I226R Protein of African Swine Fever Virus Is a Suppressor of Innate Antiviral Responses.

African swine fever is one of the most devastating swine diseases caused by African swine fever virus (ASFV). Although ASFV encodes more than 160 viral proteins, the implication of a majority of ASFV proteins in regulating host immunity is yet to be explored, and the mechanisms of immune evasion by ASFV proteins are largely unknown. Here, we report that the I226R protein of ASFV significantly suppressed innate immune responses. The ectopic expression of ASFV I226R in 293T cells significantly inhibited the activation of interferon-stimulated response element promoters triggered by Sendai virus (SeV), poly(I:C), or cyclic GMP-AMP synthase (cGAS)/STING. The I226R protein caused a significant decrease in the expression of interferons and interferon-stimulating genes in cells infected with SeV. Similar results were obtained from experiments using I226R-overexpressed PK15 and 3D4/21 cells stimulated with vesicular stomatitis virus. We observed that I226R inhibited the activation of both nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). Furthermore, it was shown that overexpression of I226R suppressed IRF3 activation and caused the degradation of NF-κB essential modulator (NEMO) protein. The I226R-induced NEMO degradation could be prevented by treatment with MG132, a proteasome inhibitor. Together, these results reveal that the ASFV I226R protein impairs antiviral responses, likely through multiple mechanisms including the suppression of NF-κB and IRF3 activation, to counteract innate immune responses during the viral infection.

Regulation and Evasion of Host Immune Response by African Swine Fever Virus.

African swine fever (ASF) is an acute lethal hemorrhagic viral disease in domestic pigs and wild boars; is widely epidemic in Africa, Europe, Asia, and Latin America; and poses a huge threat to the pig industry worldwide. ASF is caused by the infection of the ASF virus (ASFV), a cytoplasmic double-stranded DNA virus belonging to the Asfarviridae family. Here, we review how the virus regulates the host immune response and its mechanisms at different levels, including interferon modulation, inflammation, apoptosis, antigen presentation, and cellular immunity.

Glyceraldehyde-3-Phosphate Dehydrogenase Restricted in Cytoplasmic Location by Viral GP5 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Replication via Its Glycolytic Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important endemic swine pathogens, causing enormous losses in the global swine industry. Commercially available vaccines only partially prevent or counteract the virus infection and correlated losses. PRRSV's replication mechanism has not been well understood. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was screened to bind with the viral major envelope glycoprotein 5 (GP5) after PRRSV infection. The interacting sites are located within a 13-amino-acid (aa) region (aa 93 to 105) of GP5 and at Lys227 of GAPDH. Interestingly, viral GP5 restricts the translocation of GAPDH from the cytoplasm to the nucleus. Moreover, cytoplasmic GAPDH facilitates PRRSV replication by virtue of its glycolytic activity. The results suggest that PRRSV GP5 restricts GAPDH to the nucleus and exploits its glycolytic activity to stimulate virus replication. The data provide insight into the role of GAPDH in PRRSV replication and reveal a potential target for controlling viral infection. IMPORTANCE PRRSV poses a severe economic threat to the pig industry. PRRSV GP5, the major viral envelope protein, plays an important role in viral infection, pathogenicity, and immunity. However, interactions between GP5 and host proteins have not yet been well studied. Here, we show that GAPDH interacts with GP5 through binding a 13-aa sequence (aa 93 to 105) in GP5, while GP5 interacts with GAPDH at the K277 amino acid residue of GAPDH. We demonstrate that GP5 interacts with GAPDH in the cytoplasm during PPRSV infection, inhibiting GAPDH entry into the nucleus. PRRSV exploits the glycolytic activity of GAPDH to promote viral replication. These results enrich our understanding of PRRSV infection and pathogenesis and open a new avenue for antiviral prevention and PRRSV treatment strategies.

Porcine reproductive and respiratory syndrome virus Nsp4 cleaves ZAP to antagonize its antiviral activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens impacting the global swine industry. PRRSV has been recognized to modulate the host immune response through a number of mechanisms. In our previous study, we found that over-expression of ZAP, a zinc finger antiviral protein of host, could suppress PRRSV replication, but how PRRSV escape the restriction of ZAP under natural conditions was still unknown. In this study, We found PRRSV infection significantly down-regulate the endogenous ZAP protein expression in Marc-145 cells. And PRRSV nonstructural protein 4 (Nsp4), a 3C-like serine proteinase, was screened to be responsible for ZAP reduction. Nsp4 could cleave ZAP, depending on its protease activity. The anti-PRRSV activity of ZAP was antagonized by Nsp4 in Marc-145 cells. In addition, we identified a unique amino acid, serine 180 of Nsp4 was required for efficient degradation of ZAP, and the mutation at residue 180 could decrease the ability of recombinant PRRSV to degrade ZAP. Those findings reveal a manner of PRRSV Nsp4 antagonizing the antiviral activity of ZAP, and shed light on a new strategy evolved by PRRSV to escape the host defense.

The emergence of a novel recombinant porcine reproductive and respiratory syndrome virus with an amino acid insertion in GP5 protein.

As an economic devastating virus, porcine reproductive and respiratory syndrome virus (PRRSV) has spread globally, and seriously hinders the healthy development of the swine industry worldwide. In recent years, however, recombinant PRRSV strains are continuously emerging, resulting in the death of a large number of pigs in China. In this study, we reported a NADC30-like PRRSV strain GD1909, a recombinant virus, which may originate from NADC30-like and HUN4-like strains. The GP5 protein of GD1909 strain has an asparagine insertion at position 60 and has more complex glycosylation pattern. This should be helpful for a better understanding of PRRSV molecular epidemiology and the prevention of PRRSV infection in the future.

Genetic analysis of a porcine reproductive and respiratory syndrome virus 1 strain in China with new patterns of amino acid deletions in nsp2, GP3 and GP4.

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and PRRSV 2 have coexisted in China for a very long time. In this study, the complete genomic characterization of a PRRSV 1 strain named KZ2018 was conducted. The results showed that it shared 88.6% identity with Lelystad virus and 81.9-90.8% identities with other Chinese PRRSV 1 strains. Further study showed that its nsp2 protein had a unique discontinuous 6-amino acid (aa) deletion (aa357-360+aa411+aa449). Additionally, its GP3 and GP4 contained a long continuous 18-aa deletion in their overlapped region, which has never been described in other Chinese PRRSV 1 isolates. Amino acid analysis of cell epitopes revealed that GP3245-256 and GP457-68 were the most variable epitopes among different Chinese PRRSV 1 isolates. The results might enrich our knowledge of PRRSV 1 strains in China.

S100A9 regulates porcine reproductive and respiratory syndrome virus replication by interacting with the viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses to the pig industry worldwide over the last 30 years, yet the associated viral-host interactions remain poorly understood. S100A9 is a damage-associated molecular pattern of the S100 protein family. Here, we found that PRRSV infection stimulated S100A9 expression in porcine alveolar macrophages (PAMs) and Marc-145 cells. S100A9 inhibited PRRSV replication via cellular Ca2+ dependent manner. The viral nucleocapsid (N) protein co-localized with S100A9 in the cytoplasm, and directly interacted at amino acid 78 of S100A9 and amino acids 36-37 of N protein. Moreover, we also found that the mutant S100A9 (E78Q) protein exhibited decreased antiviral activity against PRRSV compared with the parent S100A9. Recombinant PRRSV rBB (36/37) with two mutations in amino acid 36-37 in the N protein exhibited greater replication than the parent PRRSV BB0907 in S100A9-overexpressed PAM and Marc-145 cells. Thus, S100A9 may restrict PRRSV proliferation by interacting with the viral N protein.

Therapeutic effect of Xanthohumol against highly pathogenic porcine reproductive and respiratory syndrome viruses.

The infection by porcine reproductive and respiratory syndrome virus (PRRSV) has a severe impact on the world swine industry. However, commercially available vaccines provide only incomplete protection against this disease. Thus, novel approaches to control PRRSV infection are essential for the robust and sustainable swine industry. In our previous study, Xanthohumol (Xn), a prenylated flavonoid extracted for hops (Humulus lupulus L), was screened from 386 natural products to inhibit PRRSV proliferation and alleviate oxidative stress induced by PRRSV via the Nrf2-HMOX1 axis in Marc-145 cells. In this study, we furtherly found that Xn could inhibit PRRSV different sub-genotype strains infection with a low IC50 value in porcine primary alveolar macrophages (PAMs). In addition, it caused decreased expression of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α in PAMs infected with PRRSV or treated with lipopolysaccharide. Animal challenge experiments showed that Xn effectively alleviated clinical signs, lung pathology, and inflammatory responses in lung tissues of pigs induced by highly pathogenic PRRSV infection. The results demonstrate that Xn is a promising therapeutic agent to combat PRRSV infections.

Xanthohumol inhibits PRRSV proliferation and alleviates oxidative stress induced by PRRSV via the Nrf2-HMOX1 axis.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent and endemic swine pathogen that causes significant economic losses in the global swine industry. Commercial vaccines provide limited protection against this virus, and no highly effective therapeutic drugs are yet available. In this study, we first screened a library of 386 natural products and found that xanthohumol (Xn), a prenylated flavonoid found in hops, displayed high anti-PRRSV activity by inhibiting PRRSV adsorption onto and internalization into cells. Transcriptome sequencing revealed that Xn treatment stimulates genes associated with the antioxidant response in the nuclear factor-erythroid 2-related factor 2 (Nrf2) signalling pathway. Xn causes increased expression of Nrf2, HMOX1, GCLC, GCLM, and NQO1 in Marc-145 cells. The action of Xn against PRRSV proliferation depends on Nrf2 in Marc-145 cells and porcine alveolar macrophages (PAMs). This finding suggests that Xn significantly inhibits PRRSV proliferation and decreases viral-induced oxidative stress by activating the Nrf2-HMOX1 pathway. This information should be helpful for developing a novel prophylactic and therapeutic strategy against PRRSV infection.

Cholesterol 25-hydroxylase negatively regulates porcine intestinal coronavirus replication by the production of 25-hydroxycholesterol.

Cholesterol 25-hydroxylase (CH25H) has been shown lately to be a host restriction factor that encodes an enzyme, which catalyzes the oxidized form of cholesterol to 25-hydroxycholesterol (25HC). A series of studies have shown that 25HC activity in hosts plays a vital role in inhibiting viral infection. In this study, we explored the antiviral effect of CH25H and 25HC on porcine epidemic diarrhea virus (PEDV), which causes high mortality rates in newborn piglets with severe diarrhea, and considerable financial loss in the swine industry worldwide. Our results showed that PEDV infection downregulated the expression of CH25H in Vero cells. An overexpression and knockdown assay indicated that CH25H has significant antiviral action against PEDV, and a CH25H mutant (CH25H-M) that lacks hydroxylase activity also retains antiviral activity to a lesser extent. Furthermore, 25HC had a broad-spectrum antiviral effect against different PEDV strains by blocking viral entry. In addition, CH25H and 25HC inhibited the replication of porcine transmissible gastroenteritis virus (TGEV). Taken together, CH25H as a natural host restriction factor could inhibit PEDV and TGEV infection.

25-Hydroxycholesterol provides antiviral protection against highly pathogenic porcine reproductive and respiratory syndrome virus in swine.

Porcine reproductive and respiratory syndrome (PRRS) is a severe respiratory disease that leads to huge economic losses in the pig industry throughout the world. Although there are several vaccines available, the protective efficacy is limited. Therefore, new control strategies to prevent PRRS virus (PRRSV) infection are urgently required. We have previously reported that CH25H and 25HC can significantly inhibit the replication of PRRSV by preventing viral entry. In the present study, we found that 25HC with a low IC50 value significantly decreased the replication of different PRRSV strains, and increased the production of IL-1ß and IL-8 in porcine primary alveolar macrophages and the lung tissue. In pigs challenged with highly pathogenic PRRSV, treatment with 25HC was associated with an obvious reduction in the level of viremia and viral load in lung samples and nasal swabs, as well as decreased lung injury and an increased survival rate. These findings suggest that 25HC could be a promising antiviral drug against PRRSV in the future.

ZAP, a CCCH-Type Zinc Finger Protein, Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication and Interacts with Viral Nsp9.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens affecting many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. PRRSV infection can cleave mitochondrial antiviral signaling protein (MAVS) and inhibit the induction of type I interferon. The antiviral effector molecules that are involved in host protective responses to PRRSV infection are not fully understood. Here, by using transcriptome sequencing, we found that a zinc finger antiviral protein, ZAP, is upregulated in MAVS-transfected Marc-145 cells and that ZAP suppresses PRRSV infection at the early stage of replication. We also found that the viral protein Nsp9, an RNA-dependent RNA polymerase (RdRp), interacts with ZAP. The interacting locations were mapped to the zinc finger domain of ZAP and N-terminal amino acids 150 to 160 of Nsp9. These findings suggest that ZAP is an effective antiviral factor for suppressing PRRSV infection, and they shed light on virus-host interaction.IMPORTANCE PRRSV continues to adversely impact the global swine industry. It is important to understand the various antiviral factors against PRRSV infection. Here, a zinc finger protein, termed ZAP, was screened from MAVS-induced antiviral genes by transcriptome sequencing, and it was found to remarkably suppress PRRSV replication and interact with PRRSV Nsp9. The zinc finger domain of ZAP and amino acids 150 to 160 of Nsp9 are responsible for the interaction. These findings expand the antiviral spectrum of ZAP and provide a better understanding of ZAP antiviral mechanisms, as well as virus-host interactions.

Encephalomyocarditis virus 2C protein antagonizes interferon-ß signaling pathway through interaction with MDA5.

Encephalomyocarditis virus (EMCV) is one of the most important picornavirus. It infects many mammalian species and causes encephalitis, myocarditis, neurologic diseases, diabetes and reproductive disorders in pigs. And it evolves mechanisms for escaping innate immune responses. But the viral pathogenesis has not been understood completely. In this study, we firstly found that EMCV protein 2C is a strong IFN-ß antagonist that interacts with MDA5 to inhibit induction of the IFN-ß signal pathway. The mutations in amino acid residue V26 of 2C decrease the inhibition of IFN-ß promoter activity and lost the ability to interact with MDA5, compared with wild type 2C protein. The rescued viruses with mutations in 2C (rV26A and rK25-3A) induced significantly higher IFN-ß mRNA and protein levels in PK-15, HEK-293A and N2a cells, compared to wild type EMCV and the repaired viruses rV26A(R) and rK25-3A(R). These data indicate that the amino acid residue V26 of EMCV 2C plays important roles in inhibiting type I IFN production by interacting with MDA5.

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83.

Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen-processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the major histocompatibility complex (MHC)-peptide complex proteins TAP1 and ERp57 and are impaired in their ability to stimulate T cell proliferation and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV nonstructural protein 1α (Nsp1α) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1α abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1α and the corresponding repaired PRRSVs. Viruses with Nsp1α mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1α stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV.IMPORTANCE PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability of MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral nonstructural protein 1 (Nsp1) is responsible for upregulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1α (L5-2A, rG45A, G48A, and L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection.

Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro.

Autophagy of cytoplasmic components plays an essential role in the pathogenic infection process. Furthermore, research suggests that autophagy is an extremely important component of the innate immune response. Our study aimed to reveal the effect of virus-induced autophagy on pseudorabies virus (PRV) replication. Our results confirmed that light chain 3 (LC3)-I was converted into LC3-II after PRV infection; this transition is considered an important indicator of autophagy. Transmission electron microscopy (TEM) revealed that PRV infection could notably increase the number of autophagosomes in mouse neuro-2a (N2a) cells. In addition, LC3-II accumulated in response to chloroquine (CQ) treatment, indicating that PRV infection induced a complete autophagic flux response. Furthermore, our analyses verified differences in the magnitude of autophagy induction by two different PRV isolates, LA and ZJ01. Subsequent analysis showed that the induction of autophagy by rapamycin facilitated PRV replication, while inhibition of autophagy by 3-methyladenine (3-MA) reduced PRV replication. These results indicated that PRV induced autophagy via the classical Beclin-1-Atg7-Atg5 pathway to enhance viral replication in N2a cells in vitro.

Vimentin modulates infectious porcine circovirus type 2 in PK-15 cells.

Porcine circovirus type 2 (PCV2) is the pathogen that causes postweaning multisystemic wasting syndrome, which leads to significant economic losses for swine farms worldwide. However, the infection mechanism of PCV2 is not completely understood yet. Vimentin is a part of the cytoskeleton network and plays an important role in several virus infections. It is not clear whether vimentin has a role in PCV2 infection nor how it affects PCV2 infection. In this study, the function of vimentin in PK-15 cells infected with PCV2 has been elucidated. We found that vimentin had a restrictive effect on the replication of PCV2 in PK-15 cells. Overexpression of vimentin by transferred pCAGGS-vimentin and down-regulation by the respective scrambled small interfering RNA showed that vimentin restricted the replication and virion production of PCV2. A special interaction between vimentin and PCV2 Cap protein was observed using laser confocal microscopy and immunoprecipitation assay. Moreover, overexpression of vimentin could decrease NF-κB activity and increase PCV2-induced caspase-3 activity in PK-15 cells. These data suggest that vimentin is involved in the replication of PCV2 and has a restrictive effect on it, which is helpful in the study of the replication mechanism of PCV2.

Cholesterol 25-hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded, positive-sense RNA virus of the Arteriviridae family, has become a global health threat for swine. Cholesterol 25-hydroxylase (CH25H) is an enzyme that catalyzes oxidation of cholesterol to 25-hydroxycholesterol (25HC). The purpose of this study was to explore the antiviral activity of CH25H against PRRSV infection. We found that CH25H was induced by interferon-α and PRRSV in Marc-145 monkey kidney cells. In addition, CH25H and 25HC significantly inhibited PRRSV infection by preventing virus entry. A CH25H mutant that exhibited decreased catalytic activity had an antiviral effect against PRRSV. Treatment with 25HC pre-infection or post-infection significantly inhibited PRRSV infection in primary porcine alveolar macrophages. Our results reveal that CH25H is an interferon-stimulated gene and its production of 25HC can be used as a natural antiviral agent to combat PRRSV infection.

A novel recombinant porcine reproductive and respiratory syndrome virus with significant variation in cell adaption and pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the swine industry worldwide. In this study, a type 2 PRRSV strain was isolated from primary porcine alveolar macrophage cells and designated as GD1404. Interestingly, this strain was unable to grow in MARC-145 cells. Analysis of the full-length genome sequence revealed that strain GD1404 was an inter-subgenotype recombinant of strains QYYZ and JXA1. The C-terminus of the GP2 protein of strain GD1404 had an amino acid deletion. Also, the ORF5a protein had 51 codons, five more than most other highly pathogenic (HP-PRRSV) strains. Phylogenetic analysis based on ORF5 gene sequences showed that strain GD1404 and five others isolated in China formed a new subgenotype represented by strain QYYZ. Challenge experiments with piglets showed that the GD1404 and HP-PRRSV BB0907 strains caused similar rates of mortality and interstitial pneumonia. However, strain GD1404 infection resulted in lower viremia and viral loads in the lungs, as compared with strain BB0907. The results of this study provide evidence of the circulation of type 2 PRRSV QYYZ-like strains in China with variations in cell adaption and pathogenic abilities.

Pathogenicity and antigenicity of a novel NADC30-like strain of porcine reproductive and respiratory syndrome virus emerged in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) has spread globally and caused huge economic loss. In recent years, a new kind of highly pathogenic NADC30-like strain has emerged in China. However, the pathogenicity and antigenicity of the virus are not well understood. In this study, PRRSV strain FJ1402 was isolated from piglets with clinical signs in Fujian Province in China in 2014. The complete genomic sequence analysis showed that it arose from recombination of North America NADC30 strain and highly pathogenic PRRSV (HP-PRRSV) in China. Experiment in piglets showed that FJ1402 had similar virulence to HP-PRRSV strain BB0907. The commercial PRRSV modified live vaccines TJM-F92 and R98 could partly provide protective efficacy against FJ1402 challenge in piglets. This should be helpful for preventing and controlling this disease in the future.

Molecular epidemiological investigation of Marek's disease virus from Guangxi, China.

The predominant field strains of Marek's disease virus in Guangxi were clearly different from the vaccine strain CVI988/Rispens based on sequencing of the envelope glycoprotein I (gI), glycoprotein E (gE) and oncogenic meq genes. These differences may be partly responsible for the most recent outbreaks in Guangxi.