Intranasal immunization with inactivated feline calicivirus particles confers robust protection against homologous virus and suppression against heterologous virus in cats.

The protective efficacy of intranasal (IN) administration of inactivated feline calicivirus (FCV) vaccine against homologous or heterologous FCV infection was investigated. Groups of cats immunized with the experimental inactivated, non-adjuvanted FCV vaccine via either the IN or subcutaneous (SC) route were exposed to homologous or highly heterologous FCV. Both the IN and SC immunization protocols established robust protection against homologous FCV infection. Although neither immunization regimen conferred protection against the heterologous strain, clinical scores and virus titres of oral swabs were lower in cats in the IN group compared to those in the SC group, accompanying a faster neutralizing antibody response against the heterologous virus in cats in the IN group. The IN group secreted more IgA specific to FCV proteins in oral washes (lavage fluids from the oral cavity) than the SC group. IN immunization with an inactivated whole FCV particle, which protects cats from homologous virus exposure and shortens the period of heterologous virus shedding, may serve as a better platform for anti-FCV vaccine.

Genetic and antigenic characterization of newly isolated bovine toroviruses from Japanese cattle.

Torovirus, a member of the Coronaviridae family, is a gastrointestinal infectious agent that has been identified in humans, cattle, pigs, and equines. Toroviruses, except equine torovirus, are difficult to propagate in cell culture; indeed, to date, only the Aichi/2004 strain of bovine torovirus (BToV) has been isolated among the human, bovine, and porcine toroviruses. In the present study, four cytopathogenic BToVs were isolated from diarrheal feces of the cattle using the HRT-18 cell line, and their genetic and antigenic properties were compared. The cytopathogenic features of BToV isolates in HRT-18 cells were similar to those of the Aichi/2004 strain. However, none of the isolates showed cytopathogenic effects in the HRT-18 cells of different origin, suggesting that one significant factor contributing to the cytopathogenicity of BToV depends on properties of the HRT-18 cells themselves. All BToVs isolated were able to agglutinate mouse, but not chicken, erythrocytes, while they lacked receptor-destroying enzyme activity. Analysis of the N terminus of the spike gene showed that three isolates, but not the Gifu-2007TI/E strain, were phylogenetically located in cluster 1 and its analogs and revealed high cross-reactivity with each other, as demonstrated by neutralization (NT) and hemagglutination inhibition (HI) assays. The Gifu-2007TI/E strain was classified close to cluster 2 and exhibited relatively low cross-reactivity with these viruses; however, the difference was not sufficient to classify BToVs into serotypes, suggesting that at least two subtypes distinguishable by the structure of the N terminus of the spike gene and that both NT and HI tests may be exist.

Detection and characterization of bovine torovirus from the respiratory tract in Japanese cattle.

Bovine torovirus (BToV), a member of the Coronaviridae family, is a causative agent of diarrhea in cattle, but it may also possess tropism for the respiratory tract. However, no surveys concerning with the relation between respiratory symptoms and the detection of BToV have been conducted in wide range. Among 311 nasal samples, BToV gene products were detected in seven samples (rBToV-1 to -7) derived only from calves with respiratory symptoms, suggesting that BToV may be a predisposing factor and/or causative agent for bovine respiratory disease. Regarding the degree of similarity between the spike and hemagglutinin-esterase coding regions, the rBToVs showed over 90.8% similarity with one another and 73.5-99.0% similarity with fecal tract-derived BToVs. rBToV-1, -2, and -3 were identical despite their being collected during different seasons; in comparison, rBToV-4 and -5 were distinct despite the fact that they were collected from the same herd, suggesting the existence of diversity among domestic rBToVs. One animal with a BToV-positive nasal sample also shed the virus in its feces, suggesting dual tropisms for BToV.

Epidemiological analysis of bovine torovirus in Japan.

Bovine torovirus (BToV), a member of the Coronaviridae family, is an established gastrointestinal infectious agent in cattle. No epidemiological research on BToV has been reported from Japan. In this study, we performed a survey to detect BToV in Japan in 2004 and 2005 using 231 fecal samples (167 from diarrheic cattle and 64 from asymptomatic cattle) that were analyzed by nested reverse transcription (RT) PCR using primers located in the consensus sequences of the reported BToV nucleocapsid (N), membrane (M), and spike (S) genes. BToV N, M, and S genes were detected in 6.5% (15/231), 6.1% (14/231), and 5.6% (13/231) of samples by nested-RT-PCR, respectively. In conclusion, detectability was improved compared to the results of the first round of RT-PCR. BToV was detected at a significantly higher rate in diarrheic samples than in asymptomatic samples (14/167 diarrheic samples [8.4%] and 1/64 asymptomatic samples [1.6%]), suggesting that BToV may act as a risk factor for diarrhea in Japanese cattle. The nucleotide sequence of M fragments from the BToV isolates including the newly identified Japanese isolates showed more than 97% identity. A similar degree of homology was observed in the N gene fragment among BToV isolates with the exception of BRV-1 and BRV-2. Domestic samples were classified into three clusters by phylogenetic analysis of the S gene fragment, which were considerably correlated with the geographic origin of the samples. BToV positive areas did not adjoin each other but were spread across a wide range, suggesting that BToV exists conventionally in Japan and is geographically differentiated. We also developed an RFLP method to distinguish these clusters using two restriction enzymes, HaeIII and AccI. This method should be useful for comparing newly acquired BToV-positive samples with the reported BToVs.

Bovine rotavirus G and P types and sequence analysis of the VP7 gene of two G8 bovine rotaviruses from Japan.

A total of 1481 fecal specimens were collected from diarrheic calves under 1 month of age on 29 dairy and beef farms in 11 prefectures in Japan during the period from 1987 to 2000. Those calves and their dams were not vaccinated against rotavirus. One hundred and forty-two bovine rotaviruses were isolated on MA-104 cell cultures and detected by latex agglutination test. They were classified into 18 G6P[1] (11.2%), 53 G6P[5] (37.3%), 15 G6P[11] (10.6%), 12 G10P[5] (8.5%), 42 G10P[11] (29.6%) and 1 G8P[11] (0.7%) by reverse transcription-polymerase chain reaction. One serotype G8 virus was untypable for the P genotype suggesting a new type of bovine origin. The least common G8 serotype viruses were isolated from the samples of farms from Niigata and Tokushima prefectures. The VP7 gene sequences of the two isolates exhibited a high degree of homology as well as previously reported G8 viruses with 93.3-98.8% identity of deduced amino acids. A phylogenetic analysis of the VP7 gene of the two G8 viruses and 13 previously reported G8 viruses by the neighbor-joining method indicated that the two newly isolated G8 rotaviruses had a common origin and they were assigned to a new disparate cluster.