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MicroRNAs (miRNAs) contribute to gene regulation at the post-transcriptional level and are capable of mRNA silencing by binding to target sites exhibiting high degrees of complementarity. Therefore, cloning host miRNA-recognition sequences into the genome of RNA viruses represents a rational strategy for manipulating viral replication. Here, we performed deep sequencing to obtain small-RNA (sRNA)-expression profiles from in vitro-cultured MARC-145 cells post infection with porcine reproductive and respiratory syndrome virus (PRRSV) and chose six candidate miRNAs of different abundance (miR-21, miR-140-3p, miR-185, miR-26a, miR-505, and miR-199a) for further study. Based on the full-length cDNA clone p7USC, we constructed a number of PRRSV mutants that provided complementary base-pairing target sites for the miRNAs in 3′ untranslated regions. Our results showed that all low- and moderate- abundant miRNA-target mutants showed similar growth properties, whereas the highest-abundant miRNA-target mutant blocked both viral transcription and replication. Discontinuous mutations in high-abundant miRNA-target sites subsequently recovered viral viability and propagation. These results demonstrated the copy number of endogenous miRNAs and the extent of sRNA complementarity were key factors to silence potential mRNA expression/translation, thereby determining PRRSV viability. Interestingly, the mutant containing miR-140-target sites (v140-t) showed strong suppression of viral replication from P1 to P3 in vitro, as shown by virus titer, plaque morphology, and qRT-PCR assays. To assess genetic stability, sequencing of v140-t (P1, P3, P5 and P10) revealed spontaneous mutations preferentially located among several nucleotides near the 3′ end of the insertion region and corresponding to the “seed region” of miR-140-3p, explaining the induced viral repression and the direction of virus evolution. This approach provided a general silencing strategy for limiting PRRSV replication by endogenous miRNAs in MARC-145 cells. [ABSTRACT FROM AUTHOR]

Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1. [ABSTRACT FROM AUTHOR]

Abstract In recent years, the outbreaks of porcine epidemic diarrhea (PED) caused by the highly virulent porcine epidemic diarrhea virus (PEDV) variants occurred frequently in China, resulting in severe economic impacts to the pork industry. In this study, we selected and analyzed the genetic evolution of 15 PEDV representative strains that were identified in fecal samples of diarrheic piglets in 10 provinces and cities during 2011–2017. The phylogenetic analysis indicated that all the 15 PEDV isolates clustered into G2 genotype associated with the current circulating strains. Compared with the genome of the prototype strain CV777, these strains had 103–120 amino acid mutations in their S proteins, most of which were in the N terminal domain of S1 (S1-NTD). We also found 37 common mutations in all these 15 strains, although these strains shared 96.9–99.7% nucleotide homology and 96.3–99.8% amino acid homology in the S protein compared with the other original pandemic strains. Computational analysis showed that these mutations may lead to remarkable changes in the conformational structure and asparagine (N)-linked glycosylation sites of S1-NTD, which may be associated with the altered pathogenicity of these variant PEDV strains. We evaluated the pathogenicity of the PEDV strain FJzz1 in piglets through oral and intramuscular infection routes. Compared with oral infection, intramuscular infection could also cause typical clinical signs but with a slightly delayed onset, confirming that the variant PEDV isolate FJzz1 was highly pathogenic to suckling piglets. In conclusion, we analyzed the genetic variation and pathogenicity of the emerging PEDV isolates of China, indicating that G2 variant PEDV strains as the main prevalent strains that may mutate continually. This study shows the necessity of monitoring the molecular epidemiology and the etiological characteristics of the epidemic PEDV isolates, which may help better control the PED outbreaks. Highlights • G2 variant PEDV strains as the main prevalent strains continue to mutate along with time. • The isolated strains were prone to mutate in the S protein with many common amino acid mutations. • These mutations may lead to remarkable changes in the conformational structure of S1-NTD and the pathogenicity of PEDV. • Both oral and intramuscular routes caused typical clinical signs to piglets. [ABSTRACT FROM AUTHOR]

Since late 2011, a pseudorabies virus (PRV) variant with increased virulence in old pigs had caused major disease outbreaks and great economic losses to the pig industry in China. The gene mutations that contributed to the increased virulence were unclear. To study the basis of the enhanced pathogenicity, an infectious bacterial artificial chromosome (BAC) clone consisting of the complete genome of the PRV variant was developed. Using homologous recombination and Cre/LoxP recombination, the recombinant virus rJS-2012-BAC carrying a BAC insertion downstream of the open reading frame (ORF) of gG was constructed. The circular genome of rJS-2012-BAC was extracted from infected Vero cells and transformed into Escherichia coli DH10B, generating the BAC clone pBAC-JS2012. The loxP sites flanking the BAC vector were used to excise the BAC sequences using Cre recombinase. The reconstituted BAC-excision virus, vJS2012 L, from pBAC-JS2012 exhibited similar biological properties to the wild-type virulent strain JS-2012. To manipulate the BAC clone pBAC-JS2012, the galK selection system was adopted to delete the gI/gE gene from pBAC-JS2012 in E. coli and to generate the gI/gE-deleted virus vJS2012-ΔgE/gI. The BAC clone, pBAC-JS2012, retained the same level of virulence as its parent strain and was easily manipulated using a galK system which would facilitate the study of the enhanced pathogenicity of the PRV variant as well as other studies on PRV. [ABSTRACT FROM AUTHOR]

Abstract: It has been shown that ORF5a protein in EAV is important but not essential for virus infectivity. In this study, we found that RNA changes in the overlapping region (1–104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for virus viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for virus viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable. [Copyright &y& Elsevier]

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is renowned for its genetic, antigenic, and pathogenic heterogeneity. As a consequence, highly pathogenic PRRSV (HP-PRRSV) has emerged and caused tremendous economic losses in the swine industry. In this study, a Chinese HP-PRRSV JX143 isolate was serially passaged in MARC-145 cells up to 100 times. We found that phenotypic changes involved with the cell adaptation process of PRRSV JX143 were characterized by higher titers, faster growth kinetics, and larger plaque sizes as the passage number increased compared with the parental virus. We found that the virulence of the JX143 strain in piglets was decreased greatly at passage 100 (JXM100). The attenuated strain JXM100 could protect piglets from lethal challenge by HP-PRRSV JX143. Genome-wide analysis showed that JXM100 contained a continuous 264 nucleotide (88 amino acids; 88aa) deletion in the nsp2 region and 75 random nucleotide mutations throughout the genome. The nucleotide changes that arose during MARC-145 passaging of HP-PRRSV JX143 provide a potential molecular basis for the observed cell-adapted phenotype in MARC-145 cells and the attenuated phenotype in vivo. We also showed that pigs inoculated with JXM100 with an 88aa deletion (del88) in nsp2 elicited a strong antibody response against the N protein but they did not develop antibody against the del88, whereas strong reactivity was observed in sera obtained from piglets infected with JX143 using the same del88-based ELISA. This suggests that del88 can be used as a genetic marker for serologically differentiating JXM100 from JX143. [Copyright &y& Elsevier]

Abstract: It has been proposed that the N-linked glycan of the minor proteins of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the production of infectious virus. In this study, we showed that N-linked glycosylation of GP2 is not essential for virus viability and none of the individual glycosylation sites in GP3 has a vital effect on the production of infectious virus. Moreover, mutations of single and double glycosylation sites in GP4 are not critically important for infectious virus recovery, triple and quadruple mutations are lethal. The bimolecular fluorescence complementation (BiFC) analysis also showed that GP4, but might be not GP2, is involved in interaction with cellular receptor CD163 and that glycosylation of GP4 might not play a vital role in the interaction with CD163. The study further revealed that none of the N-glycosylation sites in the minor proteins is critical for the susceptibility of mutants to neutralizing antibody. [Copyright &y& Elsevier]