Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines.

BACKGROUNDS: The aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. METHODS: The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines. RESULTS: Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. CONCLUSION: The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

The Asia 2 specific signal peptide region and other domains in fusion protein genes characterized Asia 1 and Asia 2 canine distemper viruses.

BACKGROUND: Although the presence of Asia 2 group of canine distemper virus (CDV) was known by the sequencing and phylogenetic analysis of hemagglutinin (H) gene, the fusion (F) protein gene sequence of Asia 2 group had not been identified. So, the sequence analysis of F gene was carried out to elucidate the genotypic varaitons among Asian isolates. RESULTS: The phylogenetic analysis of F and H gene sequences from fourteen CDV isolates obtained from diseased dogs in Japan and Thailand indicated that the F genes had a new initiation codon and extra 27 nucleotides upstream of the usual open reading frame (ORF) and the F proteins had extra 9 amino acids at the N-terminal position only in Asia 2 isolates. On the contrary, the Asia 1 isolates had three extra putative N-glycosylation sites (two sites in the signal peptide region and one site in the F1 region) except for two strains of Th12 and Ac96I (two sites in signal peptide region) adding to four putative N-glycosylation sites that were conserved among all Asian isolates and Onderstepoort strain. In addition to this difference in N-glycosylation sites, the signal peptide region had a great diversity between Asia 1 and Asia 2 isolates. Also, characteristic amino acids were detected for some strains. CONCLUSION: Asia 2 isolates were distinguished from other CDV lineages by the extra 27 nucleotide sequence. The signal peptide region of F gene gives a remarkable differentiation between Asia 1 and Asia 2 isolates. Strains Th12 and Ac96I were differentiated from other Asia 1 strains by the F protein glycosylation sites.

Detection of antibodies against Japanese encephalitis virus in raccoons, raccoon dogs and wild boars in Japan.

Japanese encephalitis virus (JEV) infects numerous animal species including humans, horses and pigs. In this study, antibodies against JEV in feral raccoons (Procyon lotor), wild boars (Sus scrofa) and raccoon dogs (Nyctereutes procyonoides) in Japan were examined. The results showed that 40.7% (22 out of 54), 64.5% (40 out of 62), 69.1% (47 out of 68) and 0% (0 out of 20) of raccoons in Hyogo, Osaka, Wakayama and Hokkaido, respectively, had virus-neutralizing antibodies against JEV. In addition, 83.3% (30 out of 36) of wild boars and 63.2% (12 out of 19) of raccoon dogs in Wakayama were seropositive for JEV. There were no significant differences in seroprevalence of JEV between males and females or between adults and juveniles in these wild animals. JEV seroprevalence was compared between 37 raccoons and 30 wild boars captured in a limited period (November 2007 to February 2008), and we found that wild boars (86.7%) were significantly more seropositive for JEV antibody than raccoons (59.5%). In conclusion, JEV was prevalent in wild mammals, indicating that the possibility of JEV infection in humans may still be high in Japan. In addition, these wild animals may be good sentinels to estimate JEV infection risk in residents, as they live near humans and are not vaccinated.

Relationship between growth behavior in vero cells and the molecular characteristics of recent isolated classified in the Asia 1 and 2 groups of canine distemper virus.

Ten recent isolates of canine distemper virus (CDV) strains were classified according to the growth ability and development of syncytial cytopathic effects (CPE) in Vero cells. Strains P94S, Ac96I, S124C, MD231, MS232, MSA5 and 095Cr were classified as Type 1 and exhibited hardly and did not develop CPE in Vero cells. Strains 007Lm, 009L and 011C were classified as Type 2 as grew well but failed to develop a syncytial CPE in Vero cells. A comparison of the phylogenetic trees of the H and P genes showed that all Type 1 strains belonged to the Asia 1 group and all Type 2 strains belonged to the Asia 2 group. Our findings suggest that the recent Asia 2 isolated of CDV in Japan, but not Asia 1 may grow in Vero cells, and their growth ability may be related with their molecular characteristics.

Automated SNPs typing system based on the Invader assay.

Typing of single nucleotide polymorphisms (SNPs) has advantages in forensic DNA identification because SNPs are abundant in genomic DNA and are easily detected using an automated high-throughput system. In this study, we examined the effectiveness of an automated multiplex SNPs typing system based on the "Invader assay". Using this system, multiplex SNPs typing is completed in 40 min without any complex procedures. The typing results were confirmed by direct sequencing, and no inconsistencies were detected between the sequencing results and the typing results. The population data of 21 SNPs from 113 Japanese indicates that the matching probability is about 1.3 x 10(-9). In our experiment, all of 21 SNPs were correctly detected from degraded samples from which typing of short tandem repeat markers was difficult.

Further Development of an Equine Cell Line that can be Propagated over 100 Times.

Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 cells, and this CPE was very similar to those seen in parental FHK-Tcl3 and primary fetal horse kidney cells. FHK-Tcl3.1 cells continue to propagate and the current passage record is over 100 times after cloning. Therefore, this cell appears to have been immortalized. FHK-Tcl3.1 cells have potential for growth and diagnosis of various equine viruses, including equine herpesviruses.

Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses.

In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.

Genomic diversity among equine herpesvirus-4 field isolates.

Infection with equine herpesvirus-4 (EHV-4) is a major cause of respiratory tract disease, equine rhinopneumonitis, in horses. Although the full sequence of EHV-4 has been reported, genomic differences among EHV-4 field isolates have not yet been characterized. In this study, the genomic diversity between 23 Japanese EHV-4 isolates was analyzed by digestion with restriction endonucleases (BamHI, BgIII, EcoRI, SacI, and SalI) and polymerase chain reaction (PCR). The restriction endonuclease digestion patterns of the EHV-4 field isolates showed distinct differences which included mobility shifts of some fragments as well as loss and/or gain of fragments. Two EHV-4 genes containing repeat sequences, ORFs 24 and 71, were amplified by PCR and the amplified fragments were compared among the field isolates. The sizes of the amplified fragments varied among epizootiologically unrelated isolates, while the fragments of related isolates had the same size. The observed genomic diversity among EHV-4 field isolates may be a useful tool for epidemiological study of equine rhinopneumonitis by EHV-4 infection.

The growth profiles of three types of canine distemper virus on Vero cells expressing canine signaling lymphocyte activation molecule.

To know growth profiles of canine distemper virus (CDV) on Vero cells stably expressing canine signaling lymphocyte activation molecule (Vero-DogSLAMtag; Vero-DST cells), the propagation of three strains of CDV was tested in Vero-DST cells in comparison with parental Vero cells. Strain MD77 could grow well in both cell lines, but demonstrated no syncytium formation or indistinguishable rounding cytopathic effects (CPE) in Vero cells. Strains Onderstepoort and KDK-1 also grew well in Vero-DST cells with apparent syncytium CPE, while they grew less or no efficiently, respectively, in Vero cells. All three CDV strains demonstrated the peak titers, in Vero-DST cells before reaching to an extensive CPE and drastic decrease of titers at/after full CPE. Immunohistochemistry revealed that viral antigens of all CDV strains were found exclusively in the syncytia in Vero-DST cells, while in Vero cells, viral antigen was identified in their single cells for strain MD77 but none for other strains. Thus, every strain of CDV could grow well in Vero-DST cells and behaved differently against Vero cells. These results would be of practical value for workers of CDV because 1) In Vero-DST cells, by observation of distinct syncytium CPE, the highest titer or the best growth of virus could be identified; 2) In Vero cells, various CDV strains could be readily classified after propagation in Vero-DST cells.

A novel genetic marker to differentiate feline herpesvirus type 1 field isolates.

Five recent field isolates of feline herpesvirus type 1 (FHV-1) were compared by digestion with a restriction endonuclease, SalI or MluI. The SalI digestion showed a potentially useful difference in one isolate 00-035 that had an approximately 3.0 kbp fragment instead of a 2.6 kbp fragment in the other strains. After cloning the 3.0 and 2.6 kbp fragments, the nucleotide sequences were analyzed. The result showed that the 3.0 kbp fragment of 00-035 included a complete open reading frame of the herpes simplex virus 1 (HSV-1) homologue of the UL17 gene and a 5'-part of UL16 gene and that only one nucleotide substitution was found in the 5'-region of UL17 gene where the SalI site of the 2.6 kbp fragment locates. Based on these nucleotide sequences, two PCR primers were designed to amplify the region around the SalI site in the UL17 gene and the PCR was carried out using 78 field isolates from various parts of Japan. The SalI digestion of the PCR products revealed an interesting profile in that the genotype without the SalI site in UL17 gene was dominant in Tottori and Yamagata prefectures (69% and 75%, respectively) but minor in the other regions of Japan (0-10%). These results suggest that the SalI digestion method described in the present study can be used as a genetic marker to differentiate some FHV-1 field isolates and this is the first report that showed different distributions of FHV-1 genotypes using the novel genetic marker.

Identification of another B-cell epitope in the type-specific region of equine herpesvirus 4 glycoprotein G.

Recently, a novel 12-mer B-cell epitope, MKNNPIYSEGSL, in the type-specific region of equine herpesvirus 1 (EHV-1) glycoprotein G (gG) was identified and used as an antigen for enzyme-linked immunosorbent assay (Maeda et al., J. Clin. Microbiol. 42:1095-1098, 2004). Although our prototype strain, TH20p, possesses two repeat sequences containing the B-cell epitope, the EHV-4 NS80567 strain has two repeat sequences that are not identical. One repeat sequence stretch contained the B-cell epitope, while the other contained the 11-mer, MKNNPVYSESL (underlining indicates a different amino acid). In this study, heterogeneity of the type-specific region was compared among Japanese EHV-4 isolates. The 11-mer peptide, MKNNPVYSESL, specifically reacted with sera from horses naturally infected with EHV-4 but not with sera from horses experimentally infected with EHV-4 TH20p. The 11-mer peptide may be another B-cell epitope in the type-specific region.

Development of an equine herpesvirus type 4-specific enzyme-linked immunosorbent assay using a B-cell epitope as an antigen.

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol. 67:6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci. 60:1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci. 62:687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione S-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.

Genetic rearrangements in the gC gene of the feline herpesvirus type 1.

In the field isolate, 91-58, of feline herpesvirus type 1 (FHV-1), one of the major immunogenic proteins was found to have different molecular masses of 75 and 130 kDa from those in the other field isolates (Maeda et al., J Vet Med Sci 57, 147-150, 1995). Immunoblot analysis using monoclonal antibodies (MAbs) indicated that the protein is glycoprotein C (gC). The gC gene of 91-58 was amplified by polymerase chain reaction (PCR) and shown to have an inserted fragment of approximately 160 base pairs (bp). Restriction endonuclease analysis of the PCR product with various restriction enzymes was carried out, indicating that the insertion located within 262 bp between Eco RV and DraI sites. Nucleotide sequence analysis indicated that the inserted fragment was 156 bp encoding 52 amino acids and composed repeat sequences. Next, five recent isolates were also examined by immunoblot analysis using anti-FHV-1 cat serum or MAbs. The result showed that one isolate, 98-064, also had the gC with different molecular weights. PCR and nucleotide sequence analyses indicated that 98-064 had an inserted sequence of 78 bp at the corresponding region identified in the gC gene of 91-58, although the inserted sequence was different from that of 91-58. These results indicated that some of FHV-1 isolates had the genetic rearrangements in the gC gene and detection of such mutations would be useful for differentiation among FHV-1 field isolates.

Experimental infection of recent field isolates of feline herpesvirus type 1.

Two field isolates of feline herpesvirus type 1 (FHV-1) designated as 00-015 and 00-035, were obtained from cats diagnosed as feline viral rhinotracheitis (FVR) in Japan. To analyze the character of recent FHV-1, these two isolates and our laboratory strain C7301 were inoculated experimentally to specific-pathogen-free cats. Although all cats showed typical FVR symptoms, more severe clinical symptoms were observed on cats infected with the isolates 00-015 and 00-035 compared with those of C7301-infected cats. Severe ocular lesions including conjunctivitis were found in the cats infected with the isolates, indicating that the recent FHV-1 has a potential to induce severe FVR symptoms including ocular lesions.