Shaqiu Zhang

Cytokine storms are primarily responsible for the rapid death of ducklings infected with duck hepatitis A virus type 1

Duck hepatitis A virus type 1 (DHAV-1) is one of the most harmful pathogens in the duck industry. The infection of adult ducks with DHAV-1 was previously shown to result in transient cytokine storms in their kidneys. To understand how DHAV-1 infection impacts the host liver, we conducted animal experiments with the virulent CH DHAV-1 strain and the attenuated CH60 commercial vaccine strain. Visual observation and standard hematoxylin and eosin staining were performed to detect pathological damage in the liver, and viral copy numbers and cytokine expression in the liver were evaluated by quantitative PCR. The CH strain (108.4 copies/mg) had higher viral titers than the CH60 strain (104.9 copies/mg) in the liver and caused ecchymotic hemorrhaging on the liver surface. Additionally, livers from ducklings inoculated with the CH strain were significantly infiltrated by numerous red blood cells, accompanied by severe cytokine storms, but similar signs were not observed in the livers of ducklings inoculated with the CH60 strain. In conclusion, the severe cytokine storm caused by the CH strain apparently induces hemorrhagic lesions in the liver, which might be a key factor in the rapid death of ducklings.

Suppression of NF-κB Activity: A Viral Immune Evasion Mechanism

Nuclear factor-κB (NF-κB) is an important transcription factor that induces the expression of antiviral genes and viral genes. NF-κB activation needs the activation of NF-κB upstream molecules, which include receptors, adaptor proteins, NF-κB (IκB) kinases (IKKs), IκBα, and NF-κB dimer p50/p65. To survive, viruses have evolved the capacity to utilize various strategies that inhibit NF-κB activity, including targeting receptors, adaptor proteins, IKKs, IκBα, and p50/p65. To inhibit NF-κB activation, viruses encode several specific NF-κB inhibitors, including NS3/4, 3C and 3C-like proteases, viral deubiquitinating enzymes (DUBs), phosphodegron-like (PDL) motifs, viral protein phosphatase (PPase)-binding proteins, and small hydrophobic (SH) proteins. Finally, we briefly describe the immune evasion mechanism of human immunodeficiency virus 1 (HIV-1) by inhibiting NF-κB activity in productive and latent infections. This paper reviews a viral mechanism of immune evasion that involves the suppression of NF-κB activation to provide new insights into and references for the control and prevention of viral diseases.

Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus

Interferon (IFN)-induced 2′-5′-oligoadenylate synthetase (OAS) proteins exhibit an extensive and efficient antiviral effect against flavivirus infection in mammals and birds. Only the 2′-5′-oligoadenylate synthetase-like (OASL) gene has been identified thus far in birds, except for ostrich, which has both OAS1 and OASL genes. In this study, we first investigated the antiviral activity of goose OASL (goOASL) protein against a duck-origin Tembusu virus (DTMUV) in duck embryo fibroblast cells (DEFs). To investigate the relationship of conserved amino acids that are related to OAS enzyme activity and ubiquitin-like (UBL) domains with the antiviral activity of goOASL, a series of mutant goOASL plasmids was constructed, including goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T. Interestingly, all these mutant proteins significantly inhibited the replication of DTMUV in DEFs in a dose-dependent manner. Immunofluorescence analysis showed that the goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T proteins were located not only in the cytoplasm where DTMUV replicates but also in the nucleus of DEFs. However, the goOASL and goOASL mutant proteins were mainly colocalized with DTMUV in the cytoplasm of infected cells. Our data indicated that goOASL could significantly inhibit DTMUV replication in vitro, while the active-site residues S64, D76, D78 and D144, which were associated with OAS enzyme activity, the UBL domains were not required for the antiviral activity of goOASL protein.

Regulated delayed attenuation enhances the immunogenicity and protection provided by recombinant Salmonella enterica serovar Typhimurium vaccines expressing serovar Choleraesuis O-polysaccharides

Regulated delayed attenuation is a well-studied strategy for retaining the immunogenicity of Salmonella-vectored vaccines. In this study, this strategy was used to optimize two previously constructed recombinant Salmonella enterica serovar Typhimurium vaccines expressing S. Choleraesuis O-polysaccharides (OPS). The novel vaccine strains SLT31 (Δasd ΔrmlB-rfbP ΔPcrp::T araC PBAD) and SLT33 (Δasd ΔrfbP ΔpagL::T araC PBADrfbP ΔPcrp::T araC PBAD) were constructed by replacement of the native crp promoter with the arabinose-dependent araC PBAD promoter. As controls, two vaccine strains with direct crp mutations were also constructed, namely, SLT30 (Δasd ΔrmlB-rfbP Δcrp) and SLT32 (Δasd ΔrfbP ΔpagL::T araC PBADrfbP Δcrp). Then, the ability to deliver the heterologous S. Choleraesuis OPS on the Asd+ plasmid pCZ1 to the mouse immune system was evaluated in the strains with or without regulated delayed attenuation. The SLT30 (pCZ1) and SLT31 (pCZ1) strains expressed only the heterologous OPS, while the SLT32 (pCZ1) and SLT33 (pCZ1) strains co-expressed the homologous and heterologous OPS. The strain SLT31 (pCZ1) or SLT33 (pCZ1), which exhibited regulated delayed attenuation, colonized mouse tissues significantly better and stimulated stronger antibody responses against S. Choleraesuis LPS post immunization than the SLT30 (pCZ1) or SLT32 (pCZ1) strain. Immunization with SLT31 (pCZ1) or SLT33 (pCZ1) resulted in a significant reduction in bacterial loads in mouse tissues and a greater degree of protection against a lethal S. Choleraesuis dose compared with the effects observed after SLT30 (pCZ1) or SLT32 (pCZ1) immunization (100% vs. 80% or 70% vs. 50%, respectively). In addition, all four vaccines conferred complete protection against S. Typhimurium challenge. Overall, our study demonstrates that regulated delayed attenuation via an araC PBAD-regulated crp gene can enhance the cross-protection by Salmonella-vectored vaccines expressing heterologous OPS, and strain SLT31 (pCZ1) is a good candidate vaccine for preventing both S. Typhimurium and S. Choleraesuis infections.

Duck plague virus Glycoprotein J is functional but slightly impaired in viral replication and cell-to-cell spread

To analyse the function of the duck plague virus (DPV) glycoprotein J homologue (gJ), two different mutated viruses, a gJ deleted mutant ΔgJ and a gJR rescue mutant gJR with US5 restored were generated. All recombinant viruses were constructed by using two-step of RED recombination system implemented on the duck plague virus Chinese virulent strain (DPV CHv) genome cloned into a bacterial artificial chromosome. DPV-mutants were characterized on non-complementing DEF cells compared with parental virus. Viral replication kinetics of intracellular and extracellular viruses revealed that the ΔgJ virus produce a 10-fold reduction of viral titers than the gJR and parental virus, which especially the production of extracellular infectivity was affected. In addition, the ΔgJ virus produced viral plaques on DEF cells that was on average approximately 11% smaller than those produced by the gJR and parental viruses. Electron microscopy confirmed that although DPV CHv without gJ could efficiently carry out viral replication, virion assembly and envelopment within infected cells, the ΔgJ virus produced and accumulated high levels of anuclear particles in the nuclear and cytoplasm. These results show that the gJ slightly impaired in viral replication, virion assembly and cell-to-cell spread, and is not essential in virion envelopment.

Author Correction: Duck plague virus Glycoprotein J is functional but slightly impaired in viral replication and cell-to-cell spread

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Analysis of the microRNA expression profiles in DEF cells infected with duck Tembusu virus

Duck Tembusu virus (DTMUV), belonging to the Flaviviridae family, is a single-stranded positive-sense RNA virus. Since April 2010, the outbreak of DTMUV in southeast provinces of China has caused great economic losses. MicroRNAs (miRNAs) play important regulatory roles in viral infection through binding to the host target genes or the viral genomes. To better understanding the molecular mechanisms of virus-host interaction, here we identified the miRNA expression profiles in DTMUV-infected and uninfected DEF cells by high-throughput sequencing. A total of 287 known and 63 novel miRNAs were identified. 48 miRNAs, including 26 known miRNAs and 22 novel miRNAs, were differentially expressed in response to DTMUV infection. Among these miRNAs, 37 miRNAs were up-regulated and 11 miRNAs were down-regulated. 9 miRNAs were randomly selected for validation by qRT-PCR experiment. The results of qRT-PCR experiment were consistent with the sequencing data. GO enrichment showed that the predicted targets of these differentially expressed miRNAs were mainly involved in the regulation of immune system, cellular process and metabolic process. KEGG pathways analysis showed that predicted target genes were involved in several signaling pathways such as Wnt signaling pathway, TGF-beta signaling pathway, mTOR signaling pathway and FoxO signaling pathway. This is the first study to evaluate changes of miRNA expression in DEF cells upon DTMUV infection. Our findings provide important clues for better understanding the DTMUV-host interaction.

Transcriptomic Characterization of a Chicken Embryo Model Infected With Duck Hepatitis A Virus Type 1

Duck hepatitis A virus type 1 (DHAV-1) is one of the most common and lethal pathogens in young ducklings. Live-attenuated DHAV vaccine (CH60 strain) developed by passaging in chicken embryos provided effective immune protection for ducklings. However, the accurate mechanism for such adaption in chicken embryos is not fully revealed. Here, we utilize RNA-sequencing to perform global transcriptional analysis of DHAV-1-innoculated embryonated livers along with histopathological and ultrastructural analysis. This study revealed that infection with DHAV-1 strain CH60 is associated with enhanced type I and II interferon responses, activated innate immune responses, elevated levels of suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3) accompanied with abnormalities in multiple metabolic pathways. Excessive inflammatory and innate immune responses induced by the CH60 strain are related to severe liver damage. Our study presents a comprehensive characterization of the transcriptome of chicken embryos infected with DHAV-CH60 and provides insight for in-depth exploration of viral adaption and virus–host interactions.

Incompatible Translation Drives a Convergent Evolution and Viral Attenuation During the Development of Live Attenuated Vaccine

Live attenuated vaccines are widely used to protect humans or animals from pathogen infections. We have previously developed a chicken embryo-attenuated Duck Hepatitis A Virus genotype 1 (DHAV-1) vaccine (CH60 strain). This study aims to understand the mechanisms that drive a virulent strain to an attenuated virus. Here, we systematically compared five DHAV-1 chicken embryo attenuated strains and 68 virulent strains. Phylogenetic analysis indicated that duck virulent strains isolated from different geographic regions of China undergo a convergent evolution in the chicken embryos. Comparative analysis indicated that the codon usage bias of the attenuated strains were shaped by chicken codons usage bias, which essentially contributed to viral adaption in the unsuitable host driven by incompatible translation. Of note, the missense mutations in coding region and mutations in untranslated regions may also contribute to viral attenuation of DHAV-1 to some extent. Importantly, we have experimentally confirmed that the expression levels of four viral proteins (2A3pro, 2A3pro, 3Cpro, and 3Dpro) in the liver and kidney of ducks infected with an attenuated strain are significantly lower than that infected with a virulent strain, despite with similar virus load. Thus, the key mechanisms of viral attenuation revealed by this study may lead to innovative and easy approaches in designing live attenuated vaccines.

Cas1 and Cas2 From the Type II-C CRISPR-Cas System of Riemerella anatipestifer Are Required for Spacer Acquisition

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide acquired genetic immunity against the entry of mobile genetic elements (MGEs). The immune defense provided by various subtypes of the CRISPR-Cas system has been confirmed and is closely associated with the formation of immunological memory in CRISPR arrays, called CRISPR adaptation or spacer acquisition. However, whether type II-C CRISPR-Cas systems are also involved in spacer acquisition remains largely unknown. This study explores and provides some definitive evidence regarding spacer acquisition of the type II-C CRISPR-Cas system from Riemerella anatipestifer (RA) CH-2 (RA-CH-2). Firstly, introducing an exogenous plasmid into RA-CH-2 triggered spacer acquisition of RA CRISPR-Cas system, and the acquisition of new spacers led to plasmid instability in RA-CH-2. Furthermore, deletion of cas1 or cas2 of RA-CH-2 abrogated spacer acquisition and subsequently stabilized the exogenous plasmid, suggesting that both Cas1 and Cas2 are required for spacer acquisition of RA-CH-2 CRISPR-Cas system, consistent with the reported role of Cas1 and Cas2 in type I-E and II-A systems. Finally, assays for studying Cas1 nuclease activity and the interaction of Cas1 with Cas2 contributed to a better understanding of the adaptation mechanism of RA CRISPR-Cas system. This is the first experimental identification of the naïve adaptation of type II-C CRISPR-Cas system.