Wenqi He

Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in sheep in the Jilin province of China.

This study investigated an acute outbreak of contagious ecthyma dermatitis that occurred in November 2008 in a herd of 180 small-tailed Han sheep in the Jilin province of China. The pathological findings of this case revealed severe vascular proliferation, viral cytopathic changes in keratinocytes by vacuolar degeneration, ballooning degeneration, and eosinophilic cytoplasmic inclusions, suggesting that the disease could have been caused by an Orf virus (ORFV) infection. Immunohistochemistry, indirect immunofluorescence (IFA), and transmission electron microscopy supported the diagnosis of ORFV infection. Finally, the pathogen of the disease was identified using polymerase chain reaction (PCR) and sequences of major envelope protein genes (ORFV 011(B2L) and ORFV 059(F1L)). The full-length ORFV 011(B2L) and ORFV 059(F1L) genes were cloned and sequenced. The nucleotide and amino acid sequences of ORFV 011(B2L) and ORFV 059(F1L) genes in these outbreaks were analyzed, and their phylogenetic trees were constructed. The phylogenetic studies of ORFV 011(B2L) genes and ORFV 059(F1L) genes showed that the ORFV-Jilin province isolate clustered in different branches and was closer to the ORFV-Mukteswar 67/04 isolate and the ORFV-OV/C2 isolate, respectively. To date, there is no report on the molecular characterization of any Orf virus with other isolates around the world in mainland China. Further, the above results may provide some insight into the genotype of the etiological agent responsible for the contagious ecthyma dermatitis outbreak in the Jilin province, and could also provide a comparative view of the central coding region genomics of parapoxvirus (PPV).

Orf virus DNA vaccines expressing ORFV 011 and ORFV 059 chimeric protein enhances immunogenicity

BackgroundORFV attenuated live vaccines have been the main prophylactic measure against contagious ecthyma in sheep and goats in the last decades, which play an important role in preventing the outbreak of the disease. However, the available vaccines do not induce lasting immunity in sheep and goats. On the other hand, variation in the terminal genome of Orf virus vaccine strains during cell culture adaptation may affect the efficacy of a vaccine. Currently, there are no more effective antiviral treatments available for contagious ecthyma.ResultsWe constructed three eukaryotic expression vectors pcDNA3.1-ORFV011, pcDNA3.1-ORFV059 and pcDNA3.1-ORFV011/ORFV059 and tested their immunogenicity in mouse model. High level expression of the recombinant proteins ORFV011, ORFV059 and ORFV011/ORFV059 was confirmed by western blotting analysis and indirect fluorescence antibody (IFA) tests. The ORFV-specific antibody titers and serum IgG1/IgG2a titers, the proliferation of lymphocytes and ORFV-specific cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α) were examined to evaluate the immune responses of the vaccinated mice. We found that mice inoculated with pcDNA3.1-ORFV 011/ORFV059 had significantly stronger immunological responses than those inoculated with pcDNA3.1-ORFV011, pcDNA3.1-ORFV059, or pcDNA3.1-ORFV011 plus pcDNA3.1-ORFV059. Compared to other vaccine plasmids immunized groups, pcDNA3.1-ORFV011/ORFV059 immunized group enhances immunogenicity.ConclusionsWe concluded that DNA vaccine pcDNA3.1-ORFV011/ORFV059 expressing ORFV011 and ORFV059 chemeric-proteins can significantly improve the potency of DNA vaccination and could be served as more effective and safe approach for new vaccines against ORFV.

Vomiting and wasting disease associated with hemagglutinating encephalomyelitis viruses infection in piglets in Jilin, China.

One coronavirus strain was isolated from brain tissues of ten piglets with evident clinical manifestations of vomiting, diarrhea and dyskinesia in Jilin province in China. Antigenic and genomic characterizations of the virus (isolate PHEV-JLsp09) were based on multiplex PCR and negative staining electron microscopy and sequence analysis of the Hemagglutinin-esterase (HE) gene. These piglets were diagnosed with Porcine hemagglutinating encephalomyelitis virus (PHEV).Necropsy was performed on the piglets. Major pathological changes included meningeal hyperemia, meningeal hemorrhage and cortical hemorrhage. Minor changes were also observed in other organs. Histopathological changes included satellitosis and neuronophagia in the cerebral cortex.Mice were infected with the isolated virus. Their histopathological changes were similar to those symptoms observed in the piglets, exhibiting typical changes for non-suppurative encephalitis. Thus, Porcine hemagglutinating encephalomyelitis virus mainly causes damage to the nervous system but also impacts other organs. This viral strain (isolate PHEV-JLsp09) found in the Siping area of Jilin Province in China is evolutionally closest to the HEV-67N stain (North American strain), indicating that this viral strain evolved from the PHEV from North America.

The evidence of Coxsackievirus B3 induced myocarditis as the cause of death in a Sichuan snub-nosed monkey (Rhinopithecus roxellana).

Background  A 5‐year‐old female Sichuan snub‐nosed monkey died at the zoological garden from infection with coxsackievirus B3.

Highly pathogenic fowlpox virus in cutaneously infected chickens, China.

We investigated an acute outbreak of the cutaneous form of fowlpox among chickens in China in November 2009. Using pathologic and virologic methods, we identified a novel type of fowlpox virus that carried an integrated genomic sequence of reticuloendotheliosis virus. This highly pathogenic virus could lead to severe ecologic effects and economic losses.

The evidence of porcine hemagglutinating encephalomyelitis virus induced nonsuppurative encephalitis as the cause of death in piglets

An acute outbreak of porcine hemagglutinating encephalomyelitis virus (PHEV) infection in piglets, characterized with neurological symptoms, vomiting, diarrhea, and wasting, occurred in China. Coronavirus-like particles were observed in the homogenized tissue suspensions of the brain of dead piglets by electron microscopy, and a wild PHEV strain was isolated, characterized, and designated as PHEV-CC14. Histopathologic examinations of the dead piglets showed characteristics of non-suppurative encephalitis, and some neurons in the cerebral cortex were degenerated and necrotic, and neuronophagia. Similarly, mice inoculated with PHEV-CC14 were found to have central nervous system (CNS) dysfunction, with symptoms of depression, arched waists, standing and vellicating front claws. Furthmore, PHEV-positive labeling of neurons in cortices of dead piglets and infected mice supported the viral infections of the nervous system. Then, the major structural genes of PHEV-CC14 were sequenced and phylogenetically analyzed, and the strain shared 95%–99.2% nt identity with the other PHEV strains available in GenBank. Phylogenetic analysis clearly proved that the wild strain clustered into a subclass with a HEV-JT06 strain. These findings suggested that the virus had a strong tropism for CNS, in this way, inducing nonsuppurative encephalitis as the cause of death in piglets. Simultaneously, the predicted risk of widespread transmission showed a certain variation among the PHEV strains currently circulating around the world. Above all, the information presented in this study can not only provide good reference for the experimental diagnosis of PHEV infection for pig breeding, but also promote its new effective vaccine development.

Identification of NCAM that interacts with the PHE-CoV spike protein.

BackgroundThe spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies.ResultsWe identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection.ConclusionsA novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

Porcine hemagglutinating encephalomyelitis virus induces apoptosis in a porcine kidney cell line via caspase-dependent pathways

Abstract Porcine hemagglutinating encephalomyelitis is an acute, highly contagious disease in piglets that is caused by the porcine hemagglutinating encephalomyelitis virus (PHEV). However, the pathogenesis of PHEV and the relationship between PHEV and the host cells are not fully understood. In this study, we investigated whether the PHEV-induced cytopathic effect (CPE) was caused by apoptosis. Replication of PHEV in a porcine kidney-derived cell line (PK-15 cells) caused an extensive CPE, leading to the destruction of the entire monolayer and the death of the infected cells. Staining with Hoechst 33,342 revealed morphological changes in the nuclei and chromatin fragmentation. In addition, PHEV caused DNA fragmentation detectable by agarose gel electrophoresis 48h post-infection, increasing with the incubation time. The percentage of apoptotic cells increased with the incubation time and reached a maximum at 96h post-infection, as determined using flow cytometry and fluorescence microscopy of cells that were stained with annexin V-FITC and propidium iodide (PI). Moreover, as is commonly observed for coronavirus infections of other animals, the activities of the effecter caspase, caspase-3, and the initiator caspases, caspase-8 and caspase-9, which are representative factors in the death receptor-mediated apoptotic pathway and the mitochondrial apoptotic pathway, respectively, were increased in PHEV-infected PK-15 cells. Moreover, the tripeptide pan-ICE (caspase) inhibitor Z-VAD-FMK blocked PHEV-induced apoptosis but did not have an effect on virus production by 96h post-infection. These results suggested that PHEV induces apoptosis in PK-15 cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental to animals because it causes cell and tissue destruction. Although the pathological characteristics of PHEV are largely unknown, apoptosis may be the pathological basis of the lesions resulting from PHEV infection.

Complete Genome Sequence of a Coxsackievirus B3 Isolated from a Sichuan Snub-Nosed Monkey

ABSTRACT Coxsackievirus B3 (CVB3) is an enterovirus in the family Picornaviridae that is significant to human health, being associated with myocarditis, aseptic meningitis, and pancreatitis, among other conditions. In addition to humans, Sichuan snub-nosed monkeys can be infected and killed by CVB3. Here, we report the first complete genome sequence of a novel coxsackievirus B3 strain, SSM-CVB3, which was isolated from a deceased Sichuan snub-nosed monkey with severe myocarditis. Our findings may aid in understanding the evolutionary characteristics and molecular pathogenesis of this virus.

A Small Region of Porcine Hemagglutinating Encephalomyelitis Virus Spike Protein Interacts with the Neural Cell Adhesion Molecule

Objective: The spike (S) protein of porcine hemagglutinating encephalomyelitis virus (PHEV) may mediate infection by binding to a cellular neural cell adhesion molecule (NCAM). This study aimed to identify the crucial domain of the S1 subunit of the S protein that interacts with NCAM. Methods: Three truncated segments (S_1-291, S_277-794 and S_548-868) of the S gene of PHEV and the NCAM gene were cloned individually into the Escherichia coli expression vectors and yeast two-hybrid expression vectors. The interaction between S_1-291, S_277-794, S_548-868 and NCAM were detected by a GST pull-down experiment and yeast two-hybrid assay. Results: Three fusion proteins (S_1-291, S_277-794 and S_548-868) were screened for their interactions with NCAM by protein-protein interaction assays. The results of these assays clarified that S_277-794 interacted with NCAM, while S_1-291 and S_548-868 did not. Conclusions: A small fragment (258-amino-acid fragment, residues 291-548) on the PHEV S protein was posited to be the minimum number of amino acids necessary to interact with NCAM. This fragment may be the receptor-binding domain that mediates PHEV binding to NCAM.