Isolation and characterization of two new herpes-like viruses from capuchin monkeys.

Two herpes-like viruses were isolated from capuchin monkey (Cebus apella) brain and (Cebus albifrons) spleen cell cultures, respectively. Both isolates induced similar cytopathic effects consisting of rounded and ballooned cells in the original monkey cell cultures and in a wide range of permissive cell types. Neutralizing antibody to each virus was present in serum from the capuchin monkey from which it was isolated, but the two viruses did not cross-react by neutralization. Fluorescein isothiocyanate conjugates of hyperimmune rabbit serum to one of the isolates showed an antigenic cross relationship between the two isolates. By electron microscopy, herpes-like virus particles were observed in the nucleus and cytoplasm of infected human diploid fibroblast cell cultures. Virus-infected cell cultures stained with acridine orange revealed small deoxyribonucleic acid-containing intranuclear inclusion bodies. Both viruses were inhibited by 5-fluorodeoxyuridine and inactivated by chloroform or exposure to 56 degrees C for 30 min. Antisera prepared against 16 prototype herpesviruses and cytomegaloviruses did not neutralize approximately 100 50% tissue culture infective doses of either capuchin isolate. Neutralizing antibody to the capuchin isolates was detected in sera from 8 of 17 capuchin monkeys but not in sera from 16 humans, 15 chimpanzees, and 10 spider, 6 rhesus, and 5 squirrel monkeys.     

Equine cytomegalovirus: structural proteins of virions and nucleocapsids

Enveloped virions and nucleocapsids of equine cytomegalovirus (ECMV; equine herpesvirus type 2) have been purified from the supernatants and the nuclear extracts of infected rabbit kidney (RK) cells, respectively, and their structural protein compositions have been analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that ECMV nucleocapsids were composed of nine proteins (average molecular weights = 148K, 52K, 49.5K, 46K, 43.5K, 38.5K, 27K, 20K, and 18K), which together constituted 89% of the total nucleocapsid protein on the basis of incorporated 3H-labeled amino acids. The 148K protein comprised 47.3% of the total protein and thus appeared to be similar in molecular weight and proportional composition to the major capsid proteins of other herpesviruses. Purified virions were composed of 37 proteins whose average molecular weights ranged from 14K to greater than 200K. Three intense glycoprotein bands (83K, 78K, and 73.5K) as well as four less intensely labeled glycoproteins were detected in [3H]glucosamine-labeled virion preparations. At least 14 structural proteins were readily detected in extracts of infected cells which had been [35S]methionine labeled late in infection, and 11 of these were immunoprecipitated by rabbit antiserum against purified virions. The protein composition of ECMV differs substantially from those of equine herpesvirus type 1 and type 3 as well as from those of other herpesviruses.     

Restriction endonuclease DNA fingerprinting of respiratory,foetal and perinatal foal isolates of equine herpesvirus type 1

DNA was prepared from 43 equine herpesvirus type 1 (EHV 1) isolates, 11 of which were from horses with respiratory disease, 22 from aborted equine foetuses, and 10 from foals that died perinatally. The restriction endonuclease DNA fingerprints of 10 of the 11 respiratory isolates, known with certainty to have been recovered from horses with respiratory disease, were entirely different from all but 3 of the 32 foetal or perinatal foal isolates. The exceptional respiratory isolate, EHV 1 Army 183, had a foetal (F) strain fingerprint but this virus cannot be said with certainty to have been isolated from the respiratory tract. The 3 exceptional foetal isolates, had respiratory (R) strain fingerprints, and were recovered from single sporadic abortions. There are no exceptions to the view that only R strains have been isolated from naturally occurring respiratory disease. Also it is clear that major epizootics of abortion (abortion storms) and of perinatal foal mortality are caused by F strains. The data together with an analysis of the epidemiological patterns, particularly in Australia, strongly support the view that F and R strains be regarded as separate species, EHV 1 and 4 respectively.     

Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure.

A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained and used to construct restriction maps for four restriction endonucleases. Hybridization experiments indicated that the EHV5.2-141 genome does not contain large terminal or internal repeats, although some evidence for very short repeated sequences in the genomic termini was obtained. Such a genome structure makes EHV5 unique among the equine herpesviruses but similar to the mouse, rat, and guinea pig cytomegaloviruses and the tupaiid herpesvirus. Sequence analysis of one of the genomic termini of EHV5.2-141 revealed the presence of a 30-bp sequence (pac-1; Deiss et al. (1986) J. Virol. 59, 605-618) which is highly conserved among herpesviruses.     

Comparison of herpesviruses isolated from reindeer,goats, and cattle by restriction endonuclease analysis

Summary A genomic comparison of bovine herpesvirus 1 (BHV-1), caprine herpesvirus (CHV-2) and reindeer herpesvirus (RHV), was performed using 5 restriction endonucleases. Cross neutralization of these three herpesviruses showed that BHV-1 and CHV-2 had a relatively low degree of cross reaction with heterologous viruses. RHV showed a higher degree of such cross reactivity. The restriction endonuclease analyses showed that the migration patterns of the DNA segments were different for the three groups of herpesviruses. The enteric caprine strain could be differentiated from genital strains using BstE II and Hpa I. The genome size of reindeer herpesvirus was estimated to be approximately 86.8×10⁶ Da (131.8 kbp), and indications of isomerization of this genome were found. It is concluded that reindeer herpesvirus is a distinct species within the familyHerpesviridae.     

Difference in growth behavior of human,swine, equine,and avian influenza viruses at a high temperature

Summary Growth characteristics of a wide range of influenza A viruses from different mammals and bird species were examined in an established line of canine kidney (MDCK) cells at an ordinary (37°C) and a high temperature (42°C). Although all viruses employed in the present study possessed a capability of replicating at 37°C, virus growth at 42°C showed considerable variation and reflected differences in the natural hosts of the isolates. All reference strains and isolates from bird species grew well in the MDCK cells maintained at 42°C, but human viruses did not, showing an asymmetrical growth behavior.In contrast to this, growth of swine and equine viruses showed growth characteristics intermediate between human and avian viruses. Of the two swine viruses examined, replication of one strain occured equally well at both temperatures and another failed to grow at 42°C. Similarly, two of the three equine viruses tested belonging to H3N8 antigenic subtypes grew at 42°C. However, the results obtained from comparison of plaque sizes and growth curves indicated that the replication of the above swine and equine viruses was restricted under a stringent temperature when compared to avian viruses. The detailed analysis of cloned viruses revealed that some of the swine and equine viruses contained two variants which are readily distinguished by growth behavior at 42°C. Genome analysis of parental and virus clones by oligonucleotide mapping and migration profiles of RNA segments did not detect any differences among the above variants exhibiting the asymmetrical growth characteristics at 42°C.     

Growth characteristics of a highly virulent,a moderately virulent,and an avirulent strain of equine arteritis virus in primary equine endothelial cells are predictive of their virulence to horses

Equine viral arteritis (EVA) is an endotheliotropic viral disease of horses caused by equine arteritis virus (EAV). Although there is only one serotype of EAV, there is marked variation in the virulence of different strains of the virus. The replication and cytopathogenicity of three well-characterized strains of EAV of different virulence to horses were compared in rabbit kidney (RK-13) and primary equine pulmonary artery endothelial cells (ECs). Viral protein expression, plaque size, and cytopathogenicity of all three viruses were similar in RK-13 cells, whereas two virulent strains of EAV were readily distinguished from an avirulent strain by their plaque morphology and cytopathogenicity in primary equine ECs. Furthermore, EAV nucleocapsid protein was detected by flow cytometric analysis significantly later in ECs infected with the avirulent than those infected with the virulent strains of EAV. Primary equine ECs provide a convenient and relevant model for in vitro characterization of the pathogenesis of EVA and the virulence determinants of EAV.     

Susceptibility to acyclovir of herpes simplex virus isolates obtained between 1977 and 1996 in Japan

The susceptibility of genital herpes to acyclovir (ACV) in immunocompetent women was examined, as was the frequency of ACV-resistant viruses by analyzing 56 clinical isolates in Japan between 1977 and 1996. The mean susceptibilities of herpes simplex virus (HSV) type 1 and type 2 were 0.13+/-0.74 and 0.42+/-0.14 microg/ml, respectively, assessed by the 50% inhibitory concentration of plaque formation. The susceptibility to ACV of clinical isolates did not changed since 1977, and also that of nine pairs of HSV-1 and HSV-2 isolates was not affected by ACV treatment. In order to characterize the change in the virus population, the quantitation of the ACV-resistant virus in 10(4) plaque forming units (PFU) of clinical isolates was adopted. The mean frequencies of ACV-resistant viruses per 10(4) PFU for all strains of HSV-1 and HSV-2 were 0.31+/-0.41 and 9.74+/-14.83, respectively, and were not influenced by ACV treatment. Additionally, the phenotypes of ACV-resistance were not influenced by ACV treatment, and more than 90% of ACV-resistant viruses were found to be thymidine kinase-deficient. This study characterized clinical isolates with respect to ACV susceptibility as a population and the quantitative and qualitative characterization of ACV-resistant virus in the virus population of clinical isolates was also studied. The susceptibility of isolates from genital lesions, the frequency of ACV-resistant viruses, and also the phenotypic characterization of ACV-resistant viruses was maintained between 1977 and 1996, even after the introduction of ACV treatment for genital herpes in Japan.     

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     

Growth phenotypes of cytomegalovirus isolates do not correlate with glycoprotein B, major immediate early genotypes or antiviral sensitivity

Human Cytomegalovirus (CMV) is generally described from in vitro experiments as a slowly replicating virus. A doubling time of one day in blood, however, has been shown in vivo. The growth phenotypes of CMV isolates and laboratory strains were studied in human fibroblasts. The viruses were found to replicate either rapidly or slowly. Comparison of CMV protein expression in lung and foreskin fibroblast cultures showed that two tissue culture adapted CMV strains (AD169 and Towne) and 3 clinical isolates belonged to the rapidly replicating viruses, whereas another 3 clinical isolates replicated slowly. CMV antigen concentrations were 6-fold and virus yields were 10-1000-fold higher for the rapidly replicating viruses than for the slow replicators. The antigen expression of two slowly replicating isolates was enhanced after 20 passages compared to the isolates at passage 5, but it was not as efficient as that of strain Towne. Slow or fast replication was related neither to major immediate early gene exon 4, and gB genotypes, nor to antiviral susceptibility. Proteins of the beta cascade may contribute to differences in the replication rate of CMV isolates.     

Latent herpesvirus infection of tests and spinal ganglia of turkeys with semen abnormalities

Summary The present study confirms that herpesviruses (LF) previously isolated from testes and buffy coat cells of male turkeys with semen abnormalities establish a latent infection in testicular cells. These experiments also present the first evidence that these herpesviruses are harbored latently in cells of spinal ganglia. One-week-old turkeys were inoculated with either the LF isolates or the prototype turkey herpesvirus FC 126; allowed to mature sexually through one breeding season and necropsied at one year of age. Persistent infections with all viral isolates were confirmed by repeated reisolation of the viruses from buffy coat cells and the development of specific precipitating antibodies. The herpesviruses were also isolated from several tissues by cocultivation on primary chick kidney cells. Primary testicular cells required subcultivation for the induction of viral replication. Latent viruses were demonstrated byin vitro explantation of testicular and spinal ganglion biopsies which, at the time of explantation, contained no detectable infectious virus, viral antigens or particles. After prolongedin vitro explantation of explants of testes and spinal ganglia tissue yielded infectious virus and viral antigens and particles were identified in outgrowing explant cells.     

Herpes simplex viruses: discrimination of types and correlation between different characteristics

Sixty strains of herpes simplex viruses from various sites of isolation were compared with one another by plaque appearance, by neutralization tests, by pock sizes on the chorioallantoic membrane of the chick embryo, and by the stabilities of their infectivity and of their thymidine kinase activities. On the basis of each of these tests (except the stability of infectivity), the strains divided into two discrete types, with no indication of intermediate strains or of the existence of more than two types. The correlation between the differentiation of these types by the various criteria was virtually complete, although one strain showed intermediate behavior on the basis of only one criterion, viz., the stability of the thymidine kinase activity. The densities of the DNAs from 12 isolates were measured; one of the genital isolates was found to be a mixture of type 1 and type 2, which was confirmed by subsequent separation of the two serotypes.Nine brain or ganglion isolates and three isolates from the mouths of patients with acute encephalitis could not be antigenically distinguished from other type 1 isolates.One of the various tests used, plaque appearance, pock size, and neutralization of infectivity were simple and discriminated efficiently. The stability of thymidine kinase activity was rapid and discriminated well between the two types. The thermolability of infectivity did not discriminate well but may be a useful confirmatory test.     

Characterization of a new canine herpes virus

Summary A cytopathogenic agent, designated CHV-BR, isolated from genital lesions in dogs has been shown to be a herpesvirus. It contained DNA, was ether-and heat-sensitive, and was unstable at low pH. Electron micrographs revealed a virion of typical herpesvirus structure. The CHV-BR isolate produced a marked cytopathic effect (CPE) in dog kidney cell cultures but failed to grow in rabbit kidney and human cell lines. The CPE in dog kidney cells consisted of extensive polykaryocytosis, and the formation of intranuclear type-A inclusion bodies. The emergence of a non-polykaryocytogenic CPE variant during serial passage of the isolate in dog cells was described. Neutralization and immunofluorescence tests showed that CHV-BR was serologically similar to the F-205 strain of canine herpesvirus (CHV) but was not related to other human and animal herpesviruses. Differences between the CHV-BR isolate and the CHV strains isolated elsewhere were discussed.     

Antigenic Relationships Among Four Herpesviruses

Common viral antigens were detected, by fluorescent-antibody studies, in cells infected with herpes simplex virus 1, squirrel monkey herpesvirus 1, bovine rhinotracheitis, and equine abortion viruses. The two primate viruses showed slight cross-neutralization.     

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.     

Comparative immunological characterization of type-specific and conserved B-cell epitopes of pinniped,felid and canid herpesviruses

Summary Murine monoclonal antibodies (MAbs) were generated against phocid herpesviruses (PhHV 2557/Han88 and 7848/Han90) isolated from European harbour seals (Phoca vitulina), and against strains of both felid (FHV strain FVR 605) and canid herpesviruses (CHV isolate 5105/Han89). MAbs were characterized with respect to certain biological properties and used to outline antigenicity profiles of isolates of PhHV (n=8), FHV (n=7) and CHV (n=3) in enzyme immunoassays employing fixed infected cells. A close antigenic relationship between herpesviruses derived from pinnipeds and terrestrial carnivores became evident: The majority of the MAbs was directed against epitopes which were expressed by at least two of the viral species tested. A number of MAbs detected epitopes which were conserved between all isolates of PhHV, FHV and CHV. A few MAbs recognized type-specific B-cell epitopes and facilitated the identification of single viral species. Moreover, the PhHV isolate 7848/Han90 was antigenically distinguishable both from seven other phocid herpesvirus isolates and from FHV or CHV. PhHV 7848/Han90 proved to be antigenically distinct from all other viruses tested when examined by cross neutralization utilizing various reconvalescent and hyperimmune sera. Although more data are needed to ensure that PhHV 7848/Han90 indeed is a new genuine seal herpesvirus, the preliminary clustering of two groups of phocid herpesvirus isolates, tentatively designated PhHV-1 (type isolate 2557/Han88) and PhHV-2 (represented by 7848/Han90), seems to be justified. By using selected MAbs an unambiguous identification and typing of herpesvirus isolates derived from marine mammals and terrestrial carnivores is significantly facilitated.     

A comparison of serological relationships among five ruminant alphaherpesviruses by ELISA

Summary Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-1.1, bovine herpesvirus-1.2, caprine herpesvirus-2, cervine (red deer) herpesvirus-1 and rangiferine (reindeer) herpesvirus-1 has been examined using rabbit hyperimmune antisera and convalescent cattle and red deer field sera. Significant cross-reactivity among all the five viruses was demonstrated. A detailed analysis showed that: (1) the two bovine herpesviruses are most closely related, (2) the cervine, caprine and rangiferine viruses are more closely related to the bovine viruses than they are to each other, (3) the cervine herpesvirus is more related to the bovine herpesvirus than to the rangiferine or caprine herpesviruses and (4) the rangiferine virus is more related to the cervine virus than to the bovine and caprine viruses. Cattle and red deer sera reacted most strongly with the bovine and cervine viruses respectively.     

Epidemiology of equine herpesvirus 2 (equine cytomegalovirus).

The epidemiology of equine herpesvirus 2 was examined by using restriction endonuclease DNA fingerprints to distinguish viruses isolated from two groups of horses. The first group consisted of three yearlings isolated from other horses but in contact with each other for 418 days, whereas the second comprised seven mares and their foals, which were sampled at monthly intervals from parturition until the foals were about 180 days old. There was a complex pattern of transmission, with 15 different viruses isolated from both groups. Four distinguishable viruses were isolated from the three yearlings by day 16 of quarantine, and by day 141 an additional two viruses were isolated. Up to five different viruses were isolated from one yearling. Although four repeat isolations of one virus from the nasal cavity of one yearling over 54 days indicated that equine herpesvirus 2 established persistent infection with constant shedding, most repeat isolations yielded distinguishable viruses. Identical viruses were isolated from the nasal cavity and leukocytes of one yearling and the nasal cavity and vagina of another, indicating that a particular equine herpesvirus 2 strain was not site specific. Although seven different viruses were isolated from the three yearlings throughout the quarantine period, two appeared to establish latent infections; one virus was not isolated until 141 days after quarantine, whereas the second was first isolated 16 days after quarantine and then for the second time, from the same horse, 402 days later. Multiple concurrent local infections were demonstrated by the isolation of two or more viruses from the same nasal swab.     

Rapid method for the identification and screening of herpesviruses by DNA fingerprinting combined with blot hybridization

Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radioactively labelled probe derived from the purified DNA of an EHV-1 reference strain was used for hybridization in order to detect the restriction fragments of different EHV-1 field isolates. This method allows the typing of many isolates within a short period of time.     

Efficiency of Plating on Chick Embryo Cells and Kinetic Neutralization of Herpesvirus hominis Strains

Since it appeared that plaque formation in monolayers of primary chick embryo cells might provide a simple technique for the typing of Herpesvirus hominis strains, 100 isolates were tested for their efficiency of plating (EOP) on chick embryo cells versus plating on human embryonic fibroblasts. EOP values varied from 10(0) to 10(-6): 88% of the strains of genital origin had an EOP equal to or greater than 10(-2), and 82% of the oral isolates had an EOP equal to or less than 10(-3). Kinetic neutralizations were done with 53 strains, including those 12 with an EOP of 10(-2) or 10(-3). An estimate of antigenic relatedness (R(a)) between strains was calculated from the neutralization results. Although the site of recovery, EOP, and R(a) generally correlated, the EOP of some oral strains did not agree with the neutralization results, and some genital strains showed type 1 EOP and R(a) values. Selection of variants with increased EOP values did not result in accompanying changes in R(a). Thus, the two markers appeared to vary independently. These data support other findings which suggest that there may be no absolute correlation between biological and antigenic markers in herpesviruses and that a larger number with more diversity of strains should be examined for more markers before a typing system is established.