Experimental transplacental transmission of canine herpesvirus in pregnant bitches during the second trimester of gestation

The effects of canine herpesvirus (CHV) on fetuses were studied after IV inoculation of pregnant bitches in the 2nd trimester of gestation. Cesarean sections were performed on 2 bitches that were inoculated with CHV on the estimated 30th day of gestation. Bitch M-1 had 2 mummified fetuses and bitch M-2 had 4 mummified and 2 dead fetuses and 3 live-born pups. Infection by CHV was confirmed histopathologically by the presence of focal areas of necrosis associated with intranuclear inclusion bodies in heart muscle sections of 1 dead fetus; CHV was not recovered from other organs. Abortion occurred between the 2nd and 3rd week after inoculation of another pregnant bitch inoculated with CHV on the estimated 30th day of gestation. Two bitches inoculated with CHV on the estimated 40th day of gestation gave birth prematurely to 10 pups. The detection of characteristic herpesviral lesions in various organs and the reisolation of CHV from the liver, spleen, kidneys, and lungs of premature pups indicated CHV infection. Transplacental infection of fetal pups by CHV resulted in their death and subsequent mummification. It appears that abortion and premature birth also may occur in pregnant bitches infected during the 2nd trimester of gestation.     

Suitability of canine herpesvirus as a vector for oral bait vaccination of foxes

Studies were conducted to evaluate the feasibility of using canine herpesvirus (CHV) as a vaccine vector for bait-delivered oral vaccination of wild foxes. To test the viability of CHV in baits, CHV was freeze-dried, incorporated into different baits, stored, and the remaining viral infectivity tested in cell culture after varying periods of time at different storage temperatures. Experimental baits (mouse carcasses) and commercial baits (FOXOFF and PROBAIT) were prepared with either liquid or freeze-dried CHV and tested in two fox trials for their capacity to induce CHV-specific antibodies following oral baiting. Freeze-drying and storage temperatures below 0 degrees C had a stabilizing effect to virus infectivity. When stored at -20 degrees C, freeze-dried CHV retained its full infectivity for up to 3 months in PROBAIT baits, the remaining infectivity in FOXOFF baits was 100-fold less. Oral baiting with CHV induced antiviral serum antibodies in all vaccinated foxes (20/20). None of the vaccinated foxes became ill or shed infectious virus into the environment although viral DNA was detected in body secretions as evaluated by PCR. The results indicate that CHV can be freeze-dried and stored over extended periods of time without loosing much of its infectivity. This is the first report of CHV being used for oral bait vaccination of foxes. It appears that CHV is well suited for use as a recombinant vector for wild canids.     

Characterization of pseudorabies virus glycoprotein B expressed by canine herpesvirus

A recombinant canine herpesvirus (CHV) which expressed glycoprotein B (gB) of pseudorabies virus (PrV) was constructed. The antigenicity of the PrV gB expressed by the recombinant CHV is similar to that of the native PrV. The expressed PrV gB was shown to be transported to the surface of infected cells as judged by an indirected immunofluorescence test. Antibodies raised in mice immunized with the recombinant CHV neutralized the infectivity of PrV in vitro. It is known that the authentic PrV gB exists as a glycoprotein complex, which consists of gBa, gBb and gBc. In MDCK cells, PrV gB expressed by the recombinant CHV was processed like authentic PrV gB, suggesting that the cleavage mechanism of PrV gB depends on a functional cleavage domain from PrV gB gene and protease from infected cells.     

Pathology of the placenta and newborn pups with suspected intrauterine infection of canine herpesvirus.

Pathologic and virologic investigations were done on the fetal placenta and on pup runts which were obtained from a bitch with a medical history of canine herpesvirus (CHV) infection. Macroscopically, the placenta was poorly developed. Small grayish white foci were observed in the placental labyrinth. Characteristic lesions of CHV infection were not prominent in the pups examined. Microscopically, however, focal degenerative and necrotizing lesions were observed in the placental labyrinth. Rarely, eosinophilic or basophilic intranuclear inclusion bodies were in the trophoblastic cells in the necrotizing lesions. In the adrenal gland of one stillborn pup, focal necrosis and hemorrhages could be seen; these irregularities were essentially the same as those seen in the newborn pups with CHV infection. Focal interstitial pneumonia was also observed in some of the pups. The CHV organism was isolated from the kidney of one pup that survived for 22 days.     

Experimental infection of European red foxes (Vulpes vulpes) with canine herpesvirus

We report on the pathogenicity of canine herpesvirus (CHV) for European red foxes. In the first experiment, we inoculated 10 adult foxes intravenously with a canine isolate of CHV. All foxes became infected and shed CHV in saliva and genital secretions for up to 14 days post-inoculation (p.i.) as evaluated by PCR and/or by virus isolation. All foxes developed clinical signs such as fever, lethargy and evidence of respiratory tract disease. Two foxes died on day 6 p.i., one on day 7 p.i., and one fox was euthanased on day 6 p.i. Tissues taken from the four dead foxes were positive for CHV by PCR. The remaining six foxes recovered after approximately 14 days p.i. Virus particles with morphology typical of herpesviruses were found by electron microscopy in the liver of an infected animal. All surviving foxes developed serum anti-CHV antibodies. In a second experiment, six foxes were dosed perorally with CHV and paired with six untreated controls. Neither the perorally dosed nor the in-contact control foxes developed clinical signs of disease. Infectious CHV was not isolated from any of the dosed or the in-contact foxes but all perorally-infected foxes and one of the in-contact foxes tested PCR-positive for CHV on several occasions p.i. All perorally-infected foxes, but none of the in-contact foxes, seroconverted. In summary, intravenous CHV inoculation caused a clinical disease in adult foxes much more severe than observed in experimentally-infected adult dogs. No clinical disease or virus spread was observed after peroral dosing although viral infection occurred as evidenced by seroconversion.     

Detection of canine herpesvirus DNA in the ganglionic neurons and the lymph node lymphocytes of latently infected dogs.

To determine the site of latent infection of canine herpesvirus (CHV), tissues from dogs convalescent from acute infection with CHV were examined for the presence of viral genome DNA by the nested polymerase chain reaction. CHV DNA was detected in the trigeminal ganglia and the retropharyngeal lymph nodes. In situ hybridization study of the tissues revealed that CHV genome persisted in the nuclei of ganglionic neurons and lymphocytes.     

Restriction endonuclease analysis of canine herpesviruses isolated in Japan

Eighteen canine herpesvirus (CHV) isolates from Japan and two reference strains were compared by restriction endonuclease analysis technique using total DNA extracts from cells infected with the viruses. In order to select the suitable restriction endonucleases for differentiation of CHV isolates, ten enzymes were used and three of them, HindIII, XbaI, and PvuII, were found to be useful for strain differentiation. With these enzymes, CHV isolates from unrelated individuals were readily differentiated from each other. In contrast, all the isolates derived from the same litter were not distinguishable on the basis of restriction cleavage patterns. However, slight mobility shifts were observed among the isolates from the same litter or the same individual. The results showed that this method provides a powerful tool for epidemiological surveys of CHV infection.     

Induction of antibodies to canine herpesvirus in mice by immunization with anti-idiotypic antibodies.

Anti-idiotypic antibodies (anti-Id Abs) were produced in rabbits after inoculation with two mouse monoclonal antibodies (mAbs) directed against canine herpesvirus (CHV) glycoproteins (gps). One of the mAbs, 12H11, was directed against an epitope on gp 145/112 of CHV which induced virus neutralizing (VN) antibodies and against a cross-reacting epitope on the gp 143/108 of feline herpes-virus type 1 (FHV-1). The other mAb, 11F7, was directed against epitopes on CHV gp47 which induce VN and hemagglutination-inhibition (HAI) antibodies. Using VN-inhibition and HAI-inhibition assays with CHV and FHV-1, the anti-Id Abs obviously inhibited the activities of autologous mAbs, suggesting that anti-Id Abs mimic the epitopes of CHV gp 145/112 or FHV-1 gp 143/108 and CHV gp47 by binding the anti-combining site of the mAbs. These anti-Id Abs, when injected into mice, elicited specific CHV-neutralizing and HAI antibody responses, and one of them also elicited a specific FHV-1-neutralizing antibody response. These data supported the idea that immunization with anti-Id Ab can induce specific VN antibody response, as has been theorized by other workers.     

Prevalence of antibodies against canine herpesvirus 1 in dogs in The Netherlands in 1997-1998

Canine herpesvirus (CHV1) is found in dogs all over the world and may spread by oronasal or sexual contact. We developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against CHV1 in dogs. The antigen used for this ELISA was prepared by purifying CHV1 virions from the medium of infected A72 cells. To investigate the prevalence of CHV1 in The Netherlands, a panel of 145 sera of dogs boarding at a kennel in Lelystad, The Netherlands, was screened using this ELISA. The dogs originated from all parts of The Netherlands and represented many different breeds. The sera were collected both at the start and at the end of the boarding period. Of the 145 paired sera 61 (42.1%) were positive, 79 (54.5%) were negative and 5 (3.4%) could not be attributed to either group. None of the negative dogs became seropositive during the boarding period, which lasted normally two to three weeks. We also tested 79 individual sera taken from dogs at various other places in The Netherlands and found that 27 (34.2%) were positive. Hence, in total 224 dog sera, collected from April 1997 to March 1998, were tested and 88 (39.3%) were found positive. We conclude that the prevalence of CHV1 seropositive dogs in The Netherlands in this period was about 40%, and that boarding at a dogs kennel did not contribute to the spread of CHV1. In addition, CHV1 has been isolated from two clinical cases of fatal haemorrhagic disease in The Netherlands.     

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.     

A serological study of canine herpesvirus-1 infection in a population of breeding bitches in Norway

Background

Canine herpesvirus-1 (CHV1) causes a fatal hemorrhagic disease in neonatal puppies and is associated with infertility in female dogs. This study was conducted to assess the status of CHV1 infection in bitches in proestrus or estrus and to investigate possible risk factors by a detailed questionnaire. Blood samples were collected from healthy bitches (n = 193) not vaccinated against CHV1, aged one year or older and admitted for estrus control to the Canine Reproductive Clinical Unit, Norwegian School of Veterinary Science. The serum samples were analysed by immunoperoxidase monolayer assay and serum titers were recorded as the reciprocal value of the highest dilution producing specific cell staining.

Results

Altogether, 85.5% of the dogs had CHV1 titers ≥ 80 and were classified as positive. Mean age for dogs included in the study was 4.2 years (95% CI 4.0-4.5), and there was no difference in age between seronegative dogs vs seropositive dogs. When grouping the seropositive dogs into three categories according to the magnitude of the titer, a total of 38.8% of the bitches displayed a weakly positive titer of 80, 44.8% had moderately positive titers of 160 or 320 and 16.4% of the dogs fell into the strongly positive category with titer of ≥640. No association was demonstrated when comparing CHV1 antibody titers to fertility parameters such as previous matings, pregnancies, whelpings, puppies born or condition of puppies. Further, there was no difference in seroprevalence between bitches that had been abroad for a period of time and dogs only living within a Norwegian environment. Samples from dogs collected in summer and fall displayed moderate to high antibody titers indicating recent infection with CHV1. Season, previous birth, and participation in competitions/shows explained 67-78% of the variation in antibody titer.

Conclusions

This study demonstrates that CHV1 infection is common in breeding bitches in the eastern part of Norway. Associations with putative risk factors were not identified. However, season, previous whelping, and participation in competitions/shows explained 67-78% of the variation in antibody titer.     

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.     

Canine herpesvirus 47 kD and mutated 41 kD glycoproteins are responsible for hemagglutination

Monoclonal antibodies (MoAbs) were used to identify the hemagglutinin of canine herpesvirus (CHV). The inhibition of viral hemagglutination (HA) activity was observed with MoAbs against 41 kD glycoprotein, while no hemagglutination-inhibition (HI) activity was observed with those against 145/112 kD and 80 kD glycoproteins, suggesting that the 41 kD glycoprotein is the hemagglutinin of plaque-selected virus of CHV YP11 strain used as immunogen for MoAb production. All of the HI MoAbs also showed HI activities against HA antigens which were prepared from cells infected with other CHV strains, namely, F-205 V and Glasgow CHV2 reference strains, eight Japanese isolates, and the original YP11 strain. However, on immunoblotting analysis, a 47 kD protein band was detected in these strains by the HI MoAbs. These data suggest that the 47 kD glycoprotein is the common molecule of the hemagglutinin among CHV strains and the plaque-selected virus of YP11 strain appears to be a mutant whose molecular weight of the hemagglutinin changed into 41 kD.     

A virus vector based on Canine Herpesvirus for vaccine applications in canids

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.     

Genome sequence of an Australian strain of canid alphaherpesvirus 1

Objective

Characterisation of a complete genome sequence of an Australian strain of canid alphaherpesvirus 1 (CHV‐1) and its phylogenetic relationship with other varicellovirus species.

Methods

Standard pathology and PCR methods were used to initially detect herpesvirus in hepatic tissue from an infected 4‐week‐old Labrador Retriever puppy. The complete CHV‐1 genome was sequenced using next‐generation sequencing technology followed by de novo and reference assembly, and genome annotation.

Results

The CHV‐1 genome was 125 kbp in length and contained 74 predicted open reading frames encoding functional proteins, all of which have counterparts in other alphaherpesviruses. Phylogenetic analysis using the DNA polymerase gene revealed that the newly sequenced CHV‐1 clustered with canid alphaherpesvirus isolated from the UK and shared a 99% overall nucleotide sequence similarity.

Conclusion

This is the first complete genome of an Australian strain of CHV‐1, which will contribute to our understanding of the genetics and evolution of herpesvirus.     

Diarrheal condition in dogs associated with viruses antigenically related to feline herpesvirus

Viruses with properties consistent with herpesvirus were isolated from dogs with diarrhea. The viruses were shown to be antigenically related to feline herpesvirus-1 (FHV-1) by virus neutralization tests. It was also observed that a canine herpesvirus (CHV) prototype, D004, and two field isolates from fatal CHV infections in 2-week-old and 6-week-old puppies were neutralized at a low level by antiserum to FHV-1. Reciprocal neutralization tests with CHV antiserum against FHV-1 were negative. These results indicated that viruses related to FHV-1 can infect the dog and that there appears to be uni-directional virus neutralization of CHV by FHV-1 antibody.     

Canine adenoviruses and herpesvirus.

Canine adenoviruses (CAVs) and canine herpesvirus (CHV) are pathogens of dogs that have been known for several decades. The two distinct types of CAVs, type 1 and type 2, are responsible for infectious canine hepatitis and infectious tracheobronchitis, respectively. In the present article, the currently available literature on CAVs and CHV is reviewed, providing a meaningful update on the epidemiologic, pathogenetic, clinical, diagnostic, and prophylactic aspects of the infections caused by these important pathogens.     

Apoptotic and proliferation indexes in canine lymphoma.

Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS.     

Seroprevalence of Canine Herpesvirus‐1 in the Belgian Dog Population in 2000

Canine herpesvirus‐1 (CHV‐1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme‐linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV‐1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV‐1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non‐breeding dogs.