The host transcriptome change involved in the inhibitory effect of exogenous interferon-γ on Getah virus replication

IntroductionGetah virus (GETV) has become a growing potential threat to the global livestock industry and public health. However, little is known about the viral pathogenesis and immune escape mechanisms, leading to ineffective control measures.MethodsIn this study, the antiviral activity of exogenous interferons (IFNs) was assessed by using western blotting (WB), real-time quantitative PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The comparative transcriptomics among mock- and GETV-infected (MOI = 0.1) ST cells with or without IFN-γ was performed by RNA-seq, and then the transcriptome profiling of GETV-infected ST cells and key pathways and putative factors involved in inhibitory effect of IFN-γ on GETV replication were analyzed by bioinformatics methods and RT-qPCR.ResultsThe results showed that treatment with IFN-γ could suppress GETV replication, and the inhibitory effect lasted for at least 48 h, while the exogenous IFN-α/ω and IFN-λ3 treatments failed to inhibit the viral infection and early replication in vitro. Furthermore, the blueprint of virus-host interaction was plotted by RNA-seq and RT-qPCR, showing systemic activation of inflammatory, apoptotic, and antiviral pathways in response to GETV infection, indicating viral hijacking and inhibition of innate host immunity such as IFN-I/III responses. Last and most importantly, activation of the JAK-STAT signaling pathway and complement and coagulation cascades may be a primary driver for IFN-γ-mediated inhibition of GETV replication.DiscussionThese findings revealed that GETV possessed the capability of viral immune escape and indicated that IFN-γ aided in the prevention and control of GETV, implying the potential molecular mechanism of suppression of GETV by IFN-γ, all of which warrant emphasis or further clarification

Expression and Evaluation of a Novel PPRV Nanoparticle Antigen Based on Ferritin Self-Assembling Technology

Peste des Petits Ruminants (PPR) is a highly pathogenic disease that is classified as a World Organization for Animal Health (OIE)-listed disease. PPRV mainly infects small ruminants such as goats and sheep. In view of the global and high pathogenicity of PPRV, in this study, we proposed a novel nanoparticle vaccine strategy based on ferritin (Fe) self-assembly technology. Using Helicobacter pylori (H. pylori) ferritin as an antigen delivery vector, a PPRV hemagglutinin (H) protein was fused with ferritin and then expressed and purified in both Escherichia coli (E. coli) and silkworm baculovirus expression systems. Subsequently, the nanoparticle antigens’ expression level, immunogenicity and protective immune response were evaluated. Our results showed that the PPRV hemagglutinin–ferritin (H-Fe) protein was self-assembled in silkworms, while it was difficult to observe the correctly folded nanoparticle in E. coli. Meanwhile, the expression level of the H-Fe protein was higher than that of the H protein alone. Furthermore, the immunogenicity and protective immune response of H-Fe nanoparticle antigens expressed by silkworms were improved compared with the H antigen alone. Particularly, the protective immune response of H-Fe antigens expressed in E. coli did not change, as opposed to the H antigen, which was probably due to the incomplete nanoparticle structure in E. coli. This study indicated that the use of ferritin nanoparticles as antigen delivery carriers could increase the expression of antigen proteins and improve the immunogenicity and immune effect of antigens

Genome sequence and organization of the Mythimna (formerly Pseudaletia) unipuncta granulovirus Hawaiian strain

Abstract Purified occlusion bodies (OBs) of Mythimna (formerly Pseudaletia) unipuncta (the true armyworm) granulovirus Hawaiian strain (MyunGV-A) were observed, showing typical GV morphological characteristics under scanning and transmission electron microscopy (EM). The genome of MyunGV-A was completely sequenced and analysed. The genome is 176,677 bp in size, with a G+C content of 39.79%. It contains 183 open reading frames (ORFs) encoding 50 or more amino acids with minimal overlap. Comparison of MyunGV-A with TnGV, XcGV, and HearGV genomes revealed extensive sequence similarity and collinearity, and the four genomes contain the same nine homologous regions (hrs) with conserved structures and locations. Three unique genes, 12 baculovirus repeated ORF (bro), 2 helicase, and 3 enhancin genes, were identified. In particular, two repeated genes (ORF39 and 49) are present in the genome, in reverse and complementarily orientations. Twenty-four OB proteins were identified from the putative protein database of MyunGV-A. In addition, MyunGV-A belongs to the Betabaculovirus group and is most closely related to TnGV (99% amino acid identity) according to a phylogenetic tree based on the combined amino acid sequences of 38 core gene contents

Genomic sequencing and analyses of HearMNPV—a new Multinucleocapsid nucleopolyhedrovirus isolated from <it>Helicoverpa armigera</it>

<p>Abstract</p> <p>Background</p> <p>HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, <it>Helicoverpa armigera</it>, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with <it>H. armigera</it> single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed.</p> <p>Methods</p> <p>The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for <it>H. armigera in vivo</it>. A random genomic library of HearMNPV was constructed according to the “partial filling-in” method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses.</p> <p>Results</p> <p>Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of <it>H. armigera</it>. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of <it>Mamestra configurata</it> NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV <it>orf66, bro</it> genes, and <it>hr</it>s are different to the corresponding parts of the MacoNPV-B genome.</p> <p>Conclusions</p> <p>HearMNPV can replicate <it>in vivo</it> in <it>H. armigera</it> and <it>in vitro</it>, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.</p

Development of a foot-and-mouth disease virus serotype A empty capsid subunit vaccine using silkworm (Bombyx mori) pupae.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID(50)). Furthermore, the 50% bovine protective dose (PD(50)) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD(50) per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases

Homology of Dipteran Bristles and Lepidopteran Scales: Requirement for the Bombyx mori achaete-scute Homologue ASH2

Lepidopteran wing scales and Drosophila bristles are considered homologous structures on the basis of the similarities in their cell lineages. However, the molecular mechanisms underlying scale development are essentially unknown as analysis of gene function in Lepidoptera is sorely limited. In this study, we used the Bombyx mori mutant scaleless (sl), which displays a nearly complete loss of wing scales, to explore the mechanism of lepidopteran wing-scale formation. We found that Bm-ASH2, one of four Bombyx achaete-scute homologs, is highly expressed in early pupal wings of wild-type silkworms, but its expression is severely reduced in sl pupal wings. Through molecular characterization of the mutant locus using luciferase and gel shift assays, genetic analysis of recombining populations, and in vivo rescue experiments, we provide evidence that a 26-bp deletion within the Bm-ASH2 promoter is closely linked to the sl locus and leads to loss of Bm-ASH2 expression and the scaleless-wings phenotype. Thus, the Bm-ASH2 appears to play a critical role in scale formation in B. mori. This finding supports the proposed homology of lepidopteran scales and dipteran bristles and provides evidence for conservation of the genetic pathway in scale/bristle development at the level of gene function

Western blot analyses of PoIFN-γ expression in silkworm larvae.

<p>The PoIFNγ-1 sample is the larval haemolymph infected with one of 24 plaques of recombinant virus stock, and the PoIFNγ-2 sample is the larval haemolymph infected with the recombinant virus acquired during cotransfection. PoIFN-γ proteins were detected in PoIFNγ-1 and PoIFNγ-2 samples as an approximately 19 kDa band. No corresponding immunoreactive protein was detected in wild-type (WT) samples.</p

Overview of the novel reBmBac expression system (refer to Airenne <i>et al</i>.).

<p>In BmBac DNA, the <i>chi</i> and <i>cat</i> genes was replaced by the <i>Copycontrol</i> origin and a <i>chl</i>^R gene. Then the essential <i>ORF1629</i> gene was partially deleted by using <i>tet</i>^R gene. The <i>chl</i>^R gene was removed by using FLP/<i>FRT</i> system. The finally acquired bacmid DNA was reBmBac.</p

Schematic for the generation of recombinant viruses and expression of interested foreign genes.

<p>Recombinant viruses containing foreign genes were generated 4–5 days after cotransfection. The recombinant protein was harvested 4–5 days after silkworm infection.</p