Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues Involved in Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a widespread viral disease that has led to huge economic losses for the global swine industry. Non-structural protein 9 (Nsp9) of PRRSV possesses essential RNA-dependent RNA polymerase (RdRp) activity for viral RNA replication. Our previous report showed that Nsp9-specific nanobody, Nb6, was able to inhibit PRRSV replication. In this study, recombinant Nsp9 and Nsp9-Nb6 complex were prepared then characterized using bio-layer interferometry (BLI) and dynamic light scattering (DLS) analyses that demonstrated high-affinity binding of Nb6 to Nsp9 to form a homogeneous complex. Small-angle X-ray scattering (SAXS) characterization analyses revealed that spatial interactions differed between Nsp9 and Nsp9-Nb6 complex molecular envelopes. Enzyme-linked immunosorbent assays (ELISAs) revealed key involvement of Nsp9 residues Ile588, Asp590, and Leu643 and Nb6 residues Tyr62, Trp105, and Pro107 in the Nsp9-Nb6 interaction. After reverse genetics-based techniques were employed to generate recombinant Nsp9 mutant viruses, virus replication efficiencies were assessed in MARC-145 cells. The results revealed impaired viral replication of recombinant viruses bearing I588A and L643A mutations as compared with replication of wild type virus, as evidenced by reduced negative-strand genomic RNA [(-) gRNA] synthesis and attenuated viral infection. Moreover, the isoleucine at position 588 of Nsp9 was conserved across PRRSV genotypes. In conclusion, structural analysis of the Nsp9-Nb6 complex revealed novel amino acid interactions involved in viral RNA replication that will be useful for guiding development of structure-based anti-PRRSV agents.

Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province, central China.

Porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) are important swine viruses that threaten the swine industry worldwide. Here, we evaluated the co-infection status of PCV2, PCV3 and PEDV in 76 enteric samples from piglets with severe diarrhea disease in Henan, China. All samples were tested by PCR/RT-PCR. Our results showed that the infection rate of PCV2, PCV3 and PEDV was 82.89%, 76.32% and 68.42%, respectively. Interestingly, most of these samples exhibited mixed infections. The co-infection rates of PCV2 and PCV3, PCV2 and PEDV, PCV3 and PEDV were 69.74%, 57.89% and 53.95%, respectively. And the triple infection rate was 48.68%. Furthermore, the genetic characteristics of PCV2 and PCV3 were analyzed based on the cap genes. Two PCV2 genotypes, PCV2b and PCV2d, were circulating in the fields. The cap gene of PCV2b and PCV2d isolates only shared 94.6%-95.0% nucleotide identities. The PCV3 isolates together with the reference strains could be divided into four clades (clade1-4), and the cap genes of these isolates have 98.6%-100% nucleotide identities to each other. Distinctive amino acid substitutions were also characterized on the cap protein of PCV2 and PCV3 isolates. Our studies provide the new knowledge on the co-infectious status of PCV2, PCV3 and PEDV in China. The results also provide insight into the genetic diversity and molecular epidemiology of PCV2 and PCV3.

Isolation of Three Novel Senecavirus A Strains and Recombination Analysis Among Senecaviruses in China.

Senecavirus A (SVA), an emerging swine picornavirus of swine, is one of the causative agents of vesicular disease which is clinically indistinguishable from foot-and-mouth disease in pigs. Here, 3 cases of vesicular disease were reported which was caused by SVA in November 2018 in Henan, China. Three new SVA strains were identified and conducted a genetically evolutionary analysis. The isolates shared 98.1-99.0% genomic pairwise identity to each other and had the highest similarity, of 98.3-98.7%, with the American strain KS15-01, respectively. Phylogenetic analysis indicated that the Chinese prevalent strains could be clearly divided into cluster 1, cluster 2, and cluster 3. Furthermore, one isolate (HeNNY-1/2018) and two previously reported strains (HB-CH-2016 and SVA/CHN/10/2017) were identified as recombinants using several algorithms. It revealed that the recombination among SVA strains has occurred in China since 2016 or earlier. The findings of studies updated the prevalent status of SVA in China. Besides, the genetic evolution and recombinant events of SVA should be attracted more attentions in the future.