Complete genome sequence of a bovine ephemeral fever virus JT02L strain in mainland China.

In this study, we report the complete genome sequence of bovine ephemeral fever virus (BEFV) JT02L, which has been used in our laboratory, in mainland China, for more than a decade. The genome is 14941 nucleotide (nt), comprising a leader sequence of 50 nt, nucleoprotein (N) gene of 1328 nt, phosphoprotein (P) gene of 858 nt, matrix protein (M) gene of 691 nt, glycoprotein (G) gene of 1897 nt, non-structural glycoprotein (GNS) gene of 1785 nt, α1α2 gene of 638 nt, ß gene of 460 nt, γ gene of 400 nt, large multi-functional enzyme (L) gene of 6470 nt and a trailer sequence of 73 nt. Individual genes are separated by intergenic regions (IGRs) of 26, 44, 47, 51, 37, 39, 68 and -21 nt respectively. The overall organization is similar to an Australian BEFV isolate BB7721 but demonstrates some distinctive features including longer α3 and ß open reading frames, intact termination/polyadenylation (TTP) sequence downstream of the ß open reading frame and a longer ß-γ IGR integrated with a 38 nt AT-rich fragment. To our knowledge, this is the first report describing the complete genome of a BEFV strain of East Asian lineage, which may facilitate studies on genomic diversity among geographic strains of BEFV in China and the world.

A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

We developed a SYBR green I-based reverse-transcription quantitative PCR (RT-qPCR) assay for bovine ephemeral fever virus (BEFV). Analytical sensitivity of the assay was ~ 100 times higher than conventional RT-PCR. The precision of the RT-qPCR established for RNA standards was high, with intra-assay and inter-assay coefficients of variation of 0.23-0.89% and 0.23-1.02%, respectively. The test was highly specific for BEFV strains, with no cross-reactivity with other viruses of veterinary significance. The assay detected BEFV RNA as early as 1 d post-infection (dpi) and up to 7-8 dpi in the blood samples of experimentally infected cattle. The most stable reference gene, peptidylprolyl isomerase A (PPIA), was selected for the quantification of BEFV. Viral RNA loads reached peak level at 3-5 dpi and then decreased rapidly through 7-8 dpi. Our assay provides a reliable approach for the detection of BEFV in the early infection stage and for use in the profiling of BEFV kinetics in vivo.

Ovine viperin inhibits bluetongue virus replication.

Viral infections can lead to interferon production, which achieves its antiviral function primarily by activating the JAK/STAT pathway and inducing multiple interferon-stimulated genes (ISGs). Although considerable ISGs have been identified in antiviral researches, little is known about ISGs in bluetongue virus (BTV) infection. Viperin is the most highly induced ISG following BTV infection, which suggests that it may play a critical role in the anti-BTV immune response. The aim of this study was to characterize ovine Viperin (oViperin) and explore whether it can inhibit BTV replication. We cloned the coding sequences (CDS) of sheep Viperin, and the sequence analysis showed that oViperin displayed a high similarity with other species. oViperin has a leucine zipper in the N-terminal, a CxxxCxxC motif in the SAM domain, and a conservative C-terminus. We found that oViperin mRNA expression was significantly up-regulated in a time- and multiplicity of infection (MOI)-dependent manner following BTV infection. oViperin overexpression resulted in a significant inhibition in BTV replication, whereas an oViperin knockdown in MDOK cells increased BTV replication. This study shows for the first time, that oViperin has antiviral activity towards BTV infection and provides important information to research the interaction between BTV and oViperin.

Evaluation of the immune response afforded by a subunit vaccine candidate against bluetongue virus in mice and sheep.

Bluetongue virus (BTV), a vector-borne pathogen, is the causative agent of bluetongue disease in ruminants. In view of the recent emergence of BTV in regions previously known to be free from the disease and/or specific serotypes or strains, optimization of the currently available vaccination strategies to control the spread of vector-borne bluetongue is crucial. The main objective of the current study was to develop a subunit vaccine candidate targeting BTV-16, a strain previously isolated in China from sheep with obvious clinical signs. To this end, five polyhistidine-tagged recombinant proteins (BTV-16 VP2, VP3, VP7, NS2 and a truncated version of VP5 (VP5-41amino acids) were expressed using the baculovirus or Escherichia coli expression system for characterization of protective activity. To determine ovine and murine immune responses to the five proteins, sheep and mice were immunized twice at 4- and 2-week intervals, respectively, with one of two different protein combinations in MontanideTM ISA201 VG adjuvant or placebo. Data from the competitive enzyme linked immunosorbent assay revealed significantly higher antibody titers in immunized than control animals. Expressed VP5 and NS2 induced a protein-specific humoral response. Interestingly, a serum neutralization test against the BTV-1 serotype showed promising cross-serotype immune response by the vaccine. Based on the collective data, we suggest that these recombinant purified proteins present promising candidates for the design of effective novel vaccines against BTV.

MicroRNA expression profiling of primary sheep testicular cells in response to bluetongue virus infection.

Bluetongue virus (BTV) is a member of the genus Orbivirus within the family Reoviridae and causes a non-contagious, insect-transmitted disease in domestic and wild ruminants, mainly in sheep and occasionally in cattle and some species of deer. Virus infection can trigger the changes of the cellular microRNA (miRNA) expression profile, which play important post-transcriptional regulatory roles in gene expression and can greatly influence viral replication and pathogenesis. Here, we employed deep sequencing technology to determine which cellular miRNAs were differentially expressed in primary sheep testicular (ST) cells infected with BTV. A total of 25 known miRNAs and 240 novel miRNA candidates that were differentially expressed in BTV-infected and uninfected ST cells were identified, and 251 and 8428 predicted target genes were annotated, respectively. Nine differentially expressed miRNAs and their mRNA targets were validated by quantitative reverse transcription-polymerase chain reaction. Targets prediction and functional analysis of these regulated miRNAs revealed significant enrichment for several signaling pathways including MAPK, PI3K-Akt, endocytosis, Hippo, NF-kB, viral carcinogenesis, FoxO, and JAK-STAT signaling pathways. This study provides a valuable basis for further investigation on the roles of miRNAs in BTV replication and pathogenesis.