletion (indel) polymorphisms of NSP2 divided PRRSVs into 25 patterns, which could generate novel references for the classification of PRRSVs. The results of this study contribute to a deeper understanding of the recombination of PRRSVs and indicate the need for coordinated epidemiological investigations among countries. [ABSTRACT FROM AUTHOR]

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1aderived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses. [ABSTRACT FROM AUTHOR]

In China, a majority of the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRSV) strains were seeded by the 2006 outbreak. However, the most recently emerged (2013-2014) HP-PRRSV strain has a very different genetic background. It is a NADC30-like PRRSV strain recently introduced from North America that has undergone genetic exchange with the classic HP-PRRSV strains in China. Subsequent isolation and characterization of this variant suggest high pathogenicity, so it merits special attention in control and vaccine strategies. [ABSTRACT FROM AUTHOR]

Glycosylated protein 3 (GP3) of PRRSV is variable between different PRRSV strains, so it is helpful for subtype classifying by using distinct epitopes. In this study, two dominant linear GP3 epitopes that were recognized by highly dilute serum in an enzyme-linked immunosorbent assay (ELISA) were identified. Sequence alignments of 36 North American (NA) PRRSV isolates revealed that the epitope H87DELGFMV94 is well conserved, whereas the epitope T59RQAAAEILE68 differs in other low-virulence NA-type strains, which have at least one amino acid mutation in this region. A mutational analysis revealed that none of these mutations could be recognized by the purified antibodies directed against the corresponding epitope, indicating that the genetic variations altered the antigenicity of the antigenic region. Using ELISA, we also found that antibodies directed against the two epitopes were present in more than 45 of 50 HP-PRRS-positive pig sera, suggesting that their antigenicity is excellent in vivo. [ABSTRACT FROM AUTHOR]

In recent years, acute outbreaks of epizootic diarrhea have occurred on many swine farms in China. Although the putative causative virus of the disease was not isolated, the genomic sequence of porcine epidemic diarrhea virus (PEDV) was consistently detected from feces of diseased pigs by reverse transcription-PCR (RT-PCR). Here we report a complete genome sequence of PEDV which is apparently different from those of early PEDV circulated in Chinese swine herds. [ABSTRACT FROM AUTHOR]