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The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation and regulation of cellular signaling pathways, and assembly of infectious virus particles. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism was not elucidated. Here, we report that NS5A interacts with NF-κB essential modulator (NEMO),a regulatory subunit of the IκB kinase (IKK) complex, and that the zinc finger domain of NEMO essential for NEMO ubiquitination and IKK activation is required for the interaction of NEMO with NS5A. Viral infection or NS5A expression by itself reduced the protein level of NEMO. Mechanistic analysis revealed that NS5A mediated proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced K27-but not K48-linked polyubiquitination of NEMO. In addition, NS5A blocked k63-linked polyubiquitination of NEMO, thereby inhibiting activation of IKK and NF-κB. These findings indicate that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV inhibits host innate immunity.ImportanceClassical swine fever (CSF) is a economically important swine viral disease leading to hemorrhagic fever and immuno-suppression. In order to successfully infect and replicate in a host cell, viruses have evolved various strategies to antagonize host innate immunity. It is known that CSFV non-structural protein Npro interacts with interferon regulatory factor 3 (IRF3) and mediates its proteasomal degradation, thereby inhibiting the production of type I interferon. However, no other mechanism by which CSFV antagonizes host innate immunity has so far been reported. Here, we show that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and by blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV antagonizes host innate immunity.

Hybrid wild boar husbandry is an important component of livestock production in Northeast China. However, the current disease situation of these animals is largely unknown due to a lack of disease surveillance. The present study was conducted to determine the prevalence of several important viral diseases in the hybrid wild boar population of Northeast China. Between September 2015 to December 2016, 169 blood and 61 tissue samples were collected from apparently healthy hybrid wild boars from farms in Jilin, Inner Mongolia and Heilongjiang provinces. ELISA detected serum antibodies against classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2) and Japanese encephalitis virus (JEV), but not against African swine fever virus (ASFV), with PCV2 having the highest seropositive rate (87.2–100% in different farms). RT-PCR or PCR performed on the processed samples detected only PCV2, with 33.1% (56/169) of blood samples and 32.8% (20/61) of spleen samples being positive, respectively, indicating widespread PCV2 infection in hybrid wild boars. Phylogenetic analysis of 15 PCV2 ORF2 sequences showed that they belong to genotypes PCV2a, PCV2b and PCV2d, with nucleotide and deduced amino acid homologies of 88.5–100% and 88.1–100%, respectively.

The family Circoviridae comprises a large group of small circular single-stranded DNA viruses with several members causing severe pig and poultry diseases. In recent years the number of new viruses within the family has had an explosive increase showing a high level of genetic diversity and a broad host range. In this report we describe two more circoviruses identified from bats in Yunnan and Heilongjiang provinces in China. Full genome sequencing has revealed that these bat associated circoviruses (bat ACV) should be classified as new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses (ICTV). The most striking result is the novel finding of a 21–28 nt polythymidine (poly-T) tract in the 3′ terminal intergenic region of bat ACV isolates from Heilongjiang province. To understand its role in viral replication, a wild type bat ACV and a mutated version with the entire poly-T deleted were rescued through construction of infectious clones. Replication comparison in vitro showed that the poly-T is not essential for viral replication. Identification of additional bat ACV isolates and study of their biological characteristics will be the main task in future to understand the potential roles of bats in transmission of circoviruses to terrestrial mammals and humans.

Background There have been four strains on Manzanilla virus (MANV) identified to date. Here, we identify a novel MANV strain (DHL10M107) isolated from Culex tritaeniorhynchus Giles mosquitoes from Ruili city, Dehong prefecture, Yunnan Province, in the People’s Republic of China. Results The DHL10M107 L, M and S genes were sequenced at the nucleotide and deduced amino acid levels. The L, M and S gene sequences of DHL10M107 clustered with the MANV strains VN04-2108, TRVL3587, SA An 4165, and AV 782. DHL10M107 was most closely related to VN04-2108. Nucleotide homology ranged between 96 and 99% between DHL10M107 and VN04-2108. In terms of amino acid homology, all of the amino acid differences were in the L (96.3% homologous) and M (97.7% homologous) fragments. Conclusions DHL10M107 is likely a MANV isolated from mosquitos in the Yunnan Province. This is the first reported isolation of MANV in mainland China. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1198-5) contains supplementary material, which is available to authorized users.

RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.

Bats are considered as important animal reservoirs for many pathogenic viruses to humans. The viral metagenomic analysis was performed to study gut and lung tissues of 30 insectivorous bats collected in Yunnan Province and 26 reads were noted to group A rotavirus (RVA). Further RT-PCR screening on bat samples and in vitro viral isolation on cell cultures confirmed the presence of a novel RVA, named as RVA/Bat-tc/MYAS33/2013/G3P[10], in one of 30 Stoliczka's trident bats. The VP7 gene of this strain MYAS33 was closely related to that of an equine RVA strain from Argentina and the nucleotide sequence similarity was 93%, while its VP4 gene was a rare P[10] type and obtained the maximum sequence identity (94.8%) with that of a human strain from Thailand. The present study highlights the potential role of bats as reservoirs for RVAs.

Viral infections usually result in alterations in the host cell proteome which determine the fate of the infected cells and the progress of pathogenesis. To uncover cellular protein responses in classical swine fever virus-infected PK-15 cells, a proteomic analysis was conducted using 2D PAGE followed by MALDI-TOF-MS/MS identification. Altered expression of 35 protein spots in infected cells at 48 h p.i. were identified in 2D gels, with 21 of these being characterized by MALDI-TOF-MS/MS, including 16 upregulated proteins and 5 down-regulated proteins. Western-blot analysis confirmed the up-regulation of annexin 2 and down-regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The altered proteins could be sorted into 7 groups according to cellular function: cytoskeleton, energy metabolism, replication/transcription and translation processes, protein processing, antioxidative stress proteins, heat shock proteins and signal transduction. The altered expression of these proteins provides a response profile of PK-15 host cells to CSFV infection. Further study of these altered proteins may facilitate understanding the mechanisms of CSFV infection and pathogenesis.

Classical swine fever (CSF) is a contagious swine disease charactered by hemorrhagic fever and leukopenia,usually leading to substantial economic losses. To obtain a insight of leucopenia caused by CSFV infection, DNA microarray analyses of peripheral blood leucocytes (PBL) of the infected pigs was performed. Three health pigs were inoculated with a lethal dose of CSFV Shimen strain and their PBLs were isolated when the onset of typical clinical signs and then subjected to total RNA extraction followed by microarray analysis with Affymetrix Porcine Genome Array GeneChips. The results showed that the significant differences were observed in cellular apoptotic genes expression at 7 days post-infection (p. i.). The changes of the genes expression were confirmed by real time RT-PCR of some selected apoptosis-related genes. This study provided a valuable information for further investigating the molecular mechanism of apoptosis caused by CSFV infection.

In order to understand the replication kinetics of classical swine fever virus (CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37 degrees C in 5% CO2 environment when growing to 80% confluence, the cells were infected with CSFV strain Shimen at 100 TCID50 per well. At various time post infection (p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay (IFA), RNA replication using reverse transcription real-time PCR and viral production using titration of TCID50. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining, the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However, the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID50 demonstrated that the production of live virions increased at 8h and peaked between 48 - 72 hrs p.i. without significant lose of titer.

The fusion protein (F) and attachment glycoprotein (G) of Nipah virus (NiV) are important for the virus to infect cells and induce protective immunity. In this study, the NiV F1 and G gene fragments without the sequences of signal peptide and transmembrane domain were amplified by PCR, then cloned into E. coli expression vector pGEX-6P-1 and modified baculovirus vector, respectively. After induction by IPTG, NiV F1 and G proteins were efficiently expressed in E. coli when analyzed by SDS-PAGE, both showing good reactivity with the rabbit antiserum anti-NiV serum in Western blot. The expression of NiV F1 and G in baculovirus system were also detected by indirect immunofluorescent assay (IFA) of fixed Sf9 cells monolayer infected with the recombinant baculoviruses expressing F1 and G. Furthermore the anti-F1 and anti-G hyperimmune sera were prepared by immunization of rabbits respectively with purified E. coli-expressed F1 and G proteins. Western blot and IFA as well as ELISA showed that antisera against both protein had high titers with good reactivity and specificity. The present study has provided a base for development of diagnostic reagents for detection of NiV infection.

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.

The biosafety of DNA vaccine is one of the key questions which should be solved before it is used in the clinical trail. In order to evaluate the biosafety of DNA vaccine, the CSFV DNA vaccine was used in the studying target, two main aspects of the vaccine were explored in the study. Firstly, the possibility of integration of two kinds of DNA vaccine plasmids into pig genome was analyzed by PCR technology after the different vaccines were injected through the intramuscular introduction. The results showed that both plasmids DNA were detected as the form were not integrated into pig genome, it can be detected 30 copies plasmid DNA in 1 microg total genomic DNA as the sensibility of PCR, indicated the safety of the DNA vaccine. Afterward the environmental fecal and soil samples in the experimental pens were picked up. Then the antibiotic resistant bacteria were isolated and its resistant genes were analyzed by PCR and gene sequencing. The results demonstrated that the transfer and spreading of two DNA vaccine plasmids studied into environmental bacteria from receptor pigs were not found. The results showed that the CSFV DNA vaccine is safe to both pigs and the surrounding environment.

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.

The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic analysis suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geographic locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002–2003 epidemic. Major genetic variations in some critical genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003.