Search

Quantum dots (QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time. The porcine reproductive and respiratory syndrome virus (PRRSV) is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide. A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies. In this study, we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells. Our results indicated that the labeling method had negligible effect on viral infectivity. We also observed that prior to the entry, PRRSV vibrated on the plasma membrane, and entered the cells via endosome mediated cell entry pathway. Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell. Our results also showed that once inside the cell, PRRSV moved along the microtubule, microfilament and vimentin cytoskeletal elements. During the transport process, virus particles also made contacts with non-muscle myosin heavy chain II-A (NMHC II-A), visualized as small spheres in cytoplasm. This study can facilitate the application of QDs in virus infection imaging, especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00187-0) contains supplementary material, which is available to authorized users.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the swine industry worldwide. miRNAs are crucial regulators of gene expression and a wide range of complex interactions of miRNAs-mRNAs is possible during virus infection. However, there is no comprehensive integrated study of miRNA and mRNA networks in MARC-145 cells after infection with PRRSV. We analyzed the differential expressions, co-relations, annotations, and putative functions of miRNA and mRNA networks in PRRSV-infected MARC-145 cells. Based on the filtering criterion, 22 differentially expressed miRNAs (DEmiRs) (15 up- and 7 downregulated) were filtered out. miRNA-mRNA interaction networks were constructed. For the 18 selected miRNAs, 390 potential target genes were predicted from the differentially expressed mRNAs (DEmRs). GO and KEGG pathway annotations predicted 34 KEGG pathways, 12 of which are known to be involved in virus infection. Real-time PCR validated the RNA-seq results. Our analysis showed that miR-27a-5p and miR-21-3p downregulate the expression of two of their potential target genes-SPARC, CLIC1, and cofilin-1, COX7A2, respectively. Further experiments proved that miR-21-3p and miR-27a-5p can promote PRRSV replication significantly. It is the first report that these two miRNAs participate in the interaction of host cells with PRRSV. Our results provide insights into the role of miRNAs in response to PRRSV infection, which will aid the research for developing novel therapies against PRRSV.

Clostera anastomosis (L.) granulovirus (CaLGV) and Clostera anachoreta granulovirus (ClanGV) are both capable of infecting each other's native host insects. Despite this, we have little information on their genetic relationship. The complete nucleotide sequence of CaLGV was determined and compared with that of the genome of ClanGV. The circular, double-stranded DNA CaLGV genome (GenBank accession no. KC179784) had a G+C content of 46.7 % and was 101,818 bp in size (331 bp larger than the ClanGV genome). Overall, the CaLGV nucleotide sequence was found to be 90 % identical to that of ClanGV. It contained a total of 123 ORFs, 119 of which had ClanGV homologues, with an identical transcription direction and ORF organization. Seventy-five of the 119 ORFs showed 90 % or greater identity to their ClanGV homologues. CaLGV contained only a single identifiable homologous region (hrs)/repeat region (similar to ClanGV hr4). The mean frequency of nucleotide substitutions in the CaLGV/ClanGV coding regions was 8.33 %. CaLGV contained four unique ORFs (CaL23, CaL39, CaL48 and CaL92). Eight ORFs found in both CaLGV and ClanGV have no homologues in other baculoviruses. Intergenic regions of CaLGV and ClanGV occupied 6.6 % and 7 % of their respective genomes. CaLGV appears closer phylogenetically to ClanGV than to any other baculoviruses.

The complete genome of Clostera anachoreta granulovirus (ClanGV) from an important pest of poplar, Clostera anachoreta (Lepidoptera: Notodontidae), was sequenced and analyzed. The circular double-stranded genome is 101,487 bp in size with a C+G content of 44.4%. It is predicted to contain 123 open reading frames (ORFs), covering 93% of the whole genome sequence. One hundred eleven ClanGV ORFs are homologues of previously reported baculovirus genes, two ORFs only exist in nucleopolyhedroviruses (NPVs), and 10 ORFs are unique to ClanGV, accounting for 3.9% of the genome in total. The average amino acid sequence identities between ClanGV and other granulovirus (GV) homologues ranged from 55.5% (PiraGV) to 43.1% (HearGV and PsunGV). ClanGV includes 16 DNA replication- and transcription-related genes, including ie-1, dnapol, helicase, lef-1, lef-2, lef-3, lef-4, lef-5, lef-6, lef-8, lef-9, lef-10, lef-11, 39K, p47 and vlf-1. Seventeen conserved structural genes and two types of apoptosis suppression proteins (P35 and IAP) have also been identified. To date, ClanGV is the second identified GV that contains the p35 gene, which is present in eight NPVs and ChocGV. Genes of chitinase and cathepsin, which are involved in the liquefaction of the host, were found in the ClanGV genome, consistent with the typical insecticidal properties of ClanGV. In the ClanGV genome, there are four homologous repeat regions, each one of which contains only 2-3 direct repeats. Phylogenetic analysis, based on the 29 core baculovirus genes, indicates that ClanGV is closely related to PhopGV, AdorGV, CpGV, CrleGV, PiraGV and ChocGV, and this agrees with the results of homology analysis and gene parity plot analysis.

The BamHI-C restriction fragment of ClanGV genome was cloned and sequenced. Sequence analysis indicated that this region contains five important baculovirus homologous genes (chitinase, cathepsin, gp37, p49 and odv-e18) and one ORF unique to ClanGV genome. The genes, located within this restriction fragment, were compared with homologues in other baculoviruses. Comparison results indicated that ClanGV, Cydia pomonella GV and Pieris rapae GV have similar arrangement and orientation of the homologous genes. The ORFs related to these five genes were analyzed.