Transcriptional control of the equine herpesvirus 1 immediate early gene

Transient expression assays measuring induction of an equine herpesvirus 1 (EHV-1) immediate early (IE) promoter construct, pIE beta gal, were used to examine trans-induction of the IE gene (IR1) promoter by superinfection with intact EHV-1 (Kentucky A strain), uv-inactivated EHV-1, or EHV-1 stocks highly enriched for defective interfering particles (DIPs), and by cotransfection with plasmids containing EHV-1 DNA fragments. Our results confirm reports by others in that the IE promoter can be induced by both intact and uv-inactivated EHV-1 and provide evidence that DIP-rich virus stocks also possess transinducing activity. Furthermore, IE promoter activity was induced by cotransfection of the promoter construct with either of two plasmids containing restriction enzyme DNA fragments that span ORF12, the putative EHV-1 homolog of the herpes simplex virus 1 (HSV-1) alpha-trans-inducing factor (alpha-TIF) gene, UL48, or by cotransfection with an isolated DNA fragment containing only ORF12, while no transactivation was associated with plasmids linearized by restriction enzyme digestion at a single site within ORF12. These data indicate that, despite its predicted lack of an acidic carboxy terminus (a region essential in HSV-1 alpha-TIF for trans-inducing activity), the EHV-1 ORF12 product is a functional alpha-TIF.     

Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1)

Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.     

The equine herpesvirus 1 (EHV-1) UL3 gene, an ICP27 homolog, is necessary for full activation of gene expression directed by an EHV-1 late promoter

We have previously reported that the equine herpesvirus 1 (EHV-1) XbaI G restriction fragment (nucleotides 1436 to 7943 relative to the left terminus of the EHV-1 genome [Kentucky A strain]) is required in combination with the EHV-1 immediate-early (IE) gene to achieve significant activation of two representative EHV-1 late promoter-chloramphenicol acetyltransferase (CAT) recombinants in transient expression assays. In this report, we demonstrate that the XbaI G-encoded UL3 gene (an ICP27 homolog) provides a trans-acting factor which acts (in combination with the EHV-1 IE gene product) to increase reporter gene expression directed by an EHV-1 late promoter-CAT recombinant plasmid. We show that cloned copies of UL3 can successfully substitute for the XbaI G fragment in CAT assays and that stop codon insertion within the UL3 open reading frame inhibits the ability of UL3 to activate reporter gene expression in trans.     

Cell-mediated cytolysis of equine herpesvirus-infected cells by leukocytes from young vaccinated horses

The objective of this study was to determine whether the administration of modified-live equine herpesvirus (EHV-1) to young horses with residual maternal antibodies stimulated EHV-specific cytolytic responses, and whether these responses were crossreactive between EHV-1 and EHV-4. Eighteen clinically normal Belgian cross-foals were used in the study and were commingled in two adjacent pens. Skin biopsies were harvested from 16 foals within 24 h of birth and fibroblast cultures were established, expanded and cryopreserved. Beginning at approximately 10 weeks of age, 10 randomly chosen foals were inoculated on days 0, 21, and 43 of the study with a vaccine containing modified-live EHV-1. Blood mononuclear leukocytes were obtained on days 0, 32, and 50 for the assessment of EHV-specific cytolytic activity using 5 h and 18 h chromium release assays. EHV-1-specific antibodies were assessed by enzyme-linked immunosorbent assay using serum collected on days -21, 0, 32, and 50 of the study. Lymphocyte blastogenic tests and bioassays for interferon activity were conducted on day 50. After two vaccinations, mononuclear leukocytes from seven of ten vaccinated foals had cytolytic activity against autologous EHV-1 cells and leukocytes from six of ten lysed EHV-4-infected cells when tested in an 18 h assay. This activity was enhanced by exogenous interleukin 2 and was markedly reduced using target cells from unrelated horses. Cytotoxicity was not detected in a 5 h assay following in vitro stimulation of leukocytes. After three vaccinations, blood leukocytes from 6/6 vaccinated foals and 0/6 unvaccinated foals had proliferative responses EHV-1. There were no significant differences in interferon production by leukocytes from these foals. Twelve foals tested had low concentrations of (maternal) EHV-1-specific antibody prior to vaccination. Five of eight foals tested had increases in EHV-specific antibodies, while 4/4 commingled unvaccinated foals had a decrease or no change in EHV-specific antibodies. These results demonstrate cytotoxic cellular immune responses can be induced in young horses with maternal antibodies following administration of modified-live vaccine.     

AP-3: an adaptor-like protein complex with ubiquitous expression

Equine herpesvirus type 2 (EHV-2) is a slow-growing, cytopathogenic gammaherpesvirus, which is suggested to be ubiquitous in the equine population. However, its precise role as a pathogen and its tissue tropism remains uncertain. To estimate the prevalence of EHV-2 in Germany and to investigate the possible pathogenicity of the virus, peripheral blood leucocytes (PBL) from 172 horses were examined for EHV-2 DNA by a sensitive and specific nested PCR based on the EcoRI-N genomic fragment and by classical cocultivation. PBL samples from 51% of the horses were positive by PCR and virus was isolated from 31% of the horses by cocultivation. However, almost all animals were seropositive for EHV-2. This may indicate that PBL do not harbour EHV-2 indefinitely after infection. Furthermore, a correlation between clinical signs and EHV-2 as a causative agent could not be determined. Nevertheless, the prevalence of virus was high among horses with upper respiratory tract disease, abortion and severe ataxia. The products of the second round of the PCR reactions showed size polymorphism. Sequencing of the products revealed that these size differences were due to repetition of the motif (AGACAGGGGCCATGCTGGC) between 9-16 times depending on the isolate, suggesting that the nested PCR might be a useful tool for the differentiation of EHV-2 isolates.     

The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions

Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.     

High molecular weight polypeptide bands specific for equine herpesvirus 4

Serum neutralisation (SN) and immunoblotting were used in attempts to distinguish between natural infections with the closely related viruses equine herpesvirus 1 (EHV-1) and equine herpes-virus 4 (EHV-4). Horse sera (n = 323) collected in 1990 from studs with no experience of EHV-1 abortions as well as 197 sera collected in 1992 from studs with a history of EHV-1 abortions were tested by SN. The two groups differed in the proportion with measurable EHV-1 antibody, the 1992 group being significantly higher. Both groups had high proportions with EHV-4 antibody and no serum had antibody to EHV-1 alone. Pools of positive sera were prepared as probes in immunoblots. High molecular weight (> 200 kDa) polypeptide bands specific for EHV-4 were detected. No bands specific for EHV-1 only were found. The specific EHV-4 polypeptides shared some properties with gp2 of EHV-1.     

Comparative trial between an ultra-short and long protocol of luteinizing hormone-releasing hormone agonist for ovarian stimulation in in-vitro fertilization

No epidemiological typing system to differentiate among Pseudomonas pseudomallei isolates has been available. Ribotype analysis was developed and used to examine 74 clinical and 10 environmental isolates of P. pseudomallei from Thailand. Six P. pseudomallei ribotypes were identified from restriction fragment polymorphisms of EcoRI chromosomal digests. The predominant ribotype, A, was found in 59 of the isolates examined. By using patterns from hybridizations with SalI, HindIII, and PstI restriction digests, isolates of ribotype A were subdivided into a further five subtypes, giving a total of 10 differentiable P. pseudomallei types. In 23 of 34 melioidosis patients studied, multiple P. pseudomallei isolates were present. In all but one of these patients, a single ribotype of the organism was present. Isolation of two different ribotypes of P. pseudomallei from one patient, one each in sputum and urine, suggests that superinfection may have occurred. The ribotype was shown to be conserved during the course of antibiotic treatments in seven patients studied, although the antibiotic sensitivity patterns in the isolates from these patients varied. The prevalence of subtype A1 in clinical and environmental specimens suggests that this strain may be predominant in this geographical location. These results demonstrate the usefulness of the ribotyping method for epidemiological studies of P. pseudomallei.     

DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) respiratory infection. A single intramuscular injection with plasmid DNA encoding EHV-1 glycoprotein D (EHV-1 gD), including its predicted C-terminal membrane anchor sequence, induced a specific antibody response detectable by 2 weeks and maintained through 23 weeks post injection. A second injection at 4 weeks markedly enhanced the antibody response and all EHV-1 gD-injected mice developed neutralizing antibodies. A lymphocyte proliferative response to whole EHV-1 was observed and a predominance of IgG2a antibodies after DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunized with EHV-1 gD DNA were able to clear virus significantly more rapidly from lung tissue and showed reduced lung pathology, in comparison to control mice.     

The prevalence of latent Equid herpesviruses in the tissues of 40 abattoir horses

Equid herpesviruses 1 or 4 (EHV-1 or -4) were isolated by cocultivation from 60% of 40 horses examined at slaughter. The lymph nodes draining the respiratory tract were the most common source of virus. EHV-1 or EHV-4 was never isolated from the trigeminal ganglia (SLG). The polymerase chain reaction (PCR) detected virus in 87.5% of bronchial lymph nodes and a similar level in the trigeminal ganglia that were examined. By both assays approximately one third of the positive animals harboured both viruses. Equid herpesvirus 2 (EHV-2) was isolated from all but one of the horses and from > 75% of the lymph nodes draining the respiratory tract; alpha viruses were isolated only in the presence of EHV-2. The results indicate that latent EHV-1 and EHV-4 are widespread in the equine population and that the primary site of latency is the lymph nodes of the respiratory tract.     

Aberrant expression of a cytokeratin in a subset of hepatocytes during chronic WHV infection

The cell culture-adapted KyA strain of equine herpesvirus type 1 (EHV-1) has been found to be attenuated in young horses (Matsumura et al., 1996, Vet. Microbiol. 48, 353-365). The KyA strain lacks at least six genes in its genome, including those encoding glycoproteins gE and gI. To elucidate whether EHV-1 glycoproteins gE and gI play a role in viral virulence, we have constructed an EHV-1 recombinant that has the genes encoding both gE and gI deleted from its genome and its revertant. Growth properties of the deletion mutant virus in vitro were compared with those of the parent and the revertant viruses. Plaque size of the mutant virus in fetal horse kidney (FHK) cells was significantly smaller than those of the parent and the revertant viruses. In one-step growth experiments, however, the yields of infectious virus from FHK cells infected with the deletion mutant, the parent, or the revertant virus were approximately the same. The results suggested that gE and/or gI of EHV-1 promoted cell-to-cell spread of the virus, but that these glycoproteins were not involved in the process of virus maturation and release or in virus attachment and penetration. Subsequently, the virulence of mutant and revertant viruses was examined in young horses. No clinical signs were observed in six horses, including three colostrum-deprived foals inoculated intranasally with the deletion mutant virus, whereas three colostrum-deprived foals inoculated intranasally with the revertant virus manifested clinical signs typical for EHV-1 respiratory infection (i.e., pyrexia, nasal discharge, and swelling of submandibular lymph nodes). The results obtained from in vivo studies revealed that the EHV-1 mutant defective in both gE and gI genes was avirulent in young horses, suggesting that gE and/or gI of the EHV-1 have an important role in EHV-1 virulence. However, the EHV-1 mutant defective in both gE and gI genes induced only a partial protectivity in inoculated foals from manifestation of respiratory symptoms after challenge infection.     

Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B

The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections. An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses. A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA. Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site. The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.     

Typing of human Mycobacterium avium isolates in Italy by IS1245-based restriction fragment length polymorphism analysis

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.     

Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.     

Mitochondrial DNA analysis of Phialophora verrucosa

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) was examined in 32 isolates of Phialophora verrucosa (eight isolates from Japan, 10 from China, four from the USA, six from Venezuela and four from Colombia) and in three of Phialophora americana using five restriction enzymes. P. verrucosa isolates were divided into 10 mtDNA types based on RFLP patterns. Phylogeny constructed on sequence divergence of mtDNA indicated that P. verrucosa is a single species and isolates are clustered into three groups. Japan and the USA contained Group A and Group B isolates, China Group B isolates and South America Group B and Group C isolates. RFLP patterns of P. americana mtDNA were identical to those of Type 1 or Type 4 of P. verrucosa mtDNA, suggesting that both are identical. RFLP patterns of P. verrucosa were distinct from those of other dematiaceous fungi including Exophiala jeanselmei, E. moniliae, E. dermatitidis, E. spinifera, Cladophialophora (Cladosporium) carrionii, Fonsecaea pedrosoi, and Hortaea werneckii. These results indicate that RFLP analysis of mtDNA is a useful method for the identification, taxonomy, typing, epidemiology and phylogeny of P. verrucosa.     

Responses of ponies to equid herpesvirus-1 ISCOM vaccination and challenge with virus of the homologous strain

An experimental (ISCOM) vaccine previously shown to protect hamsters from lethal challenge with equid herpesvirus-1 (EHV-1), was tested in horses. Vaccination with EHV-1 ISCOMs induced serum antibodies to the major virus glycoproteins gp10, 13, 14, 17, 18 and 21/22a, whereas antibody responses to gp2 were weak or absent. High levels of virus neutralising antibody of long duration were induced, but did not prevent challenge infection with virus of the homologous strain. However, in the vaccinated ponies there was a significant reduction in clinical signs, nasal virus excretion and cell associated viraemia compared with age-matched unvaccinated controls. There was a strong correlation between pre-challenge levels of serum virus neutralising antibody and the duration and total amount of virus excreted from the nasopharynx.     

Molecular analysis by pulsed-field gel electrophoresis and antibiogram of Streptococcus pneumoniae serotype 6B isolates from selected areas within the United States

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (+/-1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.     

Molecular analysis of multiple isolates of the major serotypes of group B streptococci

Serotyping of clinical isolates is a widely used technique for epidemiologic study of group B streptococcal infections. However, serotyping cannot definitively determine epidemiologically related or unrelated isolates. We investigated the use of restriction endonuclease analysis (REA) with both conventional agarose gel electrophoresis (AGE) and pulsed-field gel electrophoresis (PFGE) in 50 isolates of the major serotypes of group B streptococci. Single digestion with HindIII and HaeIII and double digestion with HindIII and then EcoRI were used for conventional AGE, and digestion with SmaI was used for PFGE. The molecular profile of one strain was compared with those of the strains within the same serotype as well as with the profiles from strains of different serotypes. Among 10 type Ia, Ia/alpha, Ia/alpha+beta, and Ia/R1 isolates and depending on the restriction enzyme used, we found between five and six REA patterns by conventional AGE and seven by PFGE; among 4 type Ib/alpha+beta isolates we found 2 to 4 REA patterns by conventional AGE and 4 by PFGE; among 21 type II, II/alpha, II/beta, II/alpha+beta, and II/R4 isolates, we found 11 REA patterns by both AGE and PFGE; and among 14 type III, III/R1, and III/R4 isolates, we found from 7 to 12 different REA patterns by AGE and 10 by PFGE. In total, among 13 serotypes and one nontypeable strain, we found 29 to 31 REA patterns by conventional AGE and 33 by PFGE. A particular REA pattern within a serotype was different from the patterns found in the other serotypes, suggesting that REA analysis by using conventional AGE or PFGE is a sensitive method for analyzing genetic relatedness and diversity in group B streptococci and has potential value in molecular epidemiologic studies.     

Molecular analysis of glycopeptide-resistant Enterococcus faecium isolates collected from Michigan hospitals over a 6-year period

The purpose of this study was to evaluate the molecular relatedness of clinical isolates of glycopeptide-resistant Enterococcus faecium isolates collected from hospitals in Michigan. A total of 379 isolates used in this study were all vancomycin-resistant E. faecium isolates collected from 28 hospitals and three extended-care facilities over a 6-year period from 1991 to 1996. For the 379 isolates, there were 73 pulsed-field gel electrophoresis (PFGE) strain types. Within strain types, there were as many as six restriction fragment differences. Most isolates (70%) belonged to six strain types, which were designated M1 (36%), M2 (3%), M3 (18%), M4 (6%), M10 (4%), and M11 (3%). PFGE strain M1 was cultured from 135 patients in 13 hospitals during the period 1993 to 1996. Strain type M2 was cultured from 11 patients in two hospitals during the period 1991 to 1992 and was not observed after 1992. Strain type M3 was cultured from 70 patients in 10 hospitals during the period of 1994 to 1996. Both M4 and M10 were cultured from 23 patients in three hospitals and from 15 patients in two hospitals, respectively, during 1995 to 1996. M11 was cultured from 13 patients in four hospitals during 1996. A total of 23 of 28 hospitals had evidence of clonal dissemination of some isolates. Plasmid content and hybridization analysis done on 103 isolates from one hospital and two affiliated extended-care facilities indicated that the strains contained from one to eight plasmids. Mating experiments indicated transfer of vancomycin resistance from 94 of these isolates into plasmid-free E. faecium GE-1 at transfer frequencies of <10(-9) to 10(-4). Gentamicin resistance and erythromycin resistance were cotransferred at various frequencies. A probe for the vanA gene hybridized to the plasmids of 23 isolates and to the chromosomes of 72 isolates. A probe for the vanB gene hybridized to the chromosomes of 8 isolates. The results of this study suggest inter- and intrahospital dissemination of vancomycin-resistant E. faecium strains over a 6-year period in southeastern Michigan. The majority of isolates studied belonged to the same few PFGE strains, indicating that clonal dissemination was responsible for most of the spread of resistance that occurred.     

Respiratory care protocols in postanesthesia care

IS1311 is an insertion sequence from Mycobacterium avium and M. avium subsp. paratuberculosis. Using a 180 bp fragment of IS 1311 as a probe, 7-10 copies of IS1311 were revealed in strains of M. avium subsp. paratuberculosis. With a given restriction enzyme, the restriction fragment length polymorphism patterns obtained from isolates of M. avium subsp. paratuberculosis from cattle were all identical, but they differed from those of isolates from sheep, which could be separated into two types. A 1259 bp fragment of IS1311 produced by polymerase chain reaction (PCR) from two isolates of M. avium subsp.paratuberculosis from cattle and two isolates from sheep was sequenced and compared to the sequence known from M. avium. Apart from five point differences the sequences were identical. Restriction endonuclease analysis (REA) of the PCR product was used to distinguish isolates of M. avium subsp. paratuberculosis from M. avium, in addition to the conventional test for IS900. In isolates of M. avium subsp. paratuberculosis from cattle the IS1311 gene was polymorphic at position 223, which enabled isolates from sheep and cattle to be distinguished by PCR-REA. These simple tests will be used to enhance disease control programmes for Johne's disease in ruminants. The findings of this study raise interesting questions about the evolution of M. avium subsp. paratuberculosis.