Archives of Virology

Wu, Yi-Min; Hsu, Chi-Hsin; Wang, Chun-Hsiung; Liu, Wangta; Chang, Wei-hau; Lin, Chan-Shing

doi: 10.1007/s00705-008-0150-6pmid: 18626568

Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. The results of mutational analysis of DxxDxD, which is putatively involved in the coordination of calcium ions, showed that the D133N mutation significantly disrupted the assembly of VLPs, while D130N and D135N mutants produced heterogeneous VLPs with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of the D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of the D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It was deduced that the three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and that, among them, the DxxD provides a more stable coordinate of calcium ligand than DxD.

Diallo, Ibrahim; Hewitson, Glen; Jong, Amanda; Kelly, Mark; Wright, Dick; Corney, Bruce; Rodwell, Barry

doi: 10.1007/s00705-008-0158-ypmid: 18677574

Twelve nasal swabs were collected from yearling horses with respiratory distress and tested for equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) by real-time PCR targeting the glycoprotein B gene. All samples were negative for EHV-1; however, 3 were positive for EHV-4. When these samples were tested for EHV-2 and EHV-5 by PCR, all samples were negative for EHV-2 and 11 were positive for EHV-5. All three samples that were positive for EHV-4 were also positive for EHV-5. These three samples gave a limited CPE in ED cells reminiscent of EHV-4 CPE. EHV-4 CPE was obvious after 3 days and was characterised by syncytia. None of the samples produced cytopathic effect (CPE) on African green monkey kidney (Vero) cells or hamster kidney (BSR) cells. Four of the samples, which were positive in the EHV-5 PCR, produced CPE on rabbit kidney (RK13) cells and equine dermis (ED) cells. EHV-5 CPE on both cell lines was slow and was apparent after four 7-day passages. On RK13 cells, the CPE was characteristic of equid herpesvirus, with the formation of syncytia. However, in ED cells, the CPE was characterised by ring-shaped syncytia. For the first time, a case of equine respiratory disease involving dual infection with EHV-4 and EHV-5 has been reported in Queensland (Australia). This was shown by simultaneously isolating EHV-4 and EHV-5 from clinical samples. EHV5 was recovered from all samples except one, suggesting that EHV5 was more prevalent in young horses than EHV2.

Zenke, Kosuke; Kim, Ki

doi: 10.1007/s00705-008-0162-2pmid: 18641914

All of the fully sequenced iridoviruses have an ORF resembling a putative RNase III gene. However, to the best of our knowledge, functional characterization of the iridovirus-encoded RNase III has not been done. In the present study, we have characterized the putative RNase III of rock bream iridovirus (RBIV), the major cause of mass mortality of cultured rock bream Oplegnathus fasciatus in Korea. RBIV RNase III has a single N-terminal endonuclease domain followed by a C-terminal double-stranded RNA (dsRNA) binding domain. The true presence of the predicted ORF encoding RNase III in RBIV was confirmed by temporal transcription analysis of the ORF in RBIV-infected grunt fin (GF) cells. Comparing the catalytic activity to that of previously reported RNase III proteins, including Escherichia coli RNase III, the present RBIV RNase III had different features in that: (1) the dsRNA substrate was cleaved by the RBIV RNase III at high concentrations of Mg 2+ (5–20 mM) at low salt concentration (50 mM), but the enzyme activity was completely inhibited at 200 mM NaCl (within physiological ranges) irrespective of Mg 2+ concentrations (0.5–20 mM); (2) the substrate dsRNA was cleaved at low concentrations of Mn 2+ (0.5–1 mM) at low salt concentration (50 mM) and was cleaved by increasing Mn 2+ (5–20 mM) at 200 mM salt. These features of RBIV RNase III are similar to E. coli RNase III devoid of the C-terminal dsRBD region. The exact role of the RNase III in RBIV replication is not known, and further studies are needed to elucidate whether the RNase III is involved in the suppression of host RNA interference, which attacks viral mRNAs, or in the processing of viral RNAs for effective replication.

Johal, Jasjit; Gresty, Karryn; Kongsuwan, Kritaya; Walker, Peter J.

doi: 10.1007/s00705-008-0164-0pmid: 18626567

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein GNS were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed GNS protein was also located on the cell surface but did not exhibit fusogenic activity. The GNS protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant GNS but did not react with G protein antibodies. A His6-tagged, soluble form of the G protein was expressed and purified by Ni2+–NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

Dohi, Koji; Tamai, Atsushi; Mori, Masashi

doi: 10.1007/s00705-008-0165-zpmid: 18654737

A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3–1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-γ) and successfully utilized to produce HuIFN-γ in both BY-2 protoplasts and transgenic BY-2 cells.

Yu, Zhijian; Wang, Zhanhui; Chen, Jinjun; Li, Hui; Lin, Zhanzhou; Zhang, Fan; Zhou, Yuanping; Hou, Jinlin

doi: 10.1007/s00705-008-0168-9pmid: 18668195

Multiple studies have established that GTPase activity is critical for MxA to act against RNA viruses. Recently, it was shown that MxA can also restrict the replication of hepatitis B virus (HBV), a DNA virus, but the requirements for GTPase activity in inhibition of HBV by MxA remain unknown. Here, we report that GTPase-defective mutants (K83A, T103A, and L612K) can downregulate extracellullar HBsAg and HBeAg and reduce the expression of extra- and intracellular HBV DNA in HepG2 cells to levels similar to that achieved by wild-type MxA. Furthermore, TMxA and T103, two nuclear forms of wild-type MxA and a GTPase-defective mutant (T103A) could only slightly decrease the expression of extra- and intracellular HBV DNA in HepG2 cells. In conclusion, GTPase activity is not essential for MxA protein to inhibit HBV replication, and MxA may have only a minimal effect on the replicative cycle of HBV in the nucleus.

Isoda, Norikazu; Sakoda, Yoshihiro; Kishida, Noriko; Soda, Kosuke; Sakabe, Saori; Sakamoto, Ryuichi; Imamura, Takashi; Sakaguchi, Masashi; Sasaki, Takashi; Kokumai, Norihide; Ohgitani, Toshiaki; Saijo, Kazue; Sawata, Akira; Hagiwara, Junko;

Chasseigneaux, Stéphanie; Pastore, Manuela; Britton-Davidian, Janice; Manié, Elodie; Stern, Marc-Henri; Callebert, Jacques; Catalan, Josette; Casanova, Danielle; Belondrade, Maxime; Provansal, Monique; Zhang, Yonghua; Bürkle, Alexander; Laplanche, Jean-Louis; Sévenet, Nicolas;

Takahashi, Masaharu; Yamada, Kentaro; Hoshino, Yu; Takahashi, Hideyuki; Ichiyama, Koji; Tanaka, Toshinori; Okamoto, Hiroaki

doi: 10.1007/s00705-008-0179-6pmid: 18679765

Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of >10 6 copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm 3 in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.

Shi, L.-Y.; Li, M.; Yuan, L.-J.; Wang, Q.; Li, X.-M.

doi: 10.1007/s00705-008-0184-9pmid: 18696006

The complete RNA genome sequence of Tianjin strain, isolated from marmoset, has been determined. The genome was 15,384 nucleotides (nt) in length, which was identical to that of Sendai virus (SeV), and consisted of six genes in the order 3′-NP-P/C-M-F-HN-L-5′. The gene junctions contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions (IGR) similar to those of SeV. There was also an overlapping open reading frame and a conserved AnGn site (AAAAAGGG) in the P mRNA. Phylogenetic analysis based on protein sequences from other members of the family Paramyxoviridae showed that Tianjin strain belonged to the genus Respirovirus and was most closely related to SeV. Pairwise comparisons of amino acid identities and phylogenetic analysis with homologous sequences of known SeVs demonstrated that Tianjin strain represented a new evolutionary lineage of SeV.