Equine herpesvirus genomes: Heterogeneity of naturally occurring type 4 isolates and of a type 1 isolate after heterologous cell passage

Summary The restriction endonuclease DNA fingerprints of 20 low passage, epidemiologically unrelated isolates of equine herpesvirus 4 (equine rhinopneumonitis virus) showed considerable heterogeneity in certain fragments, the positions of which were assigned to quite restricted positions on the 141 kilobase (kb) genome. We note that the heterogeneity observed in the restriction endonuclease DNA fingerprints of EHV1 (equine abortion virus) and of pseudorabies virus also tend to map to these same restricted regions.The restriction endonuclease DNA fingerprints of an EHV1 strain was invariant using low (<1) multiplicity of infection during 20 passages in equine cells but when adapted to hamster cells developed an approximately 0.8 kb deletion in the unique short region of the genome between 8 and 11 passages.With 3 Figures     

Equine herpesvirus type 1 (EHV-1) induced abortions and paralysis in a Lipizzaner stud: a contribution to the classification of equine herpesviruses

Summary Out of 30 cases of abortion and perinatal deaths in a Lipizzaner stud in Austria 10 mares died after having shown central nervous system disturbances, ataxias and paralysis. The etiological agent of this abortion storm was equine herpesvirus type 1 (EHV-1). The restriction enzyme pattern of the DNA from 5 isolates recovered from fetuses has been analyzed and compared with the known reference strains of EHV-1, -2, -4 and an Austrian vaccine strain. The DNA restriction profiles of the Lipizzaner isolates as well as of the vaccine strain could be identified as being typical of abortigenic strains with minor variations. Such variations on the molecular biological level of the DNA do not justify characterization of the strains as neurovariants. The vaccine strain differed from other isolates investigated with 4 restriction endonucleases (Bam HI, Bgl II, Eco R I, Kpn I) which was due to a deletion in the unique short segment of the genome. The lack of similar DNA bands in two EHV-1 viruses, causing mild respiratory disease, as well as in the vaccine strain Prevaccinol is suggestive of lowered virulence. In contrast to one Lipizzaner isolate tested (strain Austria IV) the Austrian vaccine strain proved to be of strong neurovirulence for suckling mice.With 6 Figures     

A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1

Summary We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses. The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed inE. coli as fusion proteins [Crabb and Studdert, 1993: J Virol 67: 6332–6338). The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes. Plasma samples from 97 Thoroughbred and 174 Standardbred horses were tested, all of which were unvaccinated. All horses were strongly EHV4 ELISA positive while 30% were EHV1 ELISA positive. The type-specificity of the EHV1 gG antigen was tested in cross-absorption experiments and it was found that 96% (66 of 69) of EHV1 ELISA positive horses were true EHV1 antibody positives. It was also shown that 100% (26 of 26) horses known to have been exposed to EHV1, either by infection or immunisation with EHV1, had significant levels of antibody against the EHV1 gG antigen (i.e., all horses recognised the EHV1 epitope(s) contained within this molecule). Maintenance of EHV1 gG antibody was examined by testing sera obtained from mares four years after confirmed EHV1 abortion. Seven out of 10 of these mares remained EHV1 ELISA positive. In summary, the ELISA is highly specific and is sufficiently sensitive to detect all horses previously infected with EHV4 and most previously infected with EHV1.     

Genetic characterization of equine influenza viruses isolated in Italy between 1999 and 2005

During local respiratory disease outbreaks, occurring in 2003 and 2004 in horse training stables within race-tracks in Rome, and on a stud horse farm in Bari in 2005, four strains of equine influenza (EI) virus were isolated. All outbreaks occurred in flu-vaccinated horses. Here, we are reporting the results of the genetic characterization of these isolates, together with that of another EI virus strain isolated in 1999 from a dead foal presenting pulmonary lesions. Alignment and phylogenetic analyses were carried out using the haemagglutinin amino acid sequences. The Rome and Bari isolates were identified as members of the American lineage, closely related to other recent strains isolated in America as well as in Europe, including the latest recommended American lineage vaccine prototype A/eq/SouthAfrica/4/2003. In contrast, the Italian 1999 isolate was clustered within the European lineage. In Italy, the most recent outbreaks of EI have been caused by the currently circulating American-like strains, even in vaccinated populations, confirming that vaccines should contain an updated representative strain of this lineage. Presently, companies are still in the process of registering updated vaccines but no product is yet available on the market.     

Equine herpesvirus infections in yearlings in South-East Queensland

Twelve nasal swabs were collected from yearling horses with respiratory distress and tested for equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) by real-time PCR targeting the glycoprotein B gene. All samples were negative for EHV-1; however, 3 were positive for EHV-4. When these samples were tested for EHV-2 and EHV-5 by PCR, all samples were negative for EHV-2 and 11 were positive for EHV-5. All three samples that were positive for EHV-4 were also positive for EHV-5. These three samples gave a limited CPE in ED cells reminiscent of EHV-4 CPE. EHV-4 CPE was obvious after 3 days and was characterised by syncytia. None of the samples produced cytopathic effect (CPE) on African green monkey kidney (Vero) cells or hamster kidney (BSR) cells. Four of the samples, which were positive in the EHV-5 PCR, produced CPE on rabbit kidney (RK13) cells and equine dermis (ED) cells. EHV-5 CPE on both cell lines was slow and was apparent after four 7-day passages. On RK13 cells, the CPE was characteristic of equid herpesvirus, with the formation of syncytia. However, in ED cells, the CPE was characterised by ring-shaped syncytia. For the first time, a case of equine respiratory disease involving dual infection with EHV-4 and EHV-5 has been reported in Queensland (Australia). This was shown by simultaneously isolating EHV-4 and EHV-5 from clinical samples. EHV5 was recovered from all samples except one, suggesting that EHV5 was more prevalent in young horses than EHV2.     

Infectious causes of equine respiratory disease on Ontario standardbred racetracks.

Upper respiratory disease has been a serious problem in Standardbred horses on racetracks in Ontario, with outbreaks occurring once or twice annually in late winter and early spring seasons. To determine the causes of these epidemics, a 3-year investigation was carried out in which nasal swabs and serum samples were obtained at intervals from apparently healthy horses and from horses suffering from upper respiratory disease. The nasal swabs were used to isolate bacteria and viruses. The serum samples were examined for the presence and level of antibodies to equine influenza viruses and equine herpesvirus 1. None of the bacteria isolated were associated with the outbreaks of disease. Equine herpesvirus 2 was isolated 72 times from both diseased and apparently healthy horses. Equine herpesvirus 1 was isolated 10 times from horses with respiratory disease, both during and between epidemics. Influenza equine/1 virus was isolated seven times and influenza equine/2 was isolated once during severe outbreaks of upper respiratory disease. Serological evidence confirmed that influenza viruses were the causes of the major epidemics, with the equine/1 strain being involved most often.     

Genetic homogeneity of Taylorella equigenitalis from Norwegian trotting horses revealed by chromosomal DNA fingerprinting.

Chromosomal DNA fingerprinting indicated that Norwegian Taylorella equigenitalis strains are genetically homogeneous and similar to some Swedish isolates but different from other European strains. As contagious equine metritis is rarely a serious disease in Norwegian horses, we conclude that the dominant T. equigenitalis strain in Norway is a genetically homogeneous clone of low virulence.     

Rhodococcus equi plasmids: isolation and partial characterization.

Fifty-four strains of Rhodococcus equi from different clinical sources (mainly horses and pigs) were examined for their plasmid content by two screening methods. Plasmids were detected in 49 of 54 strains. A plasmid of approximately 80 kb was isolated from 21 of 22 isolates from horses and 20 of 28 isolates from pigs, and a 105-kb plasmid was isolated from 7 of 28 isolates from pigs. The 80-kb plasmid was significantly associated with strains of equine rather than porcine origin, and the 105-kb plasmid was significantly associated with strains of porcine origin. The type strain, ATCC 6939, consistently failed to yield a plasmid. Restriction enzyme analysis of purified plasmid DNA confirmed the relatedness of the 80-kb plasmids isolated from strains of equine and porcine origin. More differences between the restriction patterns of plasmids from strains isolated from horses and from pigs than among strains from either species were observed. Restriction enzyme analysis also showed relatedness of the 105-kb plasmid to the 80-kb plasmid. Three strains shown by others to be virulent in horses or mice possessed the 80-kb plasmid, whereas three other strains not virulent for horses or mice lacked the plasmid, although one had the 105-kb plasmid. There was a significant but not perfect association between the presence of the 80-kb plasmid and production of a diffuse 17.5-kDa thermoregulated, virulence-associated protein. Further study is needed to determine whether this plasmid is associated with virulence in R. equi.     

Equine herpes virus 2 infection in horse populations in Poland

The prevalence of Equine herpesvirus 2 (EHV-2) infections in the horse populations in Poland was investigated. Peripheral blood leukocytes (PBLs) of 139 horses were tested. The animals were divided into four groups: clinically healthy horses, horses suffering from respiratory disorders, mares with a recent abortion and horses with diagnosed ataxia. Thirty-four virus isolates were obtained from leukocytes of the tested animals by cocultivation with equine dermal cells and were identified as EHV-2 by PCR using primers for the gB gene of EHV-2 and/or primers for the sequence located upstream of the gene homologous to the equine interleukin 10 (IL-10) gene. These results indicate that EHV-2 is prevalent in horse populations in Poland. As the virus was most frequently isolated from horses with respiratory disorders its etiological importance may be considered.     

The nucleotide sequence of asinine herpesvirus 3 glycoprotein G indicates that the donkey virus is closely related to equine herpesvirus 1

Summary The nucleotide sequence of the glycoprotein G (gG) homologue of asinine herpesvirus 3 (AHV3), a respiratory alphaherpesvirus of donkeys, was determined. The AHV3 gG gene consists of 1233 base pairs (bp) and codes for a predicted protein of 411 amino acids. This is identical in size to the equine herpesvirus 1 (EHV1) gG gene and 6 amino acids longer than the equine herpesvirus 4 (EHV4) gG gene. The predicted amino acid sequence of AHV3 gG has characteristics of a class 1 membrane protein. The amino acid sequence of AHV3 gG shows 92% and 60% identity to EHV1 gG and EHV4 gG respectively. Two regions within the gG amino acid sequences of EHV1 and EHV4 were previously defined, an N-terminal constant region and an immunodominant highly variable region located toward the C-terminus. In the corresponding constant region of AHV3 gG there was 96% and 75% amino acid identity with EHV1 and EHV4 gGs respectively. In the variable region, there was 73% and 24% identity respectively. Phylogenetic analyses using the gG nucleotide sequences indicated that AHV3 is much closer in evolutionary distance to EHV1 than either virus is to EHV4. These findings provide additional support for the view that AHV3, or another closely related virus, may be the progenitor of EHV1 and has adapted to horses in relatively recent times.     

Analysis of small and large plaque variants of equine herpesvirus type 3 by restriction endeonucleases

Six of 6 equine herpesvirus type 3 (EHV3) isolates, 5 of which were epidemiologically unrelated, produced a mixture of small and large plaque variants in equine foetal kidney cells under methylcellulose. In 4 of 4 instances the cleavage site(s) generating the Bam HI A fragment of large plaque variants was distinct from the site(s) for the same fragment of small plaque variants.     

Asinine herpesvirus genomes: comparison with those of the equine herpesviruses

Summary Two previously unknown and distinct herpesviruses were isolated from donkeys. One, with the characteristics of a betaherpesvirus, was isolated from the leukocytes of an apparently healthy donkey, while the second, an alphaherpesvirus, was recovered from the nasal cavity of donkeys given high doses of corticosteroids, and caused rhinitis in two seronegative weanling donkeys when they were intranasally infected. Few, if any, restriction endonuclease fragments were shared by the donkey betaherpesvirus, equine herpesvirus 2 (EHV2) or EHV5, a second distinctly different equine betaherpesvirus, nor by the donkey alphaherpesvirus, EHV1, EHV4, or EHV3. In Southern blot analysis the donkey betaherpesvirus showed low levels of sequence similarity to both EHV2 and EHV5, while the donkey alphaherpesvirus and EHV1 shared a moderate degree of sequence similarity, less similarity with EHV4 and very low level of sequence similarity with EHV3. These two isolates appear prototypic of two previously unrecorded herpesviruses for which the names asinine herpesvirus 2 and 3 are suggested for the betaherpesvirus and the alphaherpesvirus respectively.     

Studies on equine herpesviruses

Summary A complement fixation (CF) antigen for equine herpes-1 (rhinopneumonitis) virus (EHV 1) of cell culture origin was studied, and an optimum method of preparation in a minimum volume of serum-free overlay fluid developed. Many chemical and physical treatments including fluorocarbon extraction, tweenether treatment, Carbowax concentration or ultracentrifugation (25,000g) did not further improve the quality of this antigen. Inactivation (56°C for 30 minutes) of equine serum did not affect the CF antibody titre, but removed low levels of anti-complementary activity. The use of a diluent of low ionic strength (0.1m) did not increase titres in the EHV 1-equine antibody CF system.This EHV 1 CF antigen of cell culture origin was used in a microtitre system and detected the development of antibodies in all of six young horses in Queensland following natural EHV 1 respiratory infection. Maximum serum titres (1/128) were reached 1–2 weeks after the onset of acute respiratory disease, then CF titres declined steadily but were still detectable after 12 months.Serum titres of 1/16–1/32 or higher indicated EHV 1 infection to have occurred within the previous 3–6 months.     

Species specificity of plasminogen activation and acquisition of surface-associated proteolytic activity by group C streptococci grown in plasma

Our laboratory previously demonstrated that group C streptococcal isolates from humans and horses secrete streptokinases that preferentially activate plasminogens reflecting the origin of the isolates. To analyze the significance of these findings, series of streptokinase-producing Streptococcus equisimilis isolates recovered from humans and horses were examined. Southern blot analysis revealed that chromosomal DNA of the streptococcal isolates from humans reacted exclusively with a skc(hu) probe and that chromosomal DNA of streptococcal isolates from horses reacted preferentially with an skc(eq) probe in a distinct pattern. The streptococcal isolates were examined for the ability to acquire surface-bound plasmin-like activity when grown in the presence of human or equine plasma. Each of eight isolates from humans acquired significant enzymatic activity only when grown in the presence of human plasma, while each of eight isolates from horses acquired activity only when grown in the presence of equine plasma. Analysis of bacterial and host protein requirements indicated critical roles for streptokinase, activatable plasminogen, and fibrinogen. These requirements may explain why certain streptococcal isolates cause disease only in a limited number of mammalian hosts.     

Equine Herpesviruses: Antigenic Relationships and Deoxyribonucleic Acid Densities

Equine herpesviruses with a deoxyribonucleic acid density of 1.716 to 1.717 g/cm(3) were compared with one another by the plaque-reduction test and by the rate of development of cytopathic effect as indicated by plaque size in rabbit kidney cultures. Of the 19 isolates studied, the 9 which had already been tentatively labeled equine abortion viruses were serologically similar to one another; each of them grew more quickly than did any of the other 10 isolates although the mean plaque sizes formed a series of gradations with no clear hiatus which would permit the unequivocal delineation of the abortion viruses from the slowly growing strains. The 10 slowly growing isolates showed antigenic heterogeneity even though complement was present; the neutralizing capacity of an antiserum against the heterologous strains was, in most instances, markedly less than against the homologous strains, the range of the 50% endpoints being much greater than that observed among the equine abortion viruses, or among isolates of herpes simplex type 1. There was no cross neutralization between the equine abortion viruses and any of the 10 slowly growing isolates. An extra band of deoxyribonucleic acid, at 1.723 to 1.725 g/cm(3), was present in two of the slowly growing strains when originally grown in rabbit cells, but was no longer present after passage in cat cells. This band occupied the same position as one reported in the hamster-passaged strain of equine abortion virus, and had a density similar to that of the equine genital herpesvirus. Although the taxonomic demarcation of the equine abortion viruses and the slowly growing herpesviruses from one another is still open to question, they can be conveniently labeled equine herpesviruses 1 and 2, respectively; the genital virus would be termed equine herpesvirus 3.     

Characterization and Protective Immunogenicity of the SzM Protein of Streptococcus zooepidemicus NC78 from a Clonal Outbreak of Equine Respiratory Disease

Streptococcus zooepidemicus of Lancefield group C is a highly variable tonsillar and mucosal commensal that usually is associated with opportunistic infections of the respiratory tract of vertebrate hosts. More-virulent clones have caused epizootics of severe respiratory disease in dogs and horses. The virulence factors of these strains are poorly understood. The antiphagocytic protein SeM is a major virulence factor and protective antigen of Streptococcus equi, a clonal biovar of an ancestral S. zooepidemicus strain. Although the genome of S. zooepidemicus strain H70, an equine isolate, contains a partial homolog (szm) of sem, expression of the gene has not been documented. We have identified and characterized SzM from an encapsulated S. zooepidemicus strain from an epizootic of equine respiratory disease in New Caledonia. The SzM protein of strain NC78 (SzM_NC78) has a predicted predominantly alpha-helical fibrillar structure with an LPSTG cell surface anchor motif and resistance to hot acid. A putative binding site for plasminogen is present in the B repeat region, the sequence of which shares homology with repeats of the plasminogen binding proteins of human group C and G streptococci. Equine plasminogen is activated in a dose-dependent manner by recombinant SzM_NC78. Only 23.20 and 25.46% DNA homology is shared with SeM proteins of S. equi strains CF32 and 4047, respectively, and homology ranges from 19.60 to 54.70% for SzM proteins of other S. zooepidemicus strains. As expected, SzM_NC78 reacted with convalescent-phase sera from horses with respiratory disease associated with strains of S. zooepidemicus. SzM_NC78 resembles SeM in binding equine fibrinogen and eliciting strong protective antibody responses in mice. Sera of vaccinated mice opsonized S. zooepidemicus strains NC78 and W60, the SzM protein of which shared partial amino acid homology with SzM_NC78. We conclude that SzM is a protective antigen of NC78; it was strongly reactive with serum antibodies from horses during recovery from S. zooepidemicus-associated respiratory disease.     

Equine herpesvirus type 1 modified live virus vaccines: quo vaditis?

Infections of horses with equine herpesvirus type 1 (EHV-1) have garnered new attention over the last few years. Devastating outbreaks occurring worldwide, primarily of the neurologic form of the disease, have resulted in a reassessment of the control strategies, and particularly the prophylactic measures, that are necessary to keep the infection and spread of disease in check. Most of the available EHV-1 vaccines are based on preparations of inactivated virus, which are applied monovalently for prevention of EHV-1-caused abortion in pregnant mares or as part of multivalent vaccines to prevent respiratory disease. Despite the importance of an induction of cytotoxic immune responses for protection against EHV-1-induced disease, only two modified live virus vaccine preparations, which are both based on the avirulent EHV-1 strain RacH and were developed more than 40 years ago, are commercially available. Current efforts focus on exploiting the available infectious bacterial artificial chromosome clones of various EHV-1 strains to engineer a new generation of modified live virus vaccines. Both more efficient and long-lasting anti-EHV-1 immunity and delivery of immunogens of other pathogens are attempted and within immediate reach. The improvement of modified live virus vaccines will likely be a major focus of research in the future, and will hopefully help to more completely protect horses against one of the most important and devastating viral diseases.     

Antigenic and genetic analysis of H3N8 influenza viruses isolated from horses in Japan and Mongolia, and imported from Canada and Belgium during 2007-2010

A/equine/Kanazawa/1/2007 (H3N8), A/equine/Hokkaido/I828/2008 (H3N8) and A/equine/Mongolia/1/2008 (H3N8) were isolated from infected horses. A/equine/Yokohama/aq19/2009 (H3N8) and A/equine/Yokohama/aq13/2010 (H3N8) were isolated from horses imported from Canada and Belgium examined at the Animal Quarantine Service in Yokohama, Japan. In the present study, these five isolates were genetically and antigenically analyzed. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes showed that three isolates from horses in Japan and imported from Canada belonged to the same branch, clade 1 of the Florida sublineage, while the isolates from horses in Mongolia and imported from Belgium belonged to another branch, clade 2 of the Florida sublineage. Reactivity patterns of a panel of monoclonal antibodies to the HA of A/equine/Kanazawa/1/2007 (H3N8) with the five isolates indicate that the HAs of these viruses were antigenically similar to each other and to the reference strains A/equine/La Plata/1/1993 (H3N8) and A/equine/Avesta/1/1993 (H3N8). The present findings indicate that extensive antigenic variation has not accumulated among H3N8 influenza viruses in horses.     

Serologic survey of equine rhinopneumonitis virus infection among horses in various countries

Summary Horse serum samples from 388 horses in 17 countries exhibited high incidence of antibodies for equine rhinopneumonitis virus which had been isolated and studied in USA and subsequently in Japan. Complement fixing antibody was shown in 75.1% of 386 horses for viral antigen of the virus and in 36.5% for soluble antigen. In neutralization tests two serologically distinct types of the virus were used; 85.2% of 384 horses were positive with the Kentucky D strain (American) and 81.7% of 312 horses with the H-45 strain (Japanese). The high incidences of antibodies for each of the countries studied, i. e. USSR, Norway, Sweden, Holland, Germany, Switzerland, Italy, England, South Africa, Egypt, India, Malaya, Taiwan, Japan, New Zealand, Argentine, and Canada, strongly suggest world-wide dissemination of equine rhino-pneumonitis virus or closely related viruses. The present serologic data, together with the clinical and pathologic findings reported by many authors, appear to indicate that the virus abortion, associated with acute respiratory infection, recorded in European horses is caused by equine rhinopneumonitis virus. The correlation diagrams of anti-Kentucky D and anti H-45 titers in individual horses gave evidence for circulation of at least these two serotypes of the virus in various countries.     

Restriction enzyme analysis of the virulence plasmids of VapA-positive Rhodococcus equi strains isolated from humans and horses.

Restriction enzyme digestion patterns of the large virulence plasmids of 8 human and 37 foal isolates of virulence-associated protein (VapA)-positive Rhodococcus equi strains from different sources were compared. Foal isolates came from five continents. Digestion with EcoRI divided these plasmids into three closely related types, and digestion with BamHI divided them into three major types which corresponded to the EcoRI types. The only EcoRI and BamHI type 3 plasmid was from a single foal isolate obtained from Japan. There are thus two major but related virulence plasmids in isolates from foals. Geographic differences were noted, since foal isolates with the EcoRI type 1 plasmid digestion pattern tended to come mostly from the United States, Canada, European countries, India or Zimbabwe and foal isolates with EcoRI type 2 pattern tended to come mostly from Latin American countries. Only 8 of 38 different human isolates, mostly from AIDS patients, were VapA positive, in contrast to 37 of 42 foal isolates. VapA-positive isolates from humans possessed virulence plasmids of either EcoRI type 1 or EcoRI type 2. These results confirm that only a small proportion of human patients with R. equi infections acquire foal virulent R. equi.