Virus Genes

Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-β production by impairing dsRNA and PACT binding to RIG-I Abstract Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have suggested that PDCoV infection antagonizes RIG-I-like receptor (RLR)-mediated IFN-β production to evade host innate immune defense, and PDCoV-encoded nonstructural protein nsp5 and accessory protein NS6 are associated with this process. However, whether the structural protein(s) of PDCoV also antagonize IFN-β production remains unclear. In this study, we found that PDCoV nucleocapsid (N) protein, the most abundant viral structural protein, suppressed Sendai virus (SEV)-induced IFN-β production and transcription factor IRF3 activation, but did not block IFN-β production induced by overexpressing RIG-I/MDA5. Furthermore, study revealed that PDCoV N protein interacted with RIG-I and MDA5 in an in vitro overexpression system and evident interactions between N protein and RIG-I could be detected in the context of PDCoV infection, which interfered with the binding of dsRNA and protein activator of protein kinase R (PACT) to RIG-I. Together, our results demonstrate that PDCoV N protein is an IFN antagonist and utilizes diverse strategies to attenuate RIG-I recognition and activation.

Characterization and genome analysis of a novel bacteriophage vB_SpuP_Spp16 that infects Salmonella enterica serovar pullorum Abstract A novel virulent bacteriophage vB_SpuP_Spp16 (hereafter designated Spp16) that infects Salmonella enterica serovar pullorum was isolated. Transmission electron microscopy showed that Spp16 possessed an isometric polyhedral head (60 nm in diameter) and a short tail (10 nm in length) belonging to the family Podoviridae. Its complete genome was determined to be 41,832 bp, with a 39.46% GC content by next-generation sequencing. The genome contains 53 proposed open reading frames that are involved in DNA replication and modification, transcriptional regulation, phage structural and packaging proteins and bacterial lysis. No transfer RNA genes were identified. The termini of genome were determined using our previously proposed termini identification method, which suggests that this phage has redundant termini with 421 bp direct terminal repeats. BLASTn analysis revealed the highest sequence similarity with Yersinia phage phi80-18, with a genome coverage of 33% and highest sequence identity of 69%. The phylogenetic analysis indicated that Spp16 forms a distinct branch of the subfamily Autographivirinae. Comparative genomics analysis showed that the phage Spp16 should be regarded as a new subcluster within the GAP227-like cluster in the Autographivirinae subfamily. The phage Spp16 has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that Spp16 may be a proper candidate to control diseases caused by Salmonella enterica serovar pullorum.

R7A mutation in N protein renders temperature sensitive phenotype of VSV by affecting its replication and transcription in vitro Abstract Viral genomic RNA encapsidated by nucleoprotein (N) forms functional template for the transcription and replication of vesicular stomatitis virus (VSV). The crystal structure of the N-RNA complex shows that RNA is tightly sequestered between the two lobes of the N protein. The residue (R7) in N-terminal arm of N is of great importance to the formation of functional N-RNA template. In our study, we found that single amino acid substitution (R7A) resulted in the loss of CAT expression in vitro minigenome system at 37 °C. But the R7A had little effect on CAT expression at 31 °C. Further analysis showed that R7A had great effects on the RNA synthesis and the formation of cytoplasmic inclusions of VSV only at 37 °C not at 31 °C. For the further investigation of the effect of R7A on virus replication, we checked the dominant-negative effect of NR7A in minigenome system and the single step curve of recombinant virus with R7A mutation in N protein (rVSVR7A) under 37 °C and 31 °C separately. Our results showed that the mutation of R7A within the N-terminal arm of N affected both replication and transcription and induced VSV to become temperature sensitive.

Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples Abstract Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Characterization of a new bunyavirus and its derived small RNAs in the brown citrus aphid, Aphis citricidus Abstract High-throughput sequencing is widely used for virus discovery, and many RNA viruses have been discovered and identified. A new negative-sense single-stranded RNA virus was identified in the brown citrus aphid and named Aphis citricidus bunyavirus. The genome consists of large (7037 nt), medium (3462 nt), and small (1163 nt) segments. Phylogenetic analysis and amino acid sequences identities of this virus with other bunyaviruses suggest that it is a new species belonging to the family Phenuiviridae. The small interfering RNA pathway could be involved against the infection of this virus in brown citrus aphid as supported by the viral derived small RNAs. The discovery of this virus illustrates the diversity of RNA viruses and contributes to the classification of bunyaviruses.

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2 Abstract Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3′ UTR cDNA of JEV for authentic 3′ UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

Variability in the 3′ untranslated regions of the genomes of the different tick-borne encephalitis virus subtypes Abstract Tick-borne encephalitis viruses (TBEVs) are usually divided into three major subtypes: European (TBEV-Eu), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). The TBEV-Eu strains have the longest genomes, and TBEV-FE strains have the smallest genomes. Changes in the variable region of the untranslated region (V3′ UTR) play a major role in determining the viral genome length. Analyses of the 3′ UTRs of the different subtypes of TBEV have revealed significant changes in the secondary structures of the V3′ UTR of TBEV. More complex secondary structures of the V3′ UTR regions are typical for TBEV-Eu. The Siberian strain Tomsk-PT122 was isolated from birds and has an unusual 3′ UTR. Several short fragment (24–26 nucleotides) insertions derived from the viral E (2) and NS4a (1) genes have been found in the V3′ UTR of Tomsk-PT122. Additionally, the length of the V3′ UTR increases from 21 to 37 nucleotides during passages of the C11-13 strain of TBEV-Sib into PEK, 293 and Neuro-2a cells. The elongation of the V3′ UTRs of Tomsk-PT122 and C11-13 is the first direct evidence of an intragenomic 3′ UTR modification (insertion) for TBEV. Thus, the obtained results suggest that changing the length of the V3′ UTR in the genome is typical for different TBEV subtypes and can play an essential role in effective TBEV replication in different host cells.

Signaling pathways involved in regulating apoptosis induction in host cells upon PRRSV infection Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of porcine reproductive and respiratory syndrome (PRRS), a devastating disease of swine that poses a serious threat to the swine industry worldwide. The induction of apoptosis in host cells is suggested to be the key cellular mechanism that contributes to the pathogenesis of PRRS. Various signaling pathways have been identified to be involved in regulating PRRSV-induced apoptosis. In this review, we summarize the potential signaling pathways that contribute to PRRSV-induced apoptosis, and propose the issues that need to be addressed in future studies for a better understanding of the molecular basis underlying the pathogenesis of PRRS.

Presence of Newcastle disease viruses of sub-genotypes Vc and VIn in backyard chickens and in apparently healthy wild birds from Mexico in 2017 Abstract Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99–100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3–98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000–2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated.

Full-length genome analysis of the first human G8P[14] rotavirus strain from Morocco suggests evidence of zoonotic transmission Abstract An unusual group A rotavirus (RVA) strain MAR/ma31/2011/G8P[14] was detected for the first time in Morocco in a stool sample from hospitalized child aged 18 months suffering from acute gastroenteritis and fever in 2011. Complete genome sequencing of the ma31 strain was done using the capillary sequencing technology. The analysis revealed the G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 constellation and the backbone genes: I2-R2-C2-M2-A11-N2-T6-E2-H3 are commonly found in RVA strains from artiodactyls such as cattle. The constellation was shared with another Italian zoonotic G8P[14] strains (BA01 and BA02), two Hungarian human strains (182-02 and BP1062) and a sheep RVA strain OVR762. Phylogenetic analysis of each genome segment of ma31 revealed a mixed gene configuration originated from animals and human. Comparison of the antigenic regions of VP7 and VP4 amino acid sequences between ma31 strain and selected animal and human strains bearing G8 and or P[14], showed a high level of conservation, while many substitutions was observed in comparison with RotaTeq™ and Rotarix™ vaccine strains. In contrast, alignment analysis of the four antigenic sites of VP6 revealed a high degree of conservation. These findings reveal a typical zoonotic origin of the strain and confirm a high potential for RVA zoonotic transmission between bovine and humans, allowing the generation of novel rotavirus genotypes.