STAT3 regulates antiviral immunity by suppressing excessive interferon signaling.

This study identifies interleukin-6 (IL-6)-independent phosphorylation of STAT3 Y705 at the early stage of infection with several viruses, including influenza A virus (IAV). Such activation of STAT3 is dependent on the retinoic acid-induced gene I/mitochondrial antiviral-signaling protein/spleen tyrosine kinase (RIG-I/MAVS/Syk) axis and critical for antiviral immunity. We generate STAT3Y705F/+ knockin mice that display a remarkably suppressed antiviral response to IAV infection, as evidenced by impaired expression of several antiviral genes, severe lung tissue injury, and poor survival compared with wild-type animals. Mechanistically, STAT3 Y705 phosphorylation restrains IAV pathogenesis by repressing excessive production of interferons (IFNs). Blocking phosphorylation significantly augments the expression of type I and III IFNs, potentiating the virulence of IAV in mice. Importantly, knockout of IFNAR1 or IFNLR1 in STAT3Y705F/+ mice protects the animals from lung injury and reduces viral load. The results indicate that activation of STAT3 by Y705 phosphorylation is vital for establishment of effective antiviral immunity by suppressing excessive IFN signaling induced by viral infection.

Tembusu virus infection in laying chickens: Evidence for a distinct genetic cluster with significant antigenic variation.

Tembusu virus (TMUV) associated disease is a growing cause of egg production decrease and encephalitis in domestic waterfowl, with expanding distribution. In previous studies, TMUV isolates were phylogenetically classified into two genetic lineages and different clusters with varied pathogenicity. However, little is known about the phenotypic and virulence characteristics of cluster 3 isolates within the duck TMUV lineage. In this study, the etiological agent causing egg drop in a laying chicken farm in southern China was investigated and a TMUV was isolated from pooled tissue samples. Genome sequencing and phylogenetic analysis grouped the isolate into TMUV cluster 3 with closest relation to the mosquito-origin TMUV YN12193. Cross-neutralization testing using convalescent sera revealed significant antigenic variation between the isolate and a representative strain of cluster 2.2. The experimental infection of SPF hens confirmed the ability of the isolate to replicate in multiple tissues and led to ovary damage. Additionally, high seroconversion rates (95.83%-100%) were detected in the three flocks following retrospective investigation. Our study demonstrates the occurrence of cluster 3 TMUV infection in laying chickens and that the virus exhibits significant antigenic variation compared with cluster 2 TMUV.

Amino Acid Substitutions in NS5 Contribute Differentially to Tembusu Virus Attenuation in Ducklings and Cell Cultures.

Tembusu virus (TMUV), a highly infectious pathogenic flavivirus, causes severe egg-drop and encephalitis in domestic waterfowl, while the determinants responsible for viral pathogenicity are largely unknown. In our previous studies, virulent strain JXSP2-4 had been completely attenuated by successive passages in BHK-21 cells and the avirulent strain was designated as JXSP-310. Based on the backbone of JXSP2-4, a series of chimeric viruses were generated according to the amino acid substitutions in NS5 and their infectivities were also analyzed in cell cultures and ducklings. The results showed that the viral titers of RNA-dependent RNA polymerase (RdRp) domain-swapped cheimeric mutant (JXSP-310RdRp) in cells and ducklings were both markedly decreased compared with JXSP2-4, indicating that mutations in the RdRp domain affected viral replication. There are R543K and V711A two amino acid substitutions in the RdRp domain. Further site-directed mutagenesis showed that single-point R543K mutant (JXSP-R543K) exhibited similar replication efficacy compared with JXSP2-4 in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP2-4 and higher than JXSP-310RdRp. Surprisingly, the single-point V711A mutation we introduced rapidly reverted. In addition, qRT-PCR and Western blot confirmed that the mutations in the RdRp domain significantly affected the replication of the virus. Taken together, these results show that R543K substitution in the RdRp domain impairs the in vivo growth of TMUV, but sustaining its attenuated infectivity requires the concurrent presence of the V711A mutation.

Critical role of Syk-dependent STAT1 activation in innate antiviral immunity.

The JAK/STAT1 pathway is generally activated by cytokines, providing essential antiviral defense. Here, we identify that STAT1 activation is independent of cytokines and JAKs at the early infection stage of some viruses, including influenza A virus (IAV). Instead, STAT1 is activated mainly through spleen tyrosine kinase (Syk) downstream of retinoic acid-inducible gene-I/mitochondrial antiviral-signaling protein (RIG-I/MAVS) signaling. Syk deletion profoundly impairs immediate innate immunity, as evidenced by the finding that Syk deletion attenuates tyrosine phosphorylation of STAT1 and reduces the expressions of interferon-stimulated genes (ISGs) in vitro and in vivo. The antiviral response to IAV infection is also significantly suppressed in the STAT1Y701F knockin mice. The results demonstrate that STAT1 activation is dependent on Syk rather than the cytokine-activated JAK signaling at the early stage of viral infection, which is critical for initial antiviral immunity. Our finding provides insights into the complicated mechanisms underlying host immune responses to viral infection.

Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis.

Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV.IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Senecavirus A evolution.

Senecavirus A (SVA) infection was recently confirmed in pigs in Brazil, United States of America and Canada. To better understand the molecular characteristics of isolated SVA genomes, we first reported genome-wide comprehensive analyses of codon usage and various factors that have contribute to the molecular evolution in SVA. The effective number of codons (ENC) ranged from 54.51 to 55.54 with an average of 54.87 ± 0.285, which reveals a relatively stable nucleotide composition. We found that codon usage bias of the SVA was low. Mutational pressure acted as an increasingly dominant factor for the evolution of the virus compared with the natural selection. Notably, codon usage bias was also affected by the geographic distribution and isolated time. The first systemic analysis on the codon usage bias of the SVA provides important information for the understanding of the evolution of the SVA and has fundamental and theoretical benefits.

Role of Non-coding RNAs in Interactions between Host and Influenza Virus.

Non-coding RNAs (ncRNAs) are a class of RNAs that have no potential for protein coding. Increasing numbers of studies have provided strong evidence that ncRNAs play important roles in regulation of various biologic processes, including interactions between viruses and the host. Influenza viruses remain a major, threat to human health and animal livestock. Interactions between the host and mutations of influenza viruses are very complicated. Recent data have shown that many ncRNAs play important roles in the interactions between influenza viruses and the host. Understanding the fuiction of these ncRNAs in the infection and replication of influenza viruses is very important to elucidate the pathogenesis of these viruses, and to provide strategies for the prevention and control of influenza. This review summarizes the ncRNAs that act as key regulators of interactions between the host and influenza viruses.