Evaluation of tri-combinant vaccine for feline herpesvirus, calicivirus and panleukopenia virus infections in Japanese native cats

Tri-combinant vaccine consisting of attenuated feline herpesvirus (FHV) and feline calicivirus (FCV) and inactivated feline panleukopenia virus (FPLV), were evaluated for safety and efficacy, using Japanese native cats and the viral strains isolated in Japan. Thirty-eight 9- to 12-week-old kittens were inoculated intramuscularly and subcutaneously with the vaccine. Consequently, no adverse reaction was found, and protective efficacy was confirmed by challenge tests with the virulent strains of each virus. Serum-neutralizing antibodies against FCV and FPLV were maintained for at least one year after vaccination, whereas antibody against FHV disappeared in two cases at 24 weeks after vaccination. Application of this vaccine seemed effective for control of feline viral disease in cats for experimental use.     

Identification of regions and residues in feline junctional adhesion molecule required for feline calicivirus binding and infection

The feline junctional adhesion molecule A (fJAM-A) is a functional receptor for feline calicivirus (FCV). fJAM-A is a member of the immunoglobulin superfamily (IgSF) and consists of two Ig-like extracellular domains (D1 and D2), a membrane-spanning domain, and a short cytoplasmic tail. To identify regions of fJAM-A that interact with FCV, we purified recombinant fJAM-A ectodomain and D1 and D2 domains. We found that preincubation of FCV with the ectodomain or D1 was sufficient to inhibit FCV infection in plaque reduction assays. In enzyme-linked immunosorbent assays, FCV binding to fJAM-A ectodomain was concentration dependent and saturable; however, FCV bound D1 alone weakly and was unable to bind D2. To characterize FCV binding to surface-expressed fJAM-A, we transfected truncated and chimeric forms of fJAM-A into a nonpermissive cell line and assayed binding by flow cytometry. Only D1 was necessary for FCV binding to cells; all other domains could be replaced. Using a structure-guided mutational approach, we identified three mutants of fJAM-A within D1 (D42N, K43N, and S97A) that exhibited significantly decreased capacities to bind FCV. In contrast to our finding that D1 mediated FCV binding, we found that all domains of fJAM-A were necessary to confer susceptibility to FCV infection. Furthermore, surface expression of fJAM-A was not sufficient to permit FCV infection by all of the isolates we investigated. This indicates that (i) other cellular factors are required to permit productive FCV infection and (ii) individual FCV isolates differ in the factors they require.     

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts.     

Inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide

Chlorine dioxide (ClO2) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO2 multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct99.99%) at 5 degrees C were >0.77 to <1.53 mg/liter x min and >0.80 to <1.59 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C AD40 experiments, >0.49 to <0.74 mg/liter x min and <0.12 mg/liter x min Ct99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct99.99% ranges for 5 degrees C experiments were >20.20 to <30.30 mg/liter x min and >0.68 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C FCV experiments, Ct99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter x min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15 degrees C than at 5 degrees C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct99.99% ranges and EFH model Ct99.99% values demonstrated that FCV is more resistant to ClO2 than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct99.99% values are higher than Ct99.99% values calculated from bench-scale experiments and from EFH model application.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

A bipartite sequence motif induces translation reinitiation in feline calicivirus RNA

The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.     

A nonstructural protein of feline panleukopenia virus: expression in Escherichia coli and detection of multiple forms in infected cells.

Sequences coding for the nonstructural protein NS1 of the autonomous parvovirus feline panleukopenia virus were expressed in Escherichia coli as fusion proteins. The fusion proteins were specifically bound by antisera from canine parvovirus-infected dogs. Antisera against one of the fusion proteins bound to several proteins found only in feline panleukopenia virus-infected feline cells.     

Leader of the capsid protein in feline calicivirus promotes replication of Norwalk virus in cell culture

The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3′ end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.     

Serologic and virologic surveys on feline herpesvirus and feline calicivirus infections in cats for experimental use

Serologic survey and virus isolation of feline herpesvirus (FHV) and feline calicivirus (FCV) were performed on cats used for research at the Laboratory Animal Research Centre, The University of Tsukuba, over the period from 1978 to 1981. Of the 507 mature and immature cats, 4 months old or older, 102 (20.1%) had HI antibody against FHV and 412 (81.3%) SN antibody against FCV. Some 23 (16.2%) and 76 (53.5%) kittens among 142 younger than 4 months had antibodies against FHV and FCV, respectively. Both the antibodies in kittens were considered to be maternally derived. The FCV antibody rate was especially high in cats weighing 2.5 kg (males) and 2.0 kg (females) or more, which were regarded as 8 to 10 months of age. An attempt was made to isolate the viruses from the oropharynx and conjunctiva of clinically healthy mature or immature cats and kittens. As the result, either one or both of the viruses were isolated from 31 of 75 mature and immature cats, and isolation rates of FHV and FCV were 6.7% and 36.0%, respectively. On the other hand, no virus was detectable in 16 kittens.     

虎源猫泛白细胞减少症病毒的分离鉴定

利用细胞培养方法从中国某虎园送检的腹泻虎肠内容物中分离出1 株细小病毒(TPV/ HT - 69),经系统的形态学、理化学、血清学试验、人工感染试验、PCR 扩增和VP2 基因序列分析,符合猫泛白细胞减少症病毒特征,证明该毒株为猫泛白细胞减少症病毒强毒。     

Mucosal infection and vaccination against feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8+ cells and greater levels of IL-2, IL-6, IL-10 and gamma-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats.     

Serological analysis and identification of feline calicivirus strains isolated in Sicily

The isolation and characterization of calicivirus strains from symptomatic cats are reported. The correlations between the feline calicivirus strains isolated and the vaccinal strain FCV F9 were investigated by a virus-neutralization test, suggesting a strong antigenic variability.     

Histiocytic conversion of human adult skin fibroblasts by the Snyder-Theilen feline sarcoma virus

Oncogenes are apparently involved in the transformation/neodifferentiation of human cells. We now report a novel example of transformation/neodifferentiation: the specific conversion of cultured human adult skin fibroblasts (HSF) to histiocytes (tissue macrophages (TM)) by the Snyder-Theilen feline sarcoma virus (ST: (FeSV)). The de novo conversion of cultured HSF was demonstrated in a large fraction of ST:FeSV (FeLV)-transformed foci in the presence of dexamethasone (DX). Identification of tissue macrophages in ST:FeSV(FeLV)-infected HSF cultures was established by light-and transmission electron microscopy, histochemistry, immunohistochemistry, adherence-reattachment, latex particle uptake, and secretion of bioactive interleukin-1. The ST:FeSV gene and glucocorticosteroids, or other naturally occurring hormones, may play a role in morphogenetic processes within cells from a variety of normal and diseased tissues in situ, including induction of non-bone marrow, mesenchyme-derived, tissue macrophages.     

Entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes

Feline calicivirus is a major causative agent of respiratory disease in cats. It is also one of the few cultivatable members of Caliciviridae. We have examined the entry process of feline calicivirus (FCV). An earlier study demonstrated that acidification in endosomes may be required. We have confirmed this observation and expanded upon it, demonstrating, using drugs to inhibit the various endocytic pathways and dominant-negative mutants, that FCV infects cells via clathrin-mediated endocytosis. We have also observed that FCV permeabilizes cell membranes early during infection to allow the co-entry of toxins such as alpha-sarcin. Inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked this permeabilization event, demonstrating that acidification is required for uncoating of the genome and access to the cytoplasm.     

Prevalence of feline leukemia virus infection in random source laboratory cats

Three years of quarantine data involving the prevalence of Feline Leukemia Virus (FeLV) in laboratory cats (Felis catus) was evaluated. Testing was performed using a commercially available ELISA system. Annual prevalence of infection ranged from 5.4 to 10.7% of the 937 cats tested. Results obtained with the enzyme linked immunosorbent assay (ELISA) system compared well with tests performed using an immunofluorescent antibody assay system (IFA). Seasonal periodicity was noted with higher infection rates among animals received in April. Holding period at the supplier and gender did not influence disease prevalence. The availability of a simple test system, the unacceptability of FeLV infected cats for research, the danger of transmission and a higher prevalence of infection than earlier reports indicated that supplier and user evaluation of research cats for FeLV would be prudent.     

First case of peritoneal cystic echinococcosis in a domestic cat caused by Echinococcus granulosus sensu stricto (genotype 1) associated to feline immunodeficiency virus infection

A new cystic echinococcosis case in a cat in Uruguay is reported herein. The cat was taken to a veterinary clinic in Rocha city, Uruguay, due to dyspnea, constipation and abdominal enlargement. During surgery a large quantity of cysts was retrieved from the abdominal cavity. The cysts were morphologically studied and confirmed as Echinococcus granulosus sensu stricto (genotype 1) by molecular tools using cytochrome oxidase submit 1 and small subunit ribosomal RNA gene as target genes. Moreover, for the first time a coinfection with feline immunodeficiency virus (FIV) was detected. FIV-induced immunosuppression could be a determining factor in the development of cystic echinococcosis in cats.