Kotaro Tuchiya

Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

Prevalence of swine Torque teno virus genogroups 1 and 2 in Japanese swine with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease complex

Torque teno virus (TTV) was first isolated from a human hepatitis patient in 1997. TTV was also identified in several animals, including pigs, cattle, sheep, cats and dogs. In this study, we analysed the prevalence of swine TTV genogroups 1 (TTV1) and 2 (TTV2) in Japanese swine populations with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease by using a nested polymerase chain reaction method. Of 153 serum samples from 16 different herds in Japan, TTV1 was detected in 46 samples (30%), TTV2 in 47 samples (31%) and both in 15 samples (10%). There was no significant difference in the detection rate among geographical regions. The overall prevalence rate of TTV genogroups was significantly lower in < or = 30-day-old pigs (11%) compared to that in older age groups (54-82%). These results suggest that swine TTV may be widespread in post-weaning pigs and could play aetiological roles in pig diseases in Japan. This is the first report on the prevalence of swine TTV in Japan.

Biological and immunogenic properties of rabies virus glycoprotein expressed by canine herpesvirus vector

In order to evaluate whether canine herpesvirus (CHV) could be used as a live vector for the expression of heterologous immunogenes, we constructed a recombinant canine herpesvirus (CHV) expressing glycoprotein (G protein) of rabies virus (RV). The gene of G protein was inserted within the thymidine kinase gene of CHV YP11mu strain under the control of the human cytomegalovirus immediate early promoter. The G protein expressed by the recombinant CHV was processed and transported to the cell surface as in RV infected cells, and showed the same biological activities such as low pH dependent cell fusion and hemadsorption. The antigenic authenticity of the recombinant G protein was confirmed by a panel of monoclonal antibodies specific for G protein. Dogs inoculated intransally with the recombinant CHV produced higher titres of virus neutralizing antibodies against RV than those inoculated with a commercial, inactivated rabies vaccine. These results suggest that the CHV recombinant expressing G protein can be used as a vaccine to control canine rabies and that CHV may be useful as a vector to develop live recombinant against other infectious diseases in dogs.

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections.

Multiplex PCR and multiplex RT-PCR were developed to identify nine viruses in pigs with multiple infections. These viruses are: porcine circovirus type 2 (PCV2), suid herpesvirus 1, porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus, porcine rotavirus A (PoRV-A), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and Getah virus. These methods were shown to be high specificity and sensitivity. In the clinical application, a total of 75 field samples were examined by our methods and previously reported methods for PCV2, PRRSV, TGEV, and PEDV. As a result, the detection rates of our multiplex PCR and multiplex RT-PCR were higher than those of the previously reported methods. Furthermore, it was confirmed that 24 PCV2 positive samples were co-infected with other viruses, 11 with PRRSV, 10 with PPV, 2 with PoRV-A, and 1 with TGEV by a combination of multiplex PCR and multiplex RT-PCR. PPV and PoRV-A were newly detected by multiplex PCR and multiplex RT-PCR. These results suggest that the combination of our multiplex PCR and multiplex RT-PCR is useful for rapid and accurate identification of nine major pathogenic viruses in pigs with multiple infections.

Characterization of pseudorabies virus glycoprotein gII expressed by recombinant baculovirus.

The gene encoding the complete glycoprotein gII (homologue of gB of herpes simplex virus) of pseudorabies virus (PrV) was inserted into a baculovirus transfer vector, and a recombinant virus expressing gII was isolated. Three gII-related recombinant baculovirus-expressed peptides of 100, 60, and 45 to 50 kDa were detected with a polyclonal antibody against gII; these correspond to the authentic subunits gIIa and its cleavage products gIIb and gIIc, respectively. These proteins were subjected to N-terminal sequencing, and the results showed that the protease cleavage sites were identical to those of authentic gII. The expressed gII was shown to be transported to the surface of infected cells as judged by an indirect immunofluorescence test. Antibodies raised in mice immunized with the recombinant gII neutralized the infection of PrV in vitro. Mice inoculated with the recombinant gII were completely protected from lethal challenge with PrV.

A vaccine prepared from a non-pathogenic H7N7 virus isolated from natural reservoir conferred protective immunity against the challenge with lethal dose of highly pathogenic avian influenza virus in chickens

During 2001-2004, 41 H7 influenza viruses (2 H7N1 and 39 H7N7 strains) were isolated from fecal samples of migratory ducks that flew from Siberia in the autumn of each year to Japan and Mongolia. A phylogenetic analysis of the hemagglutinin (HA) genes of the nine representative isolates revealed that they belonged to the Eurasian lineage and the deduced amino acid sequence at the cleavage site of the HAs represented apathogenic profiles. One of the H7 isolates A/duck/Mongolia/736/02 (H7N7) was chosen from these H7 isolates for the preparation of the test vaccine. To improve the growth potential of A/duck/Mongolia/736/02 (H7N7) in chicken embryos, A/duck/Hokkaido/Vac-2/04 (H7N7) was generated by genetic reassortment between A/duck/Mongolia/736/02 (H7N7) as the donor of the PB2, PB1, PA, HA, NA, and NS genes and A/duck/Hokkaido/49/98 (H9N2) as that of NP and M genes. The test vaccine was prepared as follows; A/duck/Hokkaido/Vac-2/04 (H7N7) was propagated in chicken embryos and the virus in the allantoic fluid was inactivated and adjuvanted to form an oil-in-water emulsion. The test vaccine conferred immunity to chickens, completely protecting the manifestation of clinical signs against the challenge with lethal dose of H7 highly pathogenic avian influenza virus. These results indicate that influenza viruses isolated from natural reservoirs are useful for vaccine strains.

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus.

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system.

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.

Enzyme-linked immunosorbent assay for the detection of canine coronavirus and its antibody in dogs

Abstract Two methods of enzyme-linked immunosorbent assay (ELISA) were developed for the diagnosis of canine coronavirus (CCV) infection in dogs. One ELISA, in which CCV-infected CRFK cell lysate is used as antigen, is for the detection and titration of antibody against CCV, and the other ELISA uses the double antibody sandwich method for the detection of CCV antigen. The first ELISA procedure demonstrated antibody responses in dogs inoculated with CCV, as did the virus neutralization test; the second ELISA detected specific CCV antigen in feces and organ homogenates of inoculated dogs.

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.