Equid Herpesvirus Type 1 Activates Platelets

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.     

A point mutation in a herpesvirus polymerase determines neuropathogenicity

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.     

Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.     

Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons—the possible effects of the multiple passages of EHV-1 on its neurovirulence

Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.     

Isolation and partial characterization of equine herpesvirus type 1 in Czechia

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.     

Equine Herpesviruses 1 and 4: Amplification and Differentiation by Polymerase Chain Reaction

Equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are important pathogens responsible for considerable economic losses in the horse industry. Differentiation between these 2 viruses using conventional serological methods with polyclonal antisera has been difficult. Biological differences have, however, been recognised for a long time. Both EHV-1 and EHV-4 are associated with upper respiratory disease, but disseminated infection with EHV-1 can result in neurological disorders or abortion in susceptible mares.     

Antibody Responses in Humans Infected with Newly Emerging Strains of West Nile Virus in Europe

Infection with West Nile Virus (WNV) affects an increasing number of countries worldwide. Although most human infections result in no or mild flu-like symptoms, the elderly and those with a weakened immune system are at higher risk for developing severe neurological disease. Since its introduction into North America in 1999, WNV has spread across the continental United States and caused annual outbreaks with a total of 36,000 documented clinical cases and ∼1,500 deaths. In recent years, outbreaks of neuroinvasive disease also have been reported in Europe. The WNV strains isolated during these outbreaks differ from those in North America, as sequencing has revealed that distinct phylogenetic lineages of WNV concurrently circulate in Europe, which has potential implications for the development of vaccines, therapeutics, and diagnostic tests. Here, we studied the human antibody response to European WNV strains responsible for outbreaks in Italy and Greece in 2010, caused by lineage 1 and 2 strains, respectively. The WNV structural proteins were expressed as a series of overlapping fragments fused to a carrier-protein, and binding of IgG in sera from infected persons was analyzed. The results demonstrate that, although the humoral immune response to WNV in humans is heterogeneous, several dominant peptides are recognized.     

Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice

Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, interferon gamma-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.     

Sequence characteristics of a gene in equine herpesvirus 1 homologous to glycoprotein H of herpes simplex virus.

A gene in equine herpesvirus 1 (EHV-1, equine abortion virus) homologous to the glycoprotein H gene of herpes simplex virus (HSV) was identified and characterised by its nucleotide and derived amino acid sequence. The EHV-1 gH gene is located at 0.47-0.49 map units and contains an open reading frame capable of specifying a polypeptide of 848 amino acids, including N- and C-terminal hydrophobic domains consistent with signal and membrane anchor regions respectively, and 11 potential sites for N-glycosylation. Alignment of the amino acid sequence with those published for HSV gH, varicella zoster virus gpIII, Epstein Barr virus gp85 and human cytomegalovirus p86 shows similarity of the EHV gene with the 2 other alpha-herpesviruses over most of the polypeptide, but only the C-terminal half could be aligned for all 5 viruses. The identical positioning of 6 cysteine residues and a number of highly conserved amino acid motifs supports a common evolutionary origin of this gene and is consistent with its role as an essential glycoprotein of the herpesvirus family. An origin of replication is predicted to occur at approximately 300 nucleotides downstream of the EHV-1 gH coding region, on the basis of similarity to other herpesvirus origins.     

The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor

Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional ligand for the EHV-2 E1 receptor. Chemokines are likely to play a role in the regulation of immune functions in equine hosts during EHV-2 infection and, via interaction with E1, may affect viral replication and/or escape from immune responses.     

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Evidence of respiratory tract infection induced by equine herpesvirus, type 2, in the horse.

Five horses were experimentally exposed to equine herpesvirus 2 strain LK. Two young foals developed chronic pharyngitis (98 and 232 days, respectively). Growth characteristics, cytopathic effects (CPE), inclusion body formation, ether sensitivity, and immunofluorescent analysis indicated that the virus recovered from infected animals was a herpesvirus serologically identical with, or at least antigenically related to EHV-2 strain LK. No significant complement-fixing (CF) or virus-neutralizing (VN) antibody responses were observed in adult horses while both foals demonstrated a rise in CF antibody titer. One of the two foals demonstrated a rise in VN antibody only. The results suggest that EHV-2 virus induced chronic pharyngitis, primarily the result of lymphoid proliferation, with no overt clinical signs.     

Genotyping of Indian <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains by MLVA and repetitive DNA sequence based PCRs

India experienced two plague outbreaks in Gujarat and Maharastra during 1994 and then in the Shimla district of Himachal Pradesh during 2002. Yersinia pestis strains recovered from rodents and pneumonic patients during the 1994 outbreaks, pneumonic patients from the 2002 Shimla outbreak and rodents trapped on the Deccan Plateau during a surveillance activity carried out in 1998 were characterized by MLVA, ERIC-PCR and ERIC-BOX-PCR. MLVA genotyping of Indian Y. pestis strains revealed strains of 2 Orientalis, 1 Mediaevalis and 1 Antiqua genotypes distributed in three distinct branches corresponding to their biovar. The Orientalis genotype strains recovered from the 1994 outbreaks and 1998 surveillance activity clustered in one branch while the Antiqua biovar strains from the Shimla outbreak and the Mediaevalis strain recovered from a rodent trapped on the Deccan Plateau region during surveillance formed the other branches. The Orientalis Y. pestis strains recovered from rodents and patients from the 1994 plague outbreaks exhibited similar MLVA, ERIC-PCR and ERIC-BOX-PCR profiles and these were closely related to the Orientalis strains recovered from the rodents trapped on the Deccan Plateau. These data provide evidence for the possible linkage between the Y. pestis strains resident in the endemic region and those that were associated with the 1994 plague outbreaks. Mediaevalis and Antiqua biovars also were recovered from the environmental reservoir on the Deccan Plateau and from the pneumonic patients of 2002 plague outbreak. Therefore, as in Central Asian and African regions, Antiqua and Mediaevalis biovars seem to be well established in the Indian subcontinent as well. ERIC-PCR DNA fingerprinting delineated genotypes similar to those defined by MLVA. Thus ERIC-PCR appears to have the potential to be used as a molecular marker in the molecular epidemiological investigations of plague.     

Complete genomic sequence of an equine herpesvirus type 8 Wh strain isolated from China

A new strain of equine herpesvirus type 8 (EHV-8), Wh, has been isolated from horses in China, and its complete genome has been sequenced and analyzed. The result indicates that the new strain has the same constitution and arrangement of open read frames as EHV-1 and EHV-9. This work is the first announced complete genome sequence of EHV-8.     

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland

Background

Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D₇₅₂). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1.

Methods

27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank.

Results

None of the analyzed sequences belonged to the ORF30 D₇₅₂neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N₇₅₂. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV.

Conclusions

EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.

     

Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase.

We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing. Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function. In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue. Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase. The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed.     

Potential of equine herpesvirus 1 as a vector for immunization

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells.     

Use of lambda gt11 and monoclonal antibodies to map the genes for the six major glycoproteins of equine herpesvirus 1.

To localize the genes for the major glycoproteins of equine herpesvirus 1 (EHV-1), a library of the EHV-1 genome was constructed in the lambda gt11 expression vector. Recombinant bacteriophage expressing EHV-1 glycoprotein epitopes as fusion products with beta-galactosidase were detected by immunoscreening with monoclonal antibodies specific for each of six EHV-1 glycoproteins. Seventy-four recombinant lambda gt11 clones reactive with EHV-1 monoclonal antibodies were detected among 4 X 10(5) phage screened. Phage expressing determinants on each of the six EHV-1 glycoproteins were represented in the library. Herpesviral DNA sequences contained in lambda gt11 recombinants expressing epitopes of EHV-1 glycoproteins were used as hybridization probes for mapping insert sequences on the viral genome. Genes for five EHV-1 glycoproteins (gp2, gp10, gp13, gp14, and gp21/22a) mapped to the genome L component; only one EHV-1 glycoprotein (gp17/18) was expressed from the unique S region of the genome where genes of several major glycoproteins of other herpesviruses have been located. Two glycoproteins of EHV-1, gp13 and gp14, mapped to positions colinear with genes of major glycoproteins identified in several other alphaherpesviruses (gC- and gB-like glycoproteins, respectively). The genomic locations of other EHV-1 glycoproteins indicated the existence of major glycoproteins of EHV-1 (gp2, gp10, and gp21/22a) for which no genetic homologs have yet been detected in other herpesviruses. The results confirm the general utility of the lambda gt11 expression system for localizing herpesvirus genes and suggest that the genomic positioning of several high-abundance glycoproteins of EHV-1 may be different from that of the prototype alphaherpesvirus, herpes simplex virus.