Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure.

A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 µg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.

Chlorine inactivation of human norovirus, murine norovirus and poliovirus in drinking water.

AIMS: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. METHODS AND RESULTS: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench-scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0.1 and 0.5 mg l(-1). Inactivation of MNV reached more than 4 log(10) after 120 and 0.5 min contact time to chlorine at the initial free chlorine concentrations of 0.1 and 0.5 mg l(-1), respectively. CONCLUSIONS: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.

Characterization of temperature-sensitive Akabane virus mutants and their roles in attenuation.

Akabane virus (AKAV) of the genus Orthobunyavirus in the family Bunyaviridae is an important animal pathogen; however, studies on AKAV biology are scarce. Therefore, we generated temperature-sensitive (ts) mutants of AKAV in order to study its pathogenesis. The ts AKAV mutants were generated by incubating the virulent OBE-1 strain with the chemical mutagen 5-fluorouracil. Each ts mutant was inoculated intracerebrally into mice to assess its virulence, and the genomic sequences of the attenuated mutants were also determined. Three of the twelve ts mutants studied showed a mortality rate of less than 10%. Although no mutation was detected in the S RNA segment of these three mutants, amino acid substitutions were observed in both the M and L RNA segments. Three of the mutants and the wild-type virus demonstrated a similar pattern of immunoreactivity in an ELISA with anti-Gc monoclonal antibodies. On the other hand, using a minireplicon system, the level of L protein activity of each ts mutant decreased as the temperature increased. These results suggest that the L RNA segment could be involved in the virulence of AKAV, which increases our understanding of how the viral gene products contribute to pathogenesis.

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Genetic analysis of calicivirus genomes detected in intestinal contents of piglets in Japan.

Enteric caliciviruses, noroviruses, and sapoviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this study, swine enteric caliciviruses were detected in ten samples of intestinal contents from 24 piglets in Japan by reverse transcription-polymerase chain reaction using a broadly reactive primer pair (P290/289) that targeted the highly conserved RNA polymerase regions of the enteric caliciviruses. From the positive samples, the entire viral genome of strain K7/JP and 3'-end parts of the genomes of strains K5/JP and K10/JP were cloned and sequenced. K7/JP had an RNA genome of 7144 bases, excluding its 3' poly (A) tail. The K7/JP genome possessed two open reading frames and characteristics common to sapoviruses. In phylogenetic analysis using amino acid sequences of VP1, K5/JP was demonstrated to be close to the noroviruses previously detected in pigs, and K7/JP and K10/JP were considered to be classified as a new genogroup of sapoviruses.

Enhancement of apoptosis via an extrinsic factor, TNF-alpha, in cells infected with cytopathic bovine viral diarrhea virus.

Isolates of bovine viral diarrhea virus (BVDV) are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. Calves persistently infected with ncp BVDV are known to develop lethal mucosal disease (MD) after superinfection by cp BVDV. Although the UV-irradiated supernatant of cp BVDV-infected cells has been reported to have no capacity to induce cell death, we found that it could enhance cell death through apoptosis. Up-regulation of tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) mRNAs was detected specifically in cp BVDV-infected primary cell cultures. Suppression of TNF-alpha via antisense oligonucleotide transfection or incubation with a polyclonal antibody against TNF-alpha resulted in attenuation of apoptosis induced by cp BVDV, suggesting that TNF-alpha participates in apoptosis execution. Although TNF-alpha is one of the iNOS-inducible factors, the iNOS up-regulation was not regulated by TNF-alpha. And iNOS was revealed to serve as anti-apoptotic factor, contrary to our expectation. In addition, the expression level of both TNF-alpha and iNOS mRNAs in the ncp BVDV-infected cells was kept lower than that in the mock-infected cells, suggesting that ncp BVDV reduced or interfered with the factor triggering the expression of both mRNAs. These characteristic mRNA transcriptions would help to explain why BVDV acts differently in cells as well as in vivo, depending on its biotype. To elucidate viral factors inducing TNF-alpha and iNOS may be critical to understand the mechanism of MD development, which closely correlates with cp BVDV-induced apoptosis.

A novel antigenic variant of Canine parvovirus from a Vietnamese dog.

Nine isolates of Canine parvovirus (CPV) were obtained from Vietnamese dogs and cats. One canine isolate showed a unique antigenic property which indicates a novel antigenic variant of CPV-2b when examined with hemagglutination inhibition tests using our monoclonal antibodies, 21C3 and 19D7, which were recently developed. This isolate had an amino acid substitution of residue 426, Asp to Glu, and the same substitution has recently been found in CPV from Italian dogs. This study first showed that such substitution caused an antigenic difference demonstrable by monoclonal antibodies and that a similar evolution may have occurred in CPV in Vietnam.

An occurrence of salmonella infection in cranes at the Izumi Plains, Japan.

Ten thousand or more cranes migrate from Siberia and stay at the Izumi Plains, in the northern part of Kagoshima prefecture, Japan, every winter season. Four hundred and twenty samples of cranes feces were obtained 1995 to 1997 and investigated for Salmonella. As a result, twenty-nine of Salmonella strains were isolated. All isolates were determined to be identical, Salmonella Typhimurium (04:i: 1,2). since all of them indicated the same patterns of plasmid profiling and antibiotic sensitive spectrums. The isolates showed a high pathogenicity to chicken, and most of them were isolated in the latter half of the winter season; therefore the cranes were infected with the isolates during the winter season.

Genetic analyses of feline foamy virus isolates from domestic and wild feline species in geographically distinct areas.

To know the genetic diversities and phylogenetic relationship among feline foamy virus (FeFV) isolates from domestic cats (Felis catus) and FeFV-related viruses from the Iriomote cats (Felis iriomotensis) and leopard cats (Felis bengalensis) in geographically distinct areas, we sequenced a partial gag-pol region of 17 strains and a partial env region of nine strains, and the U3 region of long terminal repeat of three strains of the viruses. FeFV-related viruses from the feral cats were quite similar to the FeFV from domestic cats in the sequenced regions. In the partial gag region, the identities of nucleotide sequences among the isolates were from 94 to 99%. In the partial env gene, the isolates were divided into two distinct genotypes (F17- and FUV-types) as reported by Winkler et al. (Virology 247 (1999) 144-151). More than 94% nucleotide identities were observed in the env region within a particular env genotype and about 75% nucleotide identities were noted between the two genotypes.

Reactivation of feline foamy virus from a chronically infected feline renal cell line by trichostatin A.

Although acute infection of feline foamy virus (FeFV) is normally highly cytopathogenic in Crandell feline kidney (CRFK) cells, a noncytopathic persistent infection was established in the cells after cocultivation of the initially infected cells with uninfected cells four times. To investigate reactivation of persistent infection, CRFK cells chronically infected with FeFV were treated with trichostatin A (TA), a histone deacetylase inhibitor. TA induced higher FeFV production from the Coleman strain carrier culture and also induced marked syncytium formation. In contrast, human foamy virus, which contains less homologous long terminal repeat (LTR) and putative internal promoter (IP) sequences, persistently infecting baby hamster kidney cells was not reactivated by TA. The Sammy-1 strain of FeFV, from which a part of the U3 region in the LTR is naturally deleted, showed less reactivation. The Coleman LTR promoter-based beta-Gal-expressing plasmid was activated in the persistently Coleman-infected cells in the presence of TA, whereas the Sammy-1 LTR was not activated. Furthermore, the amounts of Gag protein expressed did not change in the presence or absence of TA. Because the putative IP region was very similar between the two strains, the initiation by TA is relatively specific for LTR sequences, and, therefore, histone deacetylation is at least in part responsible for reactivation of FeFV from carrier cell culture.

Pathogenic potential of canine parvovirus types 2a and 2c in domestic cats.

The in vivo pathogenicity of canine parvovirus (CPV) type 2c (strain V203) and of CPV type 2a (strain V154) against cats was investigated. Our results indicate that both types of CPV have the potential to induce disease in cats.

Characterisation of cross-reactivity of virus neutralising antibodies induced by feline panleukopenia virus and canine parvoviruses.

It was recently reported that canine parvoviruses (CPV) had entered cat populations and induced disease in infected cats, while they had affected only dogs in the past. It is important to determine whether conventional feline panleukopenia virus (FPLV) vaccines protect against recent CPV infections. In this study, the cross-reactivity of virus-neutralising (VN) and haemagglutinin-inhibition (HI) antibodies in cats induced by FPLV and CPV s were examined. Lower cross-reactivities of VN and HI antibodies against each CPV strain were observed in cats experimentally inoculated with FPLV or vaccinated with an inactivated FPLV vaccine. In addition, we revealed the existence of a novel type of FPLV, which reacted weakly with antibodies induced by the conventional FPLV vaccine.

Green fluorescent protein gene insertion of Sendai Virus infection in nude mice: possibility as an infection tracer.

The green fluorescent protein (GFP) marker from jellyfish Aequorea victoria is considered to have potential use in the study of host-pathogen relationships, by tracing infections in living cells, organs and animals. We compared the pathogenicity of Sendai virus with an inserted GFP gene (GFP-SeV) with that of its wild-type (Wt-SeV) to determine the usefulness of the recombinant virus in long-term infection of BALB/c nude (nu/nu) mice. The results indicated that the presence of GFP in infected cells could be analyzed easily and sensitively. GFP helped in identifying and in understanding the cellular sites of viral replication in vitro and in vivo. However, the GFP insertion into the Wt-SeV genome, led to decreased pathogenicity, altering the in vivo viral kinetics.

Expression and processing of the canine calicivirus capsid precursor.

The ORF2 product of canine calicivirus (CaCV) was identified and its processing in mammalian cells was analysed. Immunoblot analysis revealed the presence of the 75 kDa capsid precursor in addition to a 57 kDa capsid protein and a 22 kDa N-terminal polypeptide in CaCV-infected cells treated at an elevated temperature. When the CaCV ORF2 was expressed in a transient mammalian expression system, only the 75 kDa precursor was detected in immunoblot analysis, suggesting that no post-translational processing occurred in this system. However, the precursor was processed to a 57 kDa protein and a 22 kDa polypeptide by the proteinase of feline calicivirus (FCV) when this was co-expressed with ORF2. Processing was blocked by site-directed mutagenesis of the putative cleavage site in the capsid precursor. The results indicate that the proteinase of FCV can cleave the capsid precursor of CaCV to produce the mature capsid protein and that CaCV may have a similar proteinase.

Genetic analysis of a canine calicivirus: evidence for a new clade of animal caliciviruses.

This paper describes the relationship of a canine calicivirus, named No.48, to other human and animal caliciviruses, based on phylogeny of the 3' half of its genome. It was found that No.48 constitutes a unique lineage, most closely related but distinct from feline and San Miguel sea lion caliciviruses.

Analysis of the N-terminal polypeptide of the capsid precursor protein and the ORF3 product of feline calicivirus.

The N-terminal unique polypeptide region of the capsid precursor protein of feline calicivirus (FCV) and the protein encoded by ORF3 of FCV were expressed as fusion proteins with glutathione S-transferase to analyze the expressed products in FCV-infected cells. Immunoblot analysis using a serum from a cat experimentally infected with FCV indicated relatively high immunogenicity of the N-terminal polypeptide in FCV-infected cats, as compared with the ORF3 protein. Specific antisera were prepared by immunization to mice with the fused proteins and used in immunoblot analysis. A 14 kD product corresponding to the N-terminal polypeptide and a 10 kD polypeptide of the ORF3 product were identified in the FCV-infected cells but not detected in the purified particles. No neutralization activity against FCV was detected in these antisera. The proteins identified as polypeptides of 14 kD and 10 kD in this study may have functions as non-structural proteins.

Isolation of feline parvovirus from peripheral blood mononuclear cells of cats in northern Vietnam.

Feline parvovirus (FPV) was isolated rather frequently from the peripheral blood mononuclear cells (PBMCs) of cats in northern Vietnam by coculturing with MYA-1 cells (an interleukin-2-dependent feline T lymphoblastoid cell line) or Crandell feline kidney (CRFK) cells (a feline renal cell line). Efficiency of virus isolation was higher in MYA-1 cells than in CRFK cells. Interestingly, among the 17 cats from which FPV was isolated, 9 cats were positive for virus neutralizing (VN) antibody against FPV, indicating that FPV infected PBMCs and was not eliminated from PBMCs even in the presence of VN antibodies in the cats.

Genetic analysis of the RNA polymerase gene of caliciviruses from dogs and cats.

Caliciviruses that infect animals including humans cause a specific disease syndrome in their respective hosts. Feline calicivirus (FCV) is a major pathogen of respiratory disease of cats, and human caliciviru is a causative agent of diarrhea. It has been suggested, furthermore, that FCV and newly recognized canine calicivirus (CaCV) may also be possible causes of diarrhea in these animal species. In this study nucleotide sequence of the RNA polymerase gene of two caliciviruses of canine origin, namely CaCV strain No. 48 and FCV-like strain Sapporo/283, and a number of FCV strains of respiratory and enteric origins was examined. The length of sequenced region, from the 5'LKDEL motif through the 3'YGDD motif of the gene, was 555 bp for CaCV No. 48 strain and 552 bp for the other FCV strains including Sapporo/283 strain. In phylogenetic analysis, CaCV No. 48 strain grouped as a distinct branch sharing ancestral roots with San Miguel sea lion virus, and FCVs formed one compact group in which Sapporo/283 strain was included.

Organization of the canine calicivirus genome from the RNA polymerase gene to the poly(A) tail.

In recent years a wealth of data has become available about the caliciviruses that infect humans, as well as those which infect a range of animal species, notably cats, rabbits, pigs and marine animals. However, in the two decades since the earliest reports of calicivirus infection in dogs, very little has become known about the epidemiology, pathogenicity and molecular biology of the caliciviruses that may infect canines. In 1990, a canine calicivirus (CaCV) was isolated from a 2-month-old diarrhoeic domestic dog in Japan. This virus, which can be grown in cultured cells of canine origin, has the classic 'Star of David' morphology of caliciviruses, and the one major structural protein was shown to be immunogenic in dogs. In this study, a 3.8 kb region of the genome of this CaCV isolate from the RNA polymerase gene to the 3' poly(A) tail was cloned and sequenced, and phylogenetic analysis was undertaken in order to establish the relationship of CaCV to other animal and human caliciviruses. This CaCV isolate had a nucleotide sequence, genomic organization and phylogenetic position closest to, but clearly distinct from, both feline calicivirus and San Miguel sea lion virus isolates. These findings suggest that CaCV represents a new clade of animal caliciviruses, presumably as a member of the recently proposed new genus Vesivirus.