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1.
Summary.  Insertional mutagenesis was used to construct an equine herpesvirus 1 (EHV-1) mutant in which the open reading frame for glycoprotein D was replaced by a lacZ cassette. This gD deletion mutant (ΔgD EHV-1) was unable to infect normally permissive RK cells in culture, but could be propagated in an EHV-1 gD-expressing cell line (RK/gD). Phenotypically complemented ΔgD EHV-1 was able to infect RK cells, but did not spread to form syncytial plaques as seen with wild type EHV-1 or with ΔgD EHV-1 infection of RK/gD cell cultures. Therefore EHV-1 gD is required for virus entry and for cell-cell fusion. The phenotypically complemented ΔgD EHV-1 had very low pathogenicity in a mouse model of EHV-1 respiratory disease, compared to a fully replication-competent EHV-1 reporter virus (lacZ62/63 EHV-1). Intranasal or intramuscular inoculation of mice with ΔgD EHV-1 induced protective immune responses that were similar to those elicited in mice inoculated with lacZ62/63 EHV-1 and greater than those following inoculation with UV-inactivated virus. Received January 14, 2000/Accepted April 27, 2000  相似文献   

2.
The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.  相似文献   

3.
Summary.  The equine herpesvirus 1 (EHV-1) strain HVS25A regulatory genes IE and UL5, encoding homologues of herpes simplex virus 1 (HSV-1) ICP4 and ICP27 respectively, were cloned into a eukaryotic expression vector and the DNA injected intramuscularly into mice. Antibodies produced in this way detected the IE or UL5 gene products as diffuse material in nuclei of RK13 cells transfected with the individual genes but as discrete punctate or large aggregates in RK13 cells infected with EHV-1. Western blotting on EHV-1 infected RK13 cells showed multiple IE products of 120–200 kDa and a UL5 product of 52 kDa. Inoculation with plasmids expressing EHV-1 IE or UL5 provided limited protection against EHV-1 challenge in mice as determined by increased virus clearance from lungs on day 2 post-challenge and a reduction in severity of lung histopathology. However, this protection was relatively weak compared with that provided by inoculation of DNA encoding EHV-1 glycoprotein D (gD), possibly reflecting the importance of neutralising antibody in this model. Accepted May 19, 2000 Received March 20, 2000  相似文献   

4.
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.  相似文献   

5.
Equine herpesvirus 1 glycoprotein D (EHV-1 gD) has been shown in mouse models and in the natural host to have potential as a subunit vaccine, using various expression systems that included Escherichia coli, baculovirus and plasmid DNA. With the aim of producing secreted recombinant protein, we have cloned and expressed EHV-1 gD, lacking its native signal sequence and C-terminal transmembrane region, into the methylotrophic yeast Pichia pastoris. The truncated glycoprotein D (gD) gene was placed under the control of the methanol inducible alcohol oxidase 1 promoter and directed for secretion with the Saccharomyces cerevisiae alpha-factor prepro secretion signal. SDS-PAGE and Western blot analysis of culture supernatant fluid 24 h after induction revealed gD-specific protein products between 40 and 200 kDa. After treatment with PNGase F and Endo H, three predominant bands of 34, 45 and 48 kDa were detected, confirming high mannose N-linked glycosylation of Pichia-expressed gD (Pic-gD). N-terminal sequence analysis of PNGase F-treated affinity-purified protein showed that the native signal cleavage site of gD was being recognised by P. pastoris and the 34 kDa band could be explained by internal proteolytic cleavage effected by a putative Kex2-like protease. Pic-gD, when used in a DNA prime/protein boost inoculation schedule, induced high EHV-1 ELISA and virus neutralizing antibodies and provided protection from challenge infection in BALB/c mice.  相似文献   

6.
Summary Equine herpesvirus 1 glycoprotein D (EHV-1 gD) expressed constitutively in mammalian cell lines had similar electrophoretic mobility to gD produced in EHV-1 infected cells but lacked a possibly complexed higher molecular weight form seen in the latter. Recombinant gD was N-terminally cleaved at the same site as gD in EHV-1 infected cells and expression was associated with enhanced levels of cell-cell fusion, indicating a role for EHV-1 gD in cell-to-cell transmission of virus.  相似文献   

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The abortigenic effects of equine herpesvirus 1 (EHV-1) strain HVS25A, given intranasally, were assessed in pregnant BALB/c, C57BL/6J and Quakenbush mice at day 16 of pregnancy. All EHV-1-infected BALB/c mice showed clinical signs typical of EHV-1-induced disease, together with evidence of abortion. However, although there were fetal and neonatal deaths in some C57BL/6J and Quakenbush litters, the respiratory and systemic effects of EHV-1 infection in the dams were inconsistent. BALB/c dams were then inoculated at day 15 of pregnancy with either EHV-1 or rabbit kidney (RK) cell lysate (controls) and animals were killed at days 1-5 post-inoculation (pi), i.e., before the occurrence of abortions. EHV-1-infected mice showed a significant fall in rectal temperature between days 1 and 2 pi and lost weight during the first 4 days pi, demonstrating a significant mean difference in weight gain from the control group at days 2, 3, 4 and 5 pi. Death in utero was seen in five of 90 fetuses of EHV-1-infected mice, but in no fetuses from RK-inoculated mice. On days 1, 2, 3, 4 and 5 pi, the fetuses from EHV-1-infected dams were significantly smaller than those from RK-inoculated dams. Congestion and necrosis of the middle layer of trophoblast and chorionic necrosis were observed in the placentae from EHV-1-infected dams and assessed by a scoring system. Virus was isolated rarely from the fetuses (1/73), placentae (3/72) and uteri (1/16) of EHV-1-infected dams, and only from those killed on day 1 or 2 pi. This indicates that, as in the horse, abortion caused by EHV-1 infection in mice is not necessarily a consequence of fetal infection but may be due to fetal compromise due to vascular effects on the placenta.  相似文献   

10.
The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HDeltagK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HDeltagK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting M(r)s between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.  相似文献   

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An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene. On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC). However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells. Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.  相似文献   

13.
Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.  相似文献   

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Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association, it establishes persistent infections in its natural host, the bovine, and in an experimental host, the rabbit. BoHV-4 immediate early 2 (IE2) RNA is the less abundant, spliced, 1.8 kb RNA. The predicted amino acid sequence, of the IE2 protein, reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein-Barr virus (EBV) transactivator R protein and its homologues including, herpesvirus saimiri (HVS), equine herpesvirus 2 (EHV-2), murine herpesvirus 68 and Kaposi's sarcoma-associated herpesvirus (KSHV). We examined recently the interaction of BoHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although some infectious viruses can be produced, no cytopathic effect (CPE) was observed [J. Gen. Virol. 81 (2000) 1807]. Because IE2 could play a critical role in BoHV-4 productive infection and its overexpression in RD-4 cells could switch the non-permissive RD-4 status to a permissive one. RD-4 cells expressing stably BoHV-4 IE2 gene were generated. BoHV-4 IE2 induced an increased production of infectious viral particles sufficient to obtain an apparent cytopathic effect. It is concluded that BoHV-4 IE2 is a key factor in determining the outcome of BoHV-4 infection.  相似文献   

16.
Summary Six monoclonal antibodies directed against EHV-1 glycoprotein 13 were characterized. Five antibodies neutralized EHV-1 and were directed against a single antigenic site which comprised type-specific and type cross-reactive epitopes. Inhibition of monoclonal antibody binding to this site by post-infection equine sera suggests that it is a target of host antibody during natural infection with either EHV-1 or EHV-4.  相似文献   

17.
Glycoprotein G (gG) of equine herpesvirus type 1 (EHV-1), a structural component of virions and secreted from virus-infected cells, was shown to bind to a variety of different chemokines and as such might be involved in immune modulation. Little is known, however, about its role in the replication cycle and infection of EHV-1 in vivo. Here we report on the function of gG in context of virus infection in vitro and in vivo. A gG deletion mutant of pathogenic EHV-1 strain RacL11 (vL11DeltagG) was constructed and analyzed. Deletion of gG had virtually no effect on the growth properties of vL11DeltagG in cell culture when compared to parental virus or a rescuant virus vL11DeltagGR, respectively, and virus titers and plaque formation were unaffected in the absence of the glycoprotein. Similarly, in the murine model of EHV-1 infection, no significant differences in virulence between the gG deletion mutant and RacL11 or vL11DeltagGR were found at high doses of infection. However, infection of mice at lower doses revealed that the gG deletion mutant was able to replicate to higher titers in lungs of infected mice. Additionally, these mice lost significantly more weight than those infected with RacL11 and a more pronounced inflammatory response in lungs was observed. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse.  相似文献   

18.
Summary.  Effects of infection with two different strains of equine herpes virus type 1 (EHV-1; Piber 178/83, Kentucky D) on the cytoskeleton of Vero cells were investigated immunohistochemically, and evaluated by confocal laser scanning microscopy. Twenty four hours post EHV-1 infection the assembly of the microtubulus system of Vero cells was heavily disturbed. The Golgi region was dispersed into vesicles spread throughout the cytoplasm as demonstrated by WGA lectin binding. Other cytoskeletal elements such as cytokeratin, vimentin, and filamentous actin (F-actin) were not affected by EHV-1 infection. The nature of Vero cell death after EHV-1 infection was investigated by three different methods to include all possible stages of apoptosis. All methods failed to demonstrate characteristic apoptotic features, therefore, it seems likely that necrosis is the predominant way of cell death in EHV-1 infected Vero cells. Received February 23, 1999 Accepted April 26, 1999  相似文献   

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20.
One of the consequences of equine herpesvirus 1 (EHV-1) infection in the natural host is a neurological disease that can lead to paralysis. The pathology associated with EHV-1-induced neurological disease includes vasculitis of the small blood vessels within the central nervous system and subsequent damage to the surrounding neural tissue. In a previous study, an EHV-1 recombinant KyA virus (KgI/gE/75) was generated in which the sequences encoding glycoprotein I (gI) and glycoprotein E (gE) were repaired [Frampton et al. 2002 (Virus Research 90: 287-301)] using genes of the pathogenic EHV-1 strain 89c25. In contrast to the parental KyA virus that lacks gI and gE, the recombinant KgI/gE/75 was able to spread to the brains of CBA mice after intranasal infection. Infection resulted in a meningoencephalitis characterized by lymphocytic cuffing of small blood vessels within the brain, consistent with that observed in EHV-1-infected horses exhibiting neurological signs. KgI/gE/75 was able to elicit cytopathology in the lung prior to spread to the brain. However, like the attenuated KyA strain, KgI/gE/75 did not persist in the lung and was completely cleared from lung tissue by day 5 postinfection. We propose that gI and gE are neurovirulence factors for EHV-1, and that the CBA mouse model can be extended to study neurologic sequelae resulting after EHV-1 infection.  相似文献   

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