Jianye Wang

Bovine viral diarrhea virus (BVDV) infections in pigs.

Cattle are the natural hosts of bovine viral diarrhea virus (BVDV), which causes mucosal disease, respiratory and gastrointestinal tract infections, and reproductive problems in cattle. However, BVDV can also infect goats, sheep, deer, and pigs. The prevalence of BVDV infection in pig herds has substantially increased in the last several years, causing increased economic losses to the global pig breeding industry. This article is a summary of BVDV infections in pigs, including a historical overview, clinical signs, pathology, source of infection, genetic characteristics, impacts of porcine BVDV infection for diagnosis of classical swine fever virus (CSFV), differentiation of infection with CSFV and BVDV, and future prospects of porcine BVDV infection.

Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells.

Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1) infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.

Critical role of cholesterol in bovine herpesvirus type 1 infection of MDBK cells

Abstract Cholesterol is involved in the life cycle of many viruses. Here, we examined the role of cholesterol for both viral envelope and target cell membrane for bovine herpesvirus type 1 (BoHV-1) infection. Cholesterol depletion by pretreatment of Madin–Darby bovine kidney (MDBK) cells with a cholesterol-sequestering drug methyl-β-cyclodextrin (MβCD), inhibited the production of BoHV-1 in a dose-dependent manner. This inhibitory effect was partially reversed by cholesterol replenishment, indicating that the reduction was caused by cholesterol depletion. Cholesterol depletion at the post-entry stage only had a mild effect on the virus production. However, cell membrane cholesterol depletion did not reduce the virus attachment. In addition, treatment of BoHV-1 particles with MβCD also reduced the virus infectivity significantly and the effect was partially reversed by addition of exogenous cholesterol. Taken together, these data implicated that cell membrane cholesterol mainly contributed to BoHV-1 entry into MDBK cells and the viral envelope cholesterol was also essential for the virus infectivity.

Sequencing and generation of an infectious clone of the pathogenic goose parvovirus strain LH

AbstractsIn this study, the complete genome of the virulent strain LH of goose parvovirus (GPV) was sequenced and cloned into the pBluescript II (SK) plasmid vector. Sequence alignments of the inverted terminal repeats (ITR) of GPV strains revealed a common 14-nt-pair deletion in the stem of the palindromic structure in the LH strain and three other strains isolated after 1982 when compared to three GPV strains isolated earlier than that time. Transfection of 11-day-old embryonated goose eggs with the plasmid pLH, which contains the entire genome of strain LH, resulted in successful rescue of the infectious virus. Death of embryos after transfection via the chorioallantoic membrane infiltration route occurred earlier than when transfection was done via the allantoic cavity inoculation route. The rescued virus exhibited virulence similar to that of its parental virus, as evaluated by the mortality rate in goslings. Generation of the pathogenic infectious clone provides us with a powerful tool to elucidate the molecular pathogenesis of GPV in the future.

Molecular characterization and phylogenetic analysis of an avian adeno-associated virus originating from a chicken in China

The use of adeno-associated viruses (AAVs) as vectors for gene delivery is well established; however, genomic information about avian adeno-associated virus (AAAV) and its use are still limited. In this study, an AAAV strain, YZ-1, was isolated from healthy chickens in China, and the complete genome was sequenced. The genomic DNA of YZ-1 is 4,684 nucleotides long, including two ORFs encoding the nonstructural proteins (Rep) and the structural proteins (Cap), and an inverted terminal repeat (ITR) forming a typical T-shaped palindromic structure at each end. YZ-1 was 95.0 and 92.2% identical to the other two reported AAAV strains, DA-1 and VR-865, respectively, at the nucleotide sequence level. In comparison to VR-865, frameshift mutations or deletions in the N-terminal region of the Rep78 protein or VP2 protein were observed in YZ-1 and DA-1. Phylogenetic analysis revealed that YZ-1, DA-1 and VR-865 could be classified into the avian group of the AAV family. This group and other AAVs of mammalian origin displayed almost equal divergence from pathogenic waterfowl parvoviruses, revealing that AAAV has no direct evolutionary relationship to them. This study therefore provides new genomic information about AAAV.

The activation of p38MAPK and JNK pathways in bovine herpesvirus 1 infected MDBK cells.

We have shown previously that BHV-1 infection activates Erk1/2 signaling. Here, we show that BHV-1 provoked an early-stage transient and late-stage sustained activation of JNK, p38MAPK and c-Jun signaling in MDBK cells. C-Jun phosphorylation was dependent on JNK. These early events were partially due to the viral entry process. Unexpectedly, reactive oxygen species were not involved in the later activation phase. Interestingly, only activated JNK facilitated the viral multiplication identified through both chemical inhibitor and siRNA. Collectively, this study provides insight into our understanding of early stages of BHV-1 infection.

Transfection of embryonated Muscovy duck eggs with a recombinant plasmid is suitable for rescue of infectious Muscovy duck parvovirus

For members of the family Parvoviridae, rescue of infectious virus from recombinant plasmid is usually done in cultured cells. In this study, the whole genome of the pathogenic Muscovy duck parvovirus (MDPV) strain YY was cloned into the pBluescript II (SK) vector, generating recombinant plasmid pYY. With the aid of a transfection reagent, pYY plasmid was inoculated into 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route, resulting in the successful rescue of infectious virus and death of the embryos. The rescued virus exhibited pathogenicity in Muscovy ducklings similar to that of its parental strain, as evaluated based on the mortality rate. The results demonstrate that plasmid transfection in embryonated Muscovy duck eggs is a convenient and efficacious method for rescue of infectious MDPV in comparison to transfection of primary cells, which is somewhat time-consuming and laborious.

Rescue of avian adeno-associated virus from a recombinant plasmid containing deletions in the viral inverted terminal repeats

We have previously reported the complete genome sequence of avian adeno-associated virus (AAAV) strain YZ-1, isolated from healthy chickens in China. In this study, we describe the successful rescue of infectious virions from a recombinant plasmid containing the genome of YZ-1 with deletions in the viral inverted terminal repeats (ITRs). The complete genome of YZ-1 was cloned into a bacterial plasmid by a modified “A-T” cloning method. Six recombinant plasmids were selected for further experiments. Sequence analysis indicated that the six clones shared identical internal sequences except for the various deletions within ITRs at either end of the cloned genome. The recombinant plasmid pYZ525, harboring a YZ-1 genome with a 96-nt deletion at the 5′ end, was used to transfect CEL or HEK293 cells in the presence of the CELO virus or a helper plasmid, and rescued virions were obtained by both of the methods despite the presence of the deletions. Here, for the first time, we provide evidence that a certain number of nt deletions in the ITRs are not lethal for the rescue of viable AAAV from recombinant plasmids. This study provides insight into the unique biology of AAAV and the mechanism of viral replication.

Identification of 987P Protein Receptors for Enterotoxigenic Escherichia coli

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.