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Tripartite motif protein 21 (TRIM21) is an E3 ubiquitin ligase and cytosolic antibody receptor of the TRIM family. Previous reports have indicated that TRIM21 plays an important role during viral infection. This study aimed at examining the role of TRIM21 in the replication of porcine epidemic diarrhea virus (PEDV) and showed that TRIM21 inhibits PEDV proliferation by targeting and degrading the nucleocapsid (N) protein through the proteasomal pathway. Furthermore, the endogenous expression of TRIM21 was found to be downregulated by PEDV infection in Vero and LLC-PK1 cells. Overexpression of TRIM21 inhibited PEDV replication, whereas knockdown of TRIM21 increased viral titers and N protein levels. TRIM21 was found to interact and colocalize with the N protein, and the TRIM21-mediated antiviral effect was dependent on its ubiquitin ligase activity, which engages in polyubiquitination and degradation of the N protein in a proteasome-dependent manner. Taken together, these findings provide information about the role of TRIM21 in PEDV proliferation and increase our understanding of host-virus interactions. [ABSTRACT FROM AUTHOR]

The UL24 homologous genes are conserved in alphaherpesviruses. However, the proximity of the UL24 gene and the UL23 gene encoding for thymidine kinase (TK) in the genome of suid herpesvirus 1 (SuHV-1) makes it difficult to mutate UL24 without affecting the expression of the TK gene, and thus functional studies of the UL24 gene have lagged behind. In this study, CRISPR/Cas9 and homologous recombination were adopted to generate UL24 and TK mutant viruses. Deletion of either the UL24 or the TK gene resulted in significantly reduced SuHV-1 replication and spread capacity in Vero cells. However, UL24-deleted virus still maintained a certain degree of lethality in mice, while TK-deleted viruses completely lost their lethality in mice. Similarly, neurovirulence of UL24-deleted virus in mice was not significantly affected compared to parental virus. In comparison, infection with the TK-deleted viruses resulted in significantly reduced neurovirulence and complete loss of lethality. In addition, and for the first time, viral UL24 protein was found to be expressed late during SuHV-1 infection; enhanced green fluorescence protein (eGFP) labeled UL24 protein was shown to be localized in the nucleus via heterologous expression. In conclusion, the UL24 gene of SuHV-1 encodes a nuclear-localized viral protein and acts as a minor virulence-associated factor compared to the TK gene. [ABSTRACT FROM AUTHOR]

Different strains of porcine reproductive and respiratory syndrome virus (PRRSV) have emerged and circulated in different regions of mainland China since 1996, particularly after 2006. In 2012, NADC30-like PRRSV was first isolated in Henan Province. By 2016, it had spread to most provinces in China. In the present study, the whole genomes (excluding the poly(A) tails) of 13 newly emerged NADC30-like PRRSV strains were sequenced and analyzed. Furthermore, the pathogenicity of SD53-1603, one of the 13 PRRSV strains, was assessed. Phylogenetic analysis showed that these 13 newly emerged NADC30-like PRRSV strains, together with some reference strains, formed a new subgroup (subgroup 5), characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. However, low levels of whole-genome similarity and a wide variety of recombination patterns complicated the classification of the NADC30-like PRRSV isolates. Interestingly, almost all of the recombination breakpoints found in these 13 PRRSV isolates and other NADC30-like PRRSV isolates occurred in genes encoding NSPs and/or minor structural proteins. In addition, piglets infected with the newly emerged NADC30-like strain SD53-1603 displayed clear clinical respiratory symptoms and underwent typical pathological changes. The findings may be useful for elucidating the characteristics and epidemic status of NADC30-like PRRSV in China. [ABSTRACT FROM AUTHOR]

Flavivirus cDNA clones frequently demonstrate genetic instability in transformed bacteria, which hampers the construction and manipulation of cDNAs for infectious flaviviruses. In this study, we developed a stable, full-length cDNA clone, pJEHEN, of a GI JEV strain HEN0701 using a medium-copy-number pBR322 vector and propagating cDNA clones at room temperature. The virus vJEHEN recovered from the infectious clone was indistinguishable from the parent virus HEN0701 with respect to plaque morphology, growth kinetics, and virulence characteristics. A T-to-A silent mutation of nucleotide 24 of the NS2a gene was introduced into the infectious cDNA clone to eliminate frameshifting. The rescued mutant virus vJETA did not express NS1′ in infected cells and showed reduced growth and neurovirulence in mice. This convenient method for the construction and manipulation of infectious JEV cDNA clones may be of use in further studies to improve our understanding of the molecular mechanisms responsible for JEV replication and pathogenesis. [ABSTRACT FROM AUTHOR]

Canine kobuvirus (CaKVs) was a newly described virus detected in dogs in the US and Italy. To learn more about CaKVs, 5 of 106 fecal samples from diarrhea dogs were positive with CaKVs in China, and the full genome of CaKVs strain CH-1 isolated from dog with diarrhea was sequenced. The genome consists of 8186 nucleotides, excluding the 3′ poly (A) tail, and an open reading frame that maps between nucleotide positions 601 and 7943 which encodes a 2446 amino acid polyprotein. Based on the complete amino acid sequence of polyprotein, phylogenetic analysis showed that CH-1 was grouped along with other canine kobuvirus strains detected in the USA (US-PC0082, AN211D). [ABSTRACT FROM AUTHOR]

The genetic instability of Flavivirus cDNA clones in transformed bacteria is a common phenomenon. Herein, a cDNA fragment of the nucleotide (nt) 1-2913 of the genome of a flavivirus, Japanese encephalitis virus (JEV), was used to investigate factors that caused the instability of cDNA clones. Several cDNA fragments with different 5′- or 3′-termini of the 2913-nt cDNA were obtained by PCR amplification or restriction enzyme digestion and cloned into a pCR-Blunt II-TOPO vector. All the cDNA fragments were stably propagated at 25 °C. However, the 5′-untranslated region and half of the 3′-E gene could cause the instability of the 2913-nt cDNA at 37 °C. The 5′-terminus sequences of the 2913-nt fragment were subjected to testing of the prokaryotic promoter activity by luciferase assay and Western blot. The sequences of 54-120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. These findings revealed that the high prokaryotic promoter activity of the 54-120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5′-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria. [ABSTRACT FROM AUTHOR]

The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B or/and TRS-B impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B or TRS-B did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B plays more important roles than TRS-B in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV. [ABSTRACT FROM AUTHOR]

The 3′ untranslated region (3′ UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3′ UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3′ UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3′ UTR region. Our findings indicated that the conservation of the primary sequence of the 3′ terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5′-AAUU-3′) at the 3′ proximal end of the 3′ UTR and the dinucleotide 5′-AU-3′ exerted an important regulatory effect on viral viability. Of the five 3′-terminal nucleotides of the 3′ UTR (5′-AACCA-3′), at least three, including the last dinucleotide (5′-CA-3′), were essential for maintaining viral infectivity. Taken together, the 3′-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. In the present study, we compared the phosphorylation status of Akt during infection of MARC-145 cells and porcine alveolar macrophages (PAMs) with highly pathogenic PRRSV (HP-PRRSV) strain HuN4. We observed that biphasic activation of Akt was induced in at both the early stage (5, 15 and 30 min postinfection) and the late stage (12 and 24 h postinfection) of HP-PRRSV infection of MARC-145 cells, while an early-phase activation of Akt was found exclusively in virus-infected PAMs in vitro. Analysis with the PI3K-specific inhibitor LY294002 confirmed that PI3K acted as the upstream activator for the virus-induced activation of Akt. UV-irradiation-inactivated virus still induced the early event in PAMs but not in MARC-145 cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated Akt within different cell cultures. We further demonstrated that FoxO1 and Bad, which serve as downstream targets of Akt, were phosphorylated in virus-infected MARC-145 cells. Moreover, the suppression of phosphorylated Akt with LY294002 significantly inhibited the virus-induced cytopathic effect (CPE) on MARC-145 cells, but it had a negligible effect on virus propagation. Collectively, our data provide new evidence of a novel role for the PI3K/Akt pathway in PRRSV infection of MARC-145 cells. [ABSTRACT FROM AUTHOR]

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China. [ABSTRACT FROM AUTHOR]

Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the most abundant viral structural protein with high immunogenicity. Previously, the nonessential sequences for virus infectivity were identified at both N and C terminal ends of N protein. Here, by means of reverse genetics, a marker virus (v7APMa) was generated with a mutant N protein that differs from the wild-type strains (vAPRRS, type 2 PRRSV). v7APMa shows stable inheritance in cell culture and the virologic characteristics of the marker virus in vitro showed that v7APMa replicates well as its parental strain. In the pig model, the v7APMa marker virus induced a similar level of anti-N protein antibodies and robust antibodies against the marker peptide, from 14 days post infection. In addition, a peptide-based ELISA was developed to detect the specific antibodies for the introduced 7APMa peptide. This approach, using a rationally designed marker virus, provides a new potential mutant basis for further development of PRRSV novel vaccines. [ABSTRACT FROM AUTHOR]

Proviral DNA was extracted from donkey leukocyte infected with Chinese donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), and peripheral blood lymphocytes (PBL) from a horse infected with the virulent EIAV strain Liaoning (EIAV L). The entire proviral DNA from both viruses was cloned and sequenced. The lengths of complete genomic sequences of DLA-EIAV and EIAV L provirus were 8266 bp and 8235 bp, respectively. Sequence comparison indicated that DLA-EIAV shares 97.0% and 97.5% in sequence homology with EIAV L and donkey-adapted EIAV (DA-EIAV), respectively. Lots of variations occurred in long terminal repeat ( LTR, consisting of U3, R, U5), ORF S2, and env regions between DLA-EIAV and EIAV L. The nucleotide sequence differences of the two viruses in U3, R, U5, ORF S2, and env are 13.2%, 7.5%, 5.1%, 3.9%, and 2.7%, respectively, and predicted amino acid sequence differences in env and S2 coding regions are 4.4% and 8.8%, respectively. Six conserved regions are characterized in Gp90. There is a cis-activating GATA motif in ENH of DLA-EIAV and EIAV L. Two N-linked glycosylation sites disappeared in DLA-EIAV Gp90 in comparison with that of EIAV L. A bHLH transcription factor binding consensus sequence was found in LTR of DLA-EIAV but not in EIAV L. Furthermore, there is a mutation in the stem of DLA-EIAV TAR resulting in formation of a uridine tuber. Further study is needed to uncover the relationship between sequence changes and their biological functions of DLA-EIAV and L. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death, particularly in neonatal piglets. The nucleocapsid protein (N protein) of PEDV presents strong immunogenicity and contributes to the cross-reactivity between PEDV and TGEV. However, the characterization of epitopes on the PEDV N protein remains largely unknown. Here, two monoclonal antibodies (MAbs) specific to the N protein of a PEDV strain, FJzz1/2011, were generated and screened against a partially overlapping library of 24 GST-fusion N protein-truncated constructs. We confirmed that residues 18-133 (designated NEP-D4) and residues 252-262 (designated NEP-D6) were the epitopes targeted by MAbs PN-D4 and PN-D6, respectively. Sequence analysis revealed that these two epitopes were highly conserved among PEDV strains but were significantly different from other members of the Coronavirinae subfamily. Western blot analysis showed that they could be specifically recognized by PEDV antisera but could not be recognized by TGEV hyperimmune antisera. Indirect immunofluorescence (IFA) assays confirmed no cross-reaction between these two MAbs and TGEV. In addition, the freeze-thaw cycle and protease treatment results indicated that NEP-D4 was intrinsically disordered. All these results suggest that these two novel epitopes and their cognate MAbs could serve as the basis for the development of precise diagnostic assays for PEDV. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) can impact viral infections by binding to sequences with partial complementarity on viral RNA transcripts, usually resulting in the repression of virus replication. In the present study, we identified a potential binding site for miR-130 in the 5′ untranslated region (bps 155-162) of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. We found that the delivery of multiple miR-130 family mimics, especially miR-130b, resulted in inhibition of PRRSV replication in vitro. miR-130 was effective in inhibiting the replication of multiple type 2 PRRSV strains, but not against vSHE, a classical type 1 strain. miR-130 over-expression did not induce IFN-α or TNF-α expression in either uninfected or PRRSV-infected porcine alveolar macrophages. Results from luciferase reporter assays indicated that miR-130 directly targeted the PRRSV 5′ UTR. Intranasal inoculation of piglets with miR-130b exhibited antiviral activity in vivo and partially protected piglets from an otherwise lethal challenge with HP-PRRSV strain vJX143. Overall, these results demonstrate the importance of the miR-130 family in modulating PRRSV replication and also provide a scientific basis for using cellular miRNAs in anti-PRRSV therapies. [ABSTRACT FROM AUTHOR]