Construction and characterization of an infectious cDNA clone of encephalomyocarditis virus from pigs in China

Fang, Puxian; Bai, Juan; Liu, Xing; Dong, Jing; Sun, Tao; Jiang, Ping

doi:10.1007/s00705-014-2290-1

Cited by 4 publications

(5 citation statements)

References 26 publications

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“…The ampli cation of the whole genome sequence is the basis for further study of the virus. Fang and others used the strategy of segmented cloning to construct an infectious clone of NJ08 strain [25]. Next, we plan to use the segmented cloning strategy to amplify the full length of HLJ strain and construct an infectious clone for further study of the pathogenic mechanism of EMCV.…”

Section: Discussionmentioning

confidence: 99%

Isolation, molecular and phylogenetic analysis of porcine encephalomyocarditis virus strain HLJ in China

Yin

Liu

et al. 2020

Preprint

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Background Encephalomyocarditis virus (EMCV) is a positive single-stranded small RNA virus without envelope, which can infect a variety of mammals. Swine are the most susceptible animals, which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows. Diseases caused by EMCV have a wide range of effects on the global swine industry. Methods The tissue grinding fluid of aborted fetus was inoculated on baby hamster kidney 21 (BHK-21) cells to obtain HLJ isolate by cell passage. The virus was identified by reverse transcriptase polymerase chain reaction (RT-PCR), electron microscopic observation and indirect immunofluorescence assay (IFA). HLJ strain was inoculated into mice for animal regression experiment. The whole genome of HLJ strain was amplified and sequenced. The molecular and phylogenetic analysis of its sequence was generated. Results HLJ strain caused the specific cytopathic effect (CPE) on BHK-21 cells and severe pathological changes in mice. Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5–99.7%. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage I, II, III, IV, and V based on the nucleotide sequences of the entire open reading frame (ORF) and VP1 gene. HLJ isolate was grouped into lineage I. Conclusions In this study, a strain of EMCV was isolated from an aborted fetus in Northeast China. The isolation of HLJ strain enriches the epidemiological database of EMCV.

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Section: Discussionmentioning

confidence: 99%

Isolation, molecular and phylogenetic analysis of porcine encephalomyocarditis virus strain HLJ in China

Yin

Liu

et al. 2020

Preprint

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“…HM641897), was a kind gift from Prof. Ping Jiang. 63 The pLentiCRISPR v2 was a gift from Prof. Feng Zhang (Addgene, #52961). 64 The three RTase-containing plasmids, pCMV-L1-neo RT (LINE-1), pCMV-IAP-neo TNF (IAP) and pCMV-MusD-neo TNF (MusD) were initially constructed by Prof. Thierry Heidmann lab.…”

Section: Methodsmentioning

confidence: 99%

“…The EMCV virus, a member of the family Picornaviridae , was derived from the infectious clone pCMV-rNJ08. 63 The plasmid was transfected into BHK21 cells with Lipofectamine 2000 reagent following the manufacturer’s protocol. Supernatants were harvested when most of cells exhibited cytopathic effects and passaged twice.…”

Section: Methodsmentioning

confidence: 99%

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Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells

Xing

et al. 2021

Cell Res

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Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.

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“…MH77961) sequence as template. Four separate fragments (A, B, C1, and C2), each with a unique restriction enzyme site obtained by synonymous mutation [ 67 ], were amplified using Pfu DNA Polymerase (Novoprotein, China). The resulting full-length cDNA clone, designated pCMV-rSVA, contained a Mlu I site at the junction of fragments A and B, an Afl II site at the junction of fragments B and C1, and an Asc I site at the junction of fragments C1 and C2.…”

Section: Methodsmentioning

confidence: 99%

E2 ubiquitin-conjugating enzyme UBE2L6 promotes Senecavirus A proliferation by stabilizing the viral RNA polymerase

Bai

Fan

et al. 2020

PLoS Pathog

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Senecavirus A (SVA), discovered in 2002, is an emerging pathogen of swine that has since been reported in numerous pork producing countries. To date, the mechanism of SVA replication remains poorly understood. In this study, utilizing iTRAQ analysis we found that UBE2L6, an E2 ubiquitin-conjugating enzyme, is up-regulated in SVA-infected BHK-21 cells, and that its overexpression promotes SVA replication. We determined that UBE2L6 interacts with, and ubiquitinates the RNA-dependent RNA polymerase of SVA, (the 3D protein) and this ubiquitination serves to inhibit the degradation of 3D. UBE2L6-mediated ubiquitination of 3D requires a cystine at residue 86 in UBE2L6, and lysines at residues 169 and 321 in 3D. Virus with mutations in 3D (rK169R and rK321R) exhibited significantly decreased replication compared to wild type SVA and the repaired viruses, rK169R(R) and rK321R(R). These data indicate that UBE2L6, the enzyme, targets the 3D polymerase, the substrate, during SVA infection to facilitate replication.

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Construction and characterization of an infectious cDNA clone of encephalomyocarditis virus from pigs in China

Cited by 4 publications

References 26 publications

Isolation, molecular and phylogenetic analysis of porcine encephalomyocarditis virus strain HLJ in China

Isolation, molecular and phylogenetic analysis of porcine encephalomyocarditis virus strain HLJ in China

Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells

E2 ubiquitin-conjugating enzyme UBE2L6 promotes Senecavirus A proliferation by stabilizing the viral RNA polymerase

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