The pathogenicity of Ab4p, the sequenced strain of equine herpesvirus-1, in specific pathogen-free foals.

The sequencing of the genome of equine herpesvirus-1 (EHV-1) is reported in Elizabeth A. R. Telford, Moira S. Watson, Kathryn McBride, and Andrew J. Davison, 1992, Virology, 189, 304-316. The sequence was derived using a plaque-purified clone of EHV-1 strain Ab4 (designated Ab4p). To ensure that Ab4p shares the pathogenic characteristics of parental Ab4 (hereafter Ab4), both were inoculated intranasally into foals, specifically free from EHV-1 and EHV-4. Clinical signs, including rectal temperature, were similar for both viruses. In addition, nasal shedding of virus was observed over a 1- to 2-week period postinfection, and viremia was established with both Ab4 and Ab4p. Isolation of virus from one foal following intravenous administration of steroids indicates that Ab4p can establish latency and undergo reactivation. Finally, retinal lesions were observed and these were similar to those seen with Ab4. In conclusion, several pathogenic characteristics of Ab4 are retained in the plaque-purified clone, Ab4p.     

Reinfection and reactivation of equine herpesvirus-1 in the mouse

Summary Balb/c mice were inoculated with equine herpesvirsus-1 (EHV-1) by the intranasal (i.n.) route. Mice developed respiratory signs; virus replication occurred in the respiratory tract and viraemia was detected; some mice died. Recovered mice were given a second inoculation with the same strain 5 months later. Following the second infection no mice died, however, virus replication was again observed in the respiratory tract and viraemia was detected once more. Administration of an antiviral agent during the acute infection prevented mice from developing severe clinical signs and all survived. These mice, and some that had survived an acute infection without chemotherapy, were given a variety of stimuli, for example X-irradiation or corticosteroid injection. Reappearance of infectious virus was detected in approx. 1/3 animals in either the respiratory tract or blood. We speculate on the possible sites of latency in the model.     

Analysis of specific IgM responses in secondary dengue virus infections: levels and positive rates in comparison with primary infections.

BACKGROUND: Dengue viruses are a serious cause of illness in tropical and subtropical areas of the world. Laboratory diagnosis is essential for confirmation of dengue virus infections. Detection of specific IgM by IgM-capture enzymed-linked immunoassay (ELISA) has been widely used as a main serological diagnostic technique. OBJECTIVES: The levels of specific IgM in secondary dengue virus infections were compared with those in primary infections. STUDY DESIGN: A total of 1780 samples collected from 924 confirmed dengue cases were tested for anti-dengue IgM by IgM-capture ELISA. RESULTS AND CONCLUSIONS: Specific IgM was detected in all the cases with primary dengue virus infection on disease day 9 or later. However, specific IgM cannot be detected in 28% (204/716) of the cases in secondary infections. The average titers of IgM were higher in primary infections than in secondary infections. The results confirmed that IgM detection is a reliable serological diagnostic test in primary dengue virus infections. Although IgM detection is also a useful test, other serological diagnostic tests or tests for dengue virus detection are necessary for confirmation of all the secondary dengue virus infections.     

Demonstration of equine herpesvirus-1 gene expression in the placental trophoblasts of naturally aborted equine fetuses

Equine herpesvirus-1 (EHV-1) infection was demonstrated in the lung tissue of seven aborted fetuses by immunohistochemical labelling and polymerase chain reaction. The placentas of the fetuses were also examined by non-isotopic in-situ hybridization for the EHV-1 glycoprotein B (gB) gene. Positive hybridization signals were observed in the cytoplasm of trophoblasts, especially in microcotyledons, of all seven placentas, and in villous epithelium of the allantochorion of six placentas. Despite the presence of EHV-1 RNA, EHV-1 antigens were not detected in placentas by immunohistochemical examination. The present study represents the first in-vivo demonstration of the EHV-1 gene in equine trophoblasts. The findings suggest direct cell-to-cell spread of EHV-1 from endometrial cells to trophoblasts.     

The genomic diversity among equine herpesvirus-1 strains isolated in Japan

Summary The DNAs from nine Japanese field isolates of equine herpesvirus-1 (EHV-1) were analyzed by digestion with the restriction endonuclease Bam HI and Southern hybridization. Comparing restriction profiles among the EHV-1 strains, there was no considerable difference between isolates before and after vaccine application, but some minor variations in the mobility ofBam HI fragments were observed. To identify these variable fragments, all genomic DNA sequences of the Japanese prototype of EHV-1 have been cloned asBam HI restriction fragments into the plasmid pUC-18. Physical maps of the virus DNA were constructed by a combination of Southern blot analysis and double enzyme digestion of the cloned fragments. By using these cloned fragments as probes in Southern blot analysis, the areas of heterogeneity observed among the field EHV-1 isolates were located in both terminals of U_L, the center of U_L, IR, U_S and TR regions of the genome.     

Herpesvirus ICP18.5 and DNA-binding protein genes are conserved in equine herpesvirus-1

The genome of equine herpesvirus-1 (EHV-1) contained three open reading frames (ORFs) in a 3.9 kbpBamHI-SmaI fragment at 0.38–0.41 map units in the long unique region. The most 5′ ORF encoded the carboxy terminus of a protein with 45–55 percent amino acid homology to the DNA-binding proteins (ICP8-DBP) of four other alphaherpesviruses. The middle ORF translated to a polypeptide of 775 residues with 43–55% homology to the ICP18.5 proteins. The most 3′ ORF encoded the EHV-1 glycoprotein B (gB) gene. Three mRNAs of 4.3, 4.4–4.8, and 3.5–3.9 kb (corresponding to the three sequenced ORFs) were all transcribed from the same strand. The gene order of this group was conserved in all herpesviruses examined.     

Detection of equine herpesvirus-1 in the fetal membranes of aborted equine fetuses by immunohistochemical and in-situ hybridization techniques

Formalin-fixed, paraffin wax-embedded fetal membranes from 76 cases of equine abortion were examined immunohistochemically for equine herpesvirus (EHV)-1 antigen. Of the 76 cases, 11 had been proved EHV-1-positive by diagnostic methods applied to the aborted fetuses (viral isolation in tissue culture, or immunohistochemical examination, or both). Of the 11 fetal membranes from the virus-positive animals, five gave positive results on immunohistochemical examination, and three on in-situ hybridization; the positive signals were detected in trophoblastic cells and occasionally in monocytes and endothelial cells. The distribution of virus appeared to be related to areas of (1) vacuolar degeneration and desquamation of chorionic epithelium, (2) mild lympho-histiocytic vasculitis and placentitis, and (3) increased metabolic activity of mesenchymal cells in the villi of the fetal membranes. This is the first report of EHV-1 antigen and nucleic acid detection in the trophoblasts of fetal membranes from spontaneous cases of equine abortion.     

Modulation of the serological response of specific pathogen-free (EHV-free) foals to EHV-1 by previous infection with EHV-4 or a TK-deletion mutant of EHV-1

Summary EHV-1 was inoculated into specific pathogen-free (SPF) foals in order to study uncomplicated primary responses. Infection resulted in a strong serological response recognizing EHV-1-specific antigens; this contrasts with a previous publication where a weak response was recorded in SPF animals. Antibodies to EHV-1 were readily detected by four techniques (virus neutralization, complement fixation, Western blots and immune precipitation), yet there was comparatively little cross-reaction to EHV-4 target antigen. Re-in-oculation with the same virus strain stimulated antibodies to EHV-1 but no additional antigens were recognized and antibodies cross-reacting with EHV-4 antigens were not enhanced. Having characterized the uncomplicated primary response to EHV-1 in SPF foals, further animals were exposed to either EHV-4 or a thymidine kinase-deficient mutant of EHV-1 prior to challenge with w/t EHV-1 to investigate how these infections might modulate the immune responses to EHV-1 or 4. Primary inoculation with EHV-4 or with a thymidine kinase-deficient mutant of EHV-1 produced productive infections as evidenced by virus shedding and pyrexia. In both these cases, however, in contrast to that with w/t EHV-1, the serological response was very weak. Re-infection of foals primed with either EHV-4 or TK-deficient EHV-1 with w/t EHV-1 resulted in a strong response to EHV-1 antigens detected by all four methods. In addition, in the foals given a primary inoculation with EHV-4, superinfection with EHV-1 resulted in a strong cross-reactive response to EHV-4 target antigens. The relevance of these observations to the interpretation of previously reported serological responses to EHVs in SPF and naturally reared animals is discussed.     

Human herpesvirus-6 reactivation in a longitudinal study of two HIV-1 infected patients

After primary infection, human herpesvirus-6 (HHV-6) persists in latent form and can be reactivated in immunocompromised subjects. A longitudinal study of HHV-6 infection was carried out in two HIV-1 seropositive patients to provide in vivo evidence of HHV-6 reactivation. Concomitant with a significant rise of anti-HHV-6 IgG detected by IFA, a transient increase of HHV-6 viral load was shown in PBLs by PCR. During HHV-6 reactivation it was also identified either cell-free HHV-6 by PCR in plasma or IgM antibody titers. HHV-6 reactivation was followed by a temporary decrease in CD4+ count and by a progressive dramatic loss of CD4+ during the following 18 months. HHV-6 strain characterization by PCR demonstrated that first patient (MM) initially showed the B variant, followed by reactivation and persistence of the A variant, while in the second (SG) only the A variant was detected. The evidence of HHV-6 reactivation suggests its involvement in immunologic damage underlying the disease. J. Med. Virol. 51:259–264, 1997. © 1997 Wiley-Liss, Inc.     

Multi-centric histiocytosis: Experimental induction in broiler and specific pathogen-free leghorn chickens

Seventy-five 3-day-old broiler chicks and twenty specific pathogen-free leghorn chicks were injected with 0.5 ml of a homogenate, prepared from organs from broilers diagnosed with naturally-occurring multicentric histiocytosis (MH). Equal numbers of uninjected broiler and leghorn chicks (controls) were maintained in adjacent pens. Ten weeks later, nine broilers had well-developed gross and microscopic MH lesions. The distribution and histological appearance of lesions in these experimental chicks was similar to lesions described in naturally occurring field cases. Six leghorns had gross lesions similar to those found in their broiler counterparts; however, in the leghorns, the cellular masses contained more lymphocytes and, additionally, masses were found in the gizzard musculature. One gizzard contained a sarcoma. Broiler chickens with MH weighed less than their control counterparts and were more likely to be anaemic. Sequences specific for reticuloendotheliosis viruses (REV) were found in the MH homogenate, in organs from most affected experimental leghorns and broilers, and in organs from a control broiler. However, REV were not isolated from these tissues, nor were specific antibodies for REV or avian leukosis/sarcoma viruses (ALV) found in chick serum. Leukosis/sarcoma viruses were isolated from some MH-affected experimental leghorns and broilers. Sequences specific for Marek's disease herpesvirus were not identified by polymerase chain reaction. The aetiology of MH remains unknown.     

The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1

Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable by identical primers to wild type EHV-1, yet capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR-ELISA system. A competitive PCR-ELISA system which can control for the presence of PCR inhibitors and which is capable of detecting 63 genome equivalents of EHV-1 has been developed. EHV-1 presence in infected equine tissue and cell culture material was demonstrated using this system. The entire assay can be completed within one working day and facilitates multiple sample analysis. The availability of a robust, competitive PCR-ELISA system for the detection of EHV-1 will facilitate the rapid and sensitive detection of EHV-1 and offers the potential for eliminating the occurrence of abortion storms in stud farms.     

Human herpesvirus-6 and human herpesvirus-7 infections in bone marrow transplant recipients

Human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and human herpesvirus-7 (HHV-7) DNA in peripheral blood leukocytes (PBL) of 61 bone marrow transplant recipients was monitored weekly during the first 12 weeks post-transplantation by a nested polymerase chain reaction (PCR). Thirty-seven (61%), 17 (28%), and 32 (53%) of patients had one or more PBL specimens positive for HCMV, HHV-6 or HHV-7 DNA, respectively. HHV-7 DNA in PBL during the early post-transplant period was associated with a longer time to neutrophil engraftment (mean 28.8 days vs 19.8 days; P = 0.01). In two patients who failed to engraft, HHV-6 DNA and HHV-7 DNA was detected in plasma and PBL, respectively, early in their post-transplant period. Patients with HCMV disease were more likely to have concurrent HHV-7 DNA in PBL prior to onset of disease than were patients with asymptomatic HCMV infection, suggesting that HHV-7 may be a cofactor in the progression from HCMV infection to HCMV disease. In the 17 patients (179 specimens) in whom viral DNA in plasma was studied (in addition to PBL), a positive result was found only in 3. In each, viral DNA in plasma appeared to correlate with clinically significant disease. HHV-7 DNA in plasma was associated with encephalitis in an allograft recipient. J. Med. Virol. 53:295–305, 1997. © 1997 John Wiley & Sons, Inc.     

IgG antibodies to human herpesvirus-6 in children and adults and in primary Epstein-Barr virus infections and cytomegalovirus infections [corrected

Antibody titers against human herpesvirus-6 (HHV-6) were determined in 80 healthy adults and 100 children and teenagers from Sweden to gain information on the role of the virus and its epidemiology. Based on a positive immunofluorescence titer of 1:10 and above, about 85% of the adults and children were seropositive with 60% seropositivity of children below age one year. Titers were generally higher in patients with simultaneous EBV or CMV infection, yet crossreactivity appeared essentially no problem. HHV-6 thus is ubiquitous like other herpesviruses. Primary infection seems to occur early in life, and reactivation or delayed primary infection may be associated with a variety of disorders.     

Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways

We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after viral internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection.     

Immunoglobulin M to cytomegalovirus in primary and reactivation infections in renal transplant recipients.

Two commercially available enzyme immunoassays and one assembled in house were used to measure immunoglobulin M (IgM) antibody to cytomegalovirus (CMV) in a total of 220 serum specimens from 104 renal transplant recipients. All assays included a step in which interfering IgG antibody was removed or complexed. Concordance of results between pairs of assays ranged from 84 to 96%. All sera from patients with recent seroconversion (primary CMV infection) had measurable anti-CMV IgM. Among those already seropositive to CMV when transplanted, 26 to 55% had IgM antibody posttransplant, depending on the assay. This was observed regardless of the CMV serologic status of the kidney donor, indicating that reactivation of endogenous CMV, as well as reinfection, can induce this antibody in transplant recipients. Four cadaver donors known to transmit CMV to eight recipients did not have measurable IgM antibody to CMV.     

Isolation of equine herpesvirus-1 lacking glycoprotein C from a dead neonatal foal in Japan

Summary. We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.     

Pathogenesis of equine herpesvirus‐1 infection in the mouse model

Equine herpesvirus‐1 (EHV‐1) is a major equine pathogen causing respiratory diseases, abortions and severe neurological disorders. The basis of neurological disturbances is, as in other organs, infection of endothelial cells, followed by vasculitis, thrombosis and ischaemic damage of the parenchyma. Here, a murine model was used to explore the mechanism of entry to, and spread within the brain, the cell affinity of the agent and the modulating role of the immune defence, which are all factors governing the pathogenesis of the neurological disease. Because controversial views exist about these mechanisms, we undertook a neuropathological study with intranasally infected adult mice. EHV‐1 entered the brain through the olfactory neuroepithelium and along the olfactory nerves, and spread transsynaptically in rostro‐caudal direction, using olfactory and limbic neuronal networks. Exclusively neurons were infected. The cellular immune reaction exerted a restraining effect on virus dissemination. Following nasal infection, the olfactory route was the major pathway for virus entry and dissemination, involvement of the trigeminal nerve in virus spread seems much less probable. In the adult mouse brain EHV‐1 behaves as a typical neurotropic agent, using, similarly to other herpesviruses, the neuronal networks for dissemination. Vasculitis, the predominant type of lesion in natural infection, and endothelial cell positivity for EHV‐1 were detectable only in the lung. Thus, this agent exhibits in the mouse a dual affinity: it is neurotropic in the brain, and endotheliotropic in visceral organs. Consideration of pathogenetic aspects of equine and experimental murine EHV‐1 infections also helps a better understanding of human herpetic brain disease.     

A protective effect of epidermal powder immunization in a mouse model of equine herpesvirus-1 infection

To evaluate the protective effect of epidermal powder immunization (EPI) against equine herpesvirus-1 (EHV-1) infection, we prepared a powder vaccine in which formalin-inactivated virions were embedded in water-soluble, sugar-based particles. A PowderJect device was used to immunize mice with the powder vaccine via their abdominal skin. We found that twice-immunized mice were protected against challenge with the wild-type virus. This protective effect was equivalent to or better than that observed in mice immunized with other types of vaccines, including a gene gun-mediated DNA vaccine containing the glycoprotein D (gD) gene or conventional inactivated virus vaccines introduced via intramuscular or intranasal injections. These findings indicate that the powder vaccine is a promising approach for the immunological control of EHV-1 infection, either alone or as a part of prime-boost vaccination strategies.