Qiaoya Zhang

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.

The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody

Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the heavy economic losses in pig industry in the world. A number of neutralizing epitopes have been identified in the viral structural proteins GP3, GP4, GP5 and M. In this study, the important amino acid (aa) residues of HP-PRRSV strain BB affecting neutralization susceptibility of antibody were examined using resistant strains generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse genetic technique and virus neutralization assay. HP-PRRSV strain BB was passaged under the pressure of porcine NAb serum in vitro. A resistant strain BB34s with 102 and 104 aa substitutions in GP5, which have been predicted to be the positive sites for pressure selection (Delisle et al., 2012), was cloned and identified. To determine the effect of the two aa residues on neutralization, eight recombinant PRRSV strains were generated, and neutralization assay results confirmed that the aa residues 102 and 104 in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and porcine alveolar macrophages. Alignment of GP5 sequences revealed that the variant aa residues at 102 and 104 were frequent among type 2 PRRSV strains. It may be helpful for understanding the mechanism regulating the neutralization susceptibility of PRRSV to the NAbs and monitoring the antigen variant strains in the field.

The Interferon-Induced Mx2 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication

Porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most economically important diseases of swine in the world. Current vaccination strategies provide only limited protection against PRRSV infection. Recently, myxovirus resistance 2 (Mx2) has been identified as a novel interferon (IFN)-induced, innate immunity restriction factor that inhibits some viral infections. However, the role of Mx2 in PRRSV infection is not well understood. In this study, we cloned the full-length monkey Mx2 (mMx2) complementary DNA (cDNA) from IFN-β-treated African green monkey Marc-145 cells, and found that overexpression of mMx2 inhibited PRRSV replication in Marc-145 cells. IFN-β induced expression of mMx2 in Marc-145 cells and suppressed PRRSV replication in a dose-dependent manner. Knockdown of mMx2 impaired the antiviral activity mediated by IFN-β. Confocal imaging and immunoprecipitation assays indicated that mMx2 interacted with PRRSV N protein in virus-infected cells. Furthermore, we showed that GTPase activity of mMx2 is necessary, but that the first N-terminal 51 amino acids are dispensable for antiviral activity. Finally, porcine Mx2 was also found to have the antiviral activity against PRRSV in Marc-145 cells. We conclude that mMx2 protein inhibits PRRSV replication by interaction with the viral N protein.

Emerging of two new subgenotypes of porcine reproductive and respiratory syndrome viruses in Southeast China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the leading swine pathogens and causes major economic loss to the global swine industry. In this study, a total of 49 PRRSV isolates were collected from different swine herds in seven provinces in Southeast China from 2014 to 2015. All the ORF5 genes and some Nsp2 genes were sequenced. Phylogenetic analysis showed that all the isolates belonged to the North America genotype. Among them, five isolates formed a new subgenotype IV derived from highly pathogenic PRRSV (HP-PRRSV). Six isolates formed subgenotype III, which were closely related to the NADC30 strain in the US. These isolates formed 13 putative N-linked glycosylation site (NGS) patterns based on N30, 33, 34, 35, 44 and 51. There were fewer NGSs of isolates in subgenotype IV than in subgenotype III. This indicates that the two new subgenotypes of PRRSV strains with different NGS patterns were spreading in those regions of China. The genetic diversity should be considered for the control and prevention of this disease.

The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation.

Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15–21, 42–48 and 88–94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.

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.

The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-κB and Sp1 Signaling Pathways

ABSTRACT Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with DC activation and immunosuppression of T cell proliferation when expressed as soluble CD83 (sCD83). In this study, we show that PRRSV infection strongly stimulates CD83 expression in porcine monocyte-derived DCs (MoDCs) and that the nucleocapsid (N) protein and nonstructural protein 10 (nsp10) of PRRSV enhance CD83 promoter activity via the NF-κB and Sp1 signaling pathways. R43A and K44A amino acid substitution mutants of the N protein suppress the N protein-mediated increase of CD83 promoter activity. Similarly, P192-5A and G214-3A mutants of nsp10 (with 5 and 3 alanine substitutions beginning at residues P192 and G214, respectively) abolish the nsp10-mediated induction of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A, and rG214-3A) and four revertants [rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R)] were generated. Decreased induction of CD83 in MoDCs was observed after infection by mutants rR43A, rK44A, rP192-5A, and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R). These findings suggest that PRRSV N and nsp10 play important roles in modulating CD83 signaling and shed light on the mechanism by which PRRSV modulates host immunity. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the hosts immune response and induces persistent infection. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of T cell proliferation and differentiation when expressed as soluble CD83 (sCD83). In this study, we found that PRRSV infection induces sCD83 expression in porcine MoDCs via the NF-κB and Sp1 signaling pathways. The viral nucleocapsid protein, nonstructural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of the N protein, as well as residues 192 to 196 (P192-5) and 214 to 216 (G214-3) of nsp10, play important roles in CD83 promoter activation. These findings provide new insights into the molecular mechanism of immune suppression by PRRSV.

Influence of the amino acid residues at 70 in M protein of porcine reproductive and respiratory syndrome virus on viral neutralization susceptibility to the serum antibody

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the significant economic losses in pig industry in the world. The adaptive immune responses of the host act as an important source of selective pressure in the evolutionary process of the virus. In the previous study, we confirmed that the amino acid (aa) residues at 102 and 104 sites in GP5 played an important role in escaping from the neutralizing antibodies (NAbs) against highly pathogenic PRRSV (HP-PRRSV). In this study, we further analyzed the aa mutants affecting neutralization susceptibility of NAbs in other structure proteins in NAbs resistant variants.MethodsBased on the different aa residues of the structural proteins between the resistant virus BB20s and the parent virus BB, 12 recombinant PRRSV strains containing these aa residue substitutions were constructed using reverse genetic techniques. The neutralizing antibody (NA) titers of the recombinant strains were tested on MARC-145 and porcine alveolar macrophages (PAMs). And the NAbs binding abilities of parent and rescued viruses were tested by using ELISA method.ResultsBy using the neutralization assay, it was revealed that the NA titer of N4 serum with rBB/Ms was significantly lower than that with rBB. Meanwhile, NA titer of the serum with rBB20s/M was significantly higher than that with rBB20s. The ELISA binding results showed that rBB/Ms had higher binding inability to N4 than did rBB. And alignment of M protein revealed that the variant aa residue lysine (K) at 70 was also existed in field type 2 and vaccine PRRSV strains.ConclusionsThe aa residue at 70 in M protein of PRRSV played an important role in regulating neutralization susceptibility to the porcine serum NAbs. It may be helpful for monitoring the antigen variant strains in the field and developing new vaccine against PRRSV in the future.

Monkey Viperin Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen which causes huge economic damage globally in the swine industry. Current vaccination strategies provide only limited protection against PRRSV infection. Viperin is an interferon (IFN) stimulated protein that inhibits some virus infections via IFN-dependent or IFN-independent pathways. However, the role of viperin in PRRSV infection is not well understood. In this study, we cloned the full-length monkey viperin (mViperin) complementary DNA (cDNA) from IFN-α-treated African green monkey Marc-145 cells. It was found that the mViperin is up-regulated following PRRSV infection in Marc-145 cells along with elevated IRF-1 gene levels. IFN-α induced mViperin expression in a dose- and time-dependent manner and strongly inhibits PRRSV replication in Marc-145 cells. Overexpression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry and genome replication and translation but not inhibiting assembly and release. And mViperin co-localized with PRRSV GP5 and N protein, but only interacted with N protein in distinct cytoplasmic loci. Furthermore, it was found that the 13–16 amino acids of mViperin were essential for inhibiting PRRSV replication, by disrupting the distribution of mViperin protein from the granular distribution to a homogeneous distribution in the cytoplasm. These results could be helpful in the future development of novel antiviral therapies against PRRSV infection.

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.