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.     

Identification of feline herpesvirus type 1-hemagglutinin

The crude hemagglutinin of feline herpesvirus type 1 (FHV-1), solubilized from infected fcwf-4 cells by detergents, was partially purified by three kinds of chromatographic methods. Lectin-affinity chromatography showed the hemagglutination (HA) activity in fractions, which was bound to Concanavalin A-sepharose and then eluted by alpha-methyl D-mannoside, suggesting that the hemagglutinin might include a glycoprotein. Ion-exchange and gel-exclusion chromatographies were also capable of purifying the detergent-soluble crude hemagglutinin. When peak HA fractions, which were obtained from each of the three procedures, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the gel-exclusion chromatography was the most effective method. Electrophoreic analysis also showed only one band of 59,000 (59K) molecular weight protein, which was commonly observed in the three partially purified hemagglutinins with silver staining. In addition, the 59K protein band was clearly recognized in immunoblot analysis of the infected cell lysates using infected cat serum. These observations suggest that the FHV-1 detergent-soluble hemagglutinin from infected fcwf-4 cells may be closely related to a 59K immunogenic glycoprotein.     

Clinical and immunological responses of cats to feline herpesvirus type 1 infection

Cats which were challenged with feline herpesvirus type 1 developed clinical signs typical of feline viral rhinotracheitis whether or not they had been vaccinated against the disease. However, the clinical disease was less severe and of shorter duration in the vaccinated cats. After challenge, feline herpesvirus type 1 was recovered from the nostrils, oropharynx and peripheral blood leucocytes. Leucocytosis, primarily a neutrophilia, occurred initially in all the cats and was followed after clinical recovery by a mild lymphocytosis. Intradermal skin testing with feline herpesvirus type 1 and cell control antigens produced a positive delayed type skin reaction. Histology of the affected skin 72 hours after injection showed cellular infiltration, predominantly with eosinophils and neutrophils. The severity of the reaction was greater and more prolonged in the skin of the ear than in the skin of the abdomen.     

A specific PCR to differentiate between gE negative vaccine and wildtype bovine herpesvirus type 1 strains

In the context of infectious bovine rhinotracheitis (IBR) control programmes using glycoprotein E (gE) deleted marker vaccines, a PCR assay was developed to allow the genotypic differentiation between wildtype bovine herpesvirus type 1 (BoHV-1) and gE negative strains. This assay is based on the PCR amplification of a 281 bp DNA fragment within the gE gene. The specificity of the amplification was confirmed by restriction endonuclease analysis and nucleotide sequencing of the PCR product. Its ability to determine the gE genotype of BoHV-1 strains was demonstrated on isolates coming from 20 experimental calves infected with four different BoHV-1 strains. This PCR assay may be a useful tool for monitoring the spread of live marker vaccine and the gE genotype of viral field isolates.     

Genetic relatedness of disease-associated field isolates of bovid herpesvirus type 4

Eight field isolates of bovid herpesvirus type 4 (BHV-4) were examined by restriction analysis and Southern blot hybridization with respect to their relatedness to one another and to the BHV-4 prototype strain DN-599. Isolates were obtained from cattle exhibiting a range of disease states including abortion, pneumonia, enteritis, metritis, and vaginal blisters. Initial growth studies of all 9 viruses were performed and revealed that the overall rate of virus growth was slow when compared with that of other herpesviruses. Infection with each virus also resulted in the formation of large fused cells, which in addition to the slow growth rate, indicated that the isolates were of the cytomegalovirus type. Further studies to characterize and compare the various BHV-4 isolates were undertaken by obtaining cell-free virus from infected cell populations. Viral isolates were purified and used as a source of BHV-4 DNA. Purified DNA, representing each of the 8 field isolates and the prototype strain DN-599, were each cleaved with 3 restriction enzymes and were separated by agarose-gel electrophoresis, and the resultant fragment patterns were compared. In general, genomic fragments of the field isolates corresponded to those generated by cleavage of DN-599 DNA, with the exception of the abortion-associated isolate 83-3572. Additional minor differences were also seen between DN-599 DNA and DNA from the other field isolates, but the overall restriction patterns were similar. To confirm that all isolates were members of the BHV-4 type, hybridization studies were performed using DN-599.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of a live bovine herpesvirus type 1 marker vaccine under field conditions in three countries

The performance of a live marker vaccine for bovine herpesvirus type 1 (bhv-1) was studied in the field in three European Union countries with different farming conditions. The progress in the eradication of the virus was followed in a large herd in Germany and one in Italy, and a major serological survey involving 147 farms was conducted in Hungary. Commercial batches of the same vaccine were used in all three studies. The herds were vaccinated according to agreed protocols and the animals' bhv-1 antibody status was determined at local institutes by using commercial glycoprotein B (gB)- and glycoprotein E (gE)-elisas. In all three studies, the seroprevalence of bhv-1 gE decreased progressively. Given the starting conditions and the long duration of the studies, reactivation events and virus circulation would have been more likely to have occurred if the herds had not been vaccinated.     

Post-exposure treatment of cats with mouse-cat chimeric antibodies against feline herpesvirus type 1 and feline calicivirus

In order to confirm the in vivo effectiveness of anti- feline herpesvirus type 1 (FHV-1) mouse-cat chimeric antibody (FJH2), and anti-feline calicivirus (FCV) mouse-cat chimeric antibody (F1D7), cats that had been experimentally infected with FHV-1 or FCV were administered intravenously with the chimeric antibodies, and observed for clinical manifestations. The symptoms due to FHV-1 or FCV infection in the cats administered FJH2 or F1D7 were obviously decreased when compared with those of the non-administered control cats. From these results, it was confirmed that both FJH2 and F1D7 were effective at reducing the appearance of symptoms due to FHV-1 and FCV infection, respectively.     

Intracellular localization and epitope mapping of feline herpesvirus type 1 glycoproteins.

Monoclonal antibodies (MoAbs) were used to characterize feline herpesvirus type 1 (FHV-1) glycoproteins (gp). Intracellular localization and transport of these proteins as revealed by a sequential indirect immunofluorescence assay (IFA) on fixed infected cells showed slight differences between FHV-1 gp143/108 and gp113. Antibodies against gp143/108 first showed membrane fluorescence at 4 hrs post-infection (PI) followed by a pronounced perinuclear and cytoplasmic staining from 8 hrs PI onwards. Those reacting with gp113 showed the same pattern but fluorescence did not appear until 8 hrs PI. In contrast, MoAbs against gp60 first showed para- and perinuclear staining at 12 hrs PI which became intranuclear at 16 hrs PI, followed by intracytoplasmic staining at 20 hrs PI. Sequential IFA of unfixed infected cells revealed that the three glycoproteins were expressed on the cell surface membrane as well. Topographical mapping of the functional epitopes of gp113 by ELISA additivity test indicated the presence of 2 antigenic domains--a neutralizing domain consisting of 3 overlapping epitopes and a non-neutralizing domain. On the other hand, gp143/108 contained only one antigenic site consisting of 5 similar or overlapping epitopes, one of which seemed to be a conserved region recognized by all MoAbs reacting to this protein.     

Responses of cats to nasal vaccination with a live, modified feline herpesvirus type 1

Intranasal vaccination with a cold-adapted strain of feline herpesvirus type 1 (FHV-1) two days before challenge gave partial protection, and four days before challenge gave complete protection, against feline viral rhinotracheitis. Protection at this time appeared to be specific since vaccination with FHV-1 did not affect the disease caused by the unrelated feline calicivirus. The time course of onset of protection also confirmed that the protective mechanism was likely to be specific. However, six days after vaccination only low levels of FHV-specific IgA and IgM antibody and of interferon were found in serum and nasal washings. In lymphocyte transformation assays neither peripheral blood lymphocytes nor tonsil lymphocytes gave a significant proliferative response in the presence of FHV antigen. Pathogenesis experiments demonstrated that the tonsil and nasal turbinates were the most important sites of virulent FHV-1 replication. Vaccination significantly reduced levels of infectious virus found in both sites. The results provide evidence that no one mechanism is responsible for protection following vaccination but local specific responses are more likely to be involved.     

Update on pathogenesis, diagnosis, and treatment of feline herpesvirus type 1

Feline herpesvirus type 1 (FHV-1) infection, but not necessarily chronic or recurrent disease, is common throughout domestic cat populations worldwide. Knowledge of a few essential virological facts permits practitioners to provide appropriate advice to owners of individual pet cats infected with this virus and to assist in the management of shelters and other multicat households in which the virus is enzootic. This article discusses pathogenesis, diagnostic techniques, and clinical signs considered characteristic of infection with FHV-1. Treatment options are considered under the broad categories of supportive care, antiviral agents, and adjunctive therapies.     

A field trial to assess the effect of vaccination against feline herpesvirus, feline calicivirus and feline panleucopenia virus in 6-week-old kittens.

A trivalent (feline panleucopenia, feline herpesvirus, feline calicivirus), modified live, commercially available cat vaccine was used at either 6, 9 and 12 weeks of age (early schedule) or 9 and 12 weeks of age (conventional schedule), and the serological response to vaccination was assessed. The level of maternally derived antibody present at 6 weeks of age was also established. The use of early vaccination at 6 weeks of age induced an antibody response to each virus by 9 weeks of age in a significant proportion of kittens compared with unvaccinated littermates. There was no difference between the conventionally and early-vaccinated groups in terms of antibody response to any antigen by 12 and 15 weeks of age.     

An improved method for hemagglutinin extraction from feline herpesvirus type 1-infected cell line

Hemagglutination (HA) activity of feline herpesvirus type 1 (FHV-1) propagated in feline lung cell culture and two established feline cell lines, CRFK and fcwf-4, was investigated. Intra- and extracellular crude samples obtained from those infected cell cultures did not show HA activity. However, when treated with tween 80-ether, HA activity appeared. There was no correlation between virus infectivity titers and the HA titers at various harvesting times, and besides, hemagglutinins were found in intracellular samples at the early stage of infection. By ultrasonic destruction of the infected fcwf-4 cells, high titer hemagglutinins were obtained. High titer hemagglutinins were also extracted successfully from infected fcwf-4 cell membranes by solubilization with any of the three detergents: Triton X-100, DOC, and CHAPS. The optimal concentrations of each detergent for solubilizing hemagglutinin were 0.05 (v/v)%, 0.5 (w/v)%, and 0.1-0.2 (w/v)%, respectively. The HA activities of both the ultrasonic-treated hemagglutinin and the detergent-soluble hemagglutinin from infected fcwf-4 cells were inhibited specifically by anti-FHV-1 sera. Therefore, either hemagglutinin could be used as HA antigen for the hemagglutination-inhibition test.     

Persistent cutaneous ulcers associated with feline herpesvirus type 1 infection in a cheetah.

Persistent cutaneous ulcers developed in a female cheetah cub after an episode of rhinotracheitis. When they were 3 weeks old, the cub and a male littermate developed mucopurulent oculonasal discharge consistent with feline herpesvirus type 1 infection (feline viral rhinotracheitis). The male cub was weaned and its lesions resolved. The female cub remained with the dam until the cub was 3 months old, at which time plaque-like lesions developed on the eye margins and muzzle. These plaques regressed over the next month and were replaced with cutaneous ulcers ranging from 1 to 10 mm in diameter. Feline herpesvirus type 1 was isolated from biopsy specimens collected from the ulcers. Cutaneous ulcers are uncommon manifestations of feline herpesvirus infections and have not been reported in other exotic fields. A proposed susceptibility to viral infections related to low genetic diversity has been proposed in cheetahs, and may be involved in the pathogenesis of persistent herpetic ulcers.     

Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.     

Use of serologic tests to predict resistance to feline herpesvirus 1, feline calicivirus,and feline parvovirus infection in cats

OBJECTIVE: To determine whether detection of virus-specific serum antibodies correlates with resistance to challenge with virulent feline herpesvirus 1 (FHV-1), feline calicivirus (FCV), and feline parvovirus (FPV) in cats and to determine percentages of client-owned cats with serum antibodies to FHV-1, FCV, and FPV. DESIGN: Prospective experimental study. ANIMALS: 72 laboratory-reared cats and 276 client-owned cats. PROCEDURES: Laboratory-reared cats were vaccinated against FHV-1, FCV, and FPV, using 1 of 3 commercial vaccines, or maintained as unvaccinated controls. Between 9 and 36 months after vaccination, cats were challenged with virulent virus. Recombinant-antigen ELISA for detection of FHV-1-, FCV-, and FPV-specific antibodies were developed, and results were compared with results of hemagglutination inhibition (FPV) and virus neutralization (FHV-1 and FCV) assays and with resistance to viral challenge. RESULTS: For vaccinated laboratory-reared cats, predictive values of positive results were 100% for the FPV and FCV ELISA and 90% for the FHV-1 ELISA. Results of the FHV-1, FCV, and FPV ELISA were positive for 195 (70.7%), 255 (92.4%), and 189 (68.5%), respectively, of the 276 client-owned cats. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for cats that have been vaccinated, detection of FHV-1-, FCV-, and FPV-specific antibodies is predictive of whether cats are susceptible to disease, regardless of vaccine type or vaccination interval. Because most client-owned cats had detectable serum antibodies suggestive of resistance to infection, use of arbitrary booster vaccination intervals is likely to lead to unnecessary vaccination of some cats.     

Genotyping of a microsatellite locus to differentiate clinical Ostreid herpesvirus 1 specimens

Ostreid herpesvirus 1 (OsHV-1) is a DNA virus belonging to the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 has been associated with mortality outbreaks in different bivalve species including the Pacific cupped oyster, Crassostrea gigas. Since 2008, massive mortality events have been reported among C. gigas in Europe in relation to the detection of a variant of OsHV-1, called μVar. Since 2009, this variant has been mainly detected in France. These results raise questions about the emergence and the virulence of this variant. The search for association between specific virus genetic markers and clinical symptoms is of great interest and the characterization of the genetic variability of OsHV-1 specimens is an area of growing interest. Determination of nucleotide sequences of PCR-amplified virus DNA fragments has already been used to characterize OsHV-1 specimens and virus variants have thus been described. However, the virus DNA sequencing approach is time-consuming in the high-scale format. Identification and genotyping of highly polymorphic microsatellite loci appear as a suitable approach. The main objective of the present study was the development of a genotyping method in order to characterise clinical OsHV-1 specimens by targeting a particular microsatellite locus located in the ORF4 area. Genotyping results were compared to sequences already available. An excellent correlation was found between the detected genotypes and the corresponding sequences showing that the genotyping approach allowed an accuraté discrimination between virus specimens.