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1.
Virus Res ; 339: 199262, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-37931881

RESUMO

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.


Assuntos
Infecções por Herpesviridae , Herpesvirus Equídeo 1 , Herpesvirus Equídeo 4 , Doenças dos Cavalos , Humanos , Animais , Cavalos , Herpesvirus Equídeo 1/genética , Células HEK293 , Anticorpos Antivirais , Anticorpos Neutralizantes , Infecções por Herpesviridae/veterinária , Glicoproteínas , Herpesvirus Equídeo 4/genética
2.
J Virol Methods ; 310: 114615, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36087793

RESUMO

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail.


Assuntos
Infecções por Herpesviridae , Herpesviridae , Herpesvirus Equídeo 1 , Herpesvirus Equídeo 4 , Doenças dos Cavalos , Cavalos , Animais , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/veterinária , Doenças dos Cavalos/diagnóstico , DNA Viral/genética , Herpesviridae/genética , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética
3.
Viruses ; 14(4)2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35458443

RESUMO

Equid herpesvirus 2 (EHV-2) and 5 (EHV-5) are two γ-herpesviruses that are commonly detected from horses worldwide, based on several cross-sectional molecular surveys. Comparatively few studies examined the dynamics of γ-herpesvirus infection over time in a group of horses. The aim of the current study was to investigate the dynamics of EHV-2/5 infections among mares and their foals at three Polish national studs with different breeds of horses: Arabians, Thoroughbreds and Polish Konik horses. Nasal swabs were collected from each of 38 mare-foal pairs monthly for a period of 6 to 8 months. Virus-specific quantitative PCR assays were used to determine the viral load of EHV-2 and EHV-5 in each sample. All 76 horses sampled were positive for EHV-2 or EHV-5 on at least one sampling occasion. The majority (73/76, 96%) were infected with both EHV-2 and EHV-5. In general, the mean load of viral DNA was higher in samples from foals than from mares, but similar for EHV-2 and EHV-5 at most sampling occasions. There was, however, a considerable variability in the viral DNA load between samples collected at different times from the same foal, as well as between samples from different foals. The latter was more apparent for EHV-2 than for EHV-5. All foals became infected with both viruses early in life, before weaning, and remained positive on all, or most, subsequent samplings. The virus shedding by mares was more intermittent, indicating the existence of age-related differences. Overall, the data presented extend our knowledge of EHV-2/5 epidemiology among mares and foals.


Assuntos
Infecções por Herpesviridae , Herpesvirus Equídeo 1 , Herpesvirus Equídeo 4 , Doenças dos Cavalos , Rhadinovirus , Animais , Estudos Transversais , DNA Viral/genética , Feminino , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Cavalos , Cinética , Polônia/epidemiologia , Rhadinovirus/genética
4.
J Vet Diagn Invest ; 30(6): 924-928, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30239276

RESUMO

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.


Assuntos
Infecções por Herpesviridae/veterinária , Doenças dos Cavalos/diagnóstico , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Doenças Respiratórias/veterinária , Animais , Primers do DNA , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/isolamento & purificação , Doenças dos Cavalos/virologia , Cavalos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Doenças Respiratórias/diagnóstico , Doenças Respiratórias/virologia , Sensibilidade e Especificidade
5.
BMC Genomics ; 18(1): 887, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29157201

RESUMO

BACKGROUND: The varicelloviruses comprise a genus within the alphaherpesvirus subfamily, and infect both humans and other mammals. Recently, next-generation sequencing has been used to generate genomic sequences of several members of the Varicellovirus genus. Here, currently available varicellovirus genomic sequences were used for phylogenetic, recombination, and genetic distance analysis. RESULTS: A phylogenetic network including genomic sequences of individual species, was generated and suggested a potential restriction between the ungulate and non-ungulate viruses. Intraspecies genetic distances were higher in the ungulate viruses (pseudorabies virus (SuHV-1) 1.65%, bovine herpes virus type 1 (BHV-1) 0.81%, equine herpes virus type 1 (EHV-1) 0.79%, equine herpes virus type 4 (EHV-4) 0.16%) than non-ungulate viruses (feline herpes virus type 1 (FHV-1) 0.0089%, canine herpes virus type 1 (CHV-1) 0.005%, varicella-zoster virus (VZV) 0.136%). The G + C content of the ungulate viruses was also higher (SuHV-1 73.6%, BHV-1 72.6%, EHV-1 56.6%, EHV-4 50.5%) compared to the non-ungulate viruses (FHV-1 45.8%, CHV-1 31.6%, VZV 45.8%), which suggests a possible link between G + C content and intraspecies genetic diversity. Varicellovirus clade nomenclature is variable across different species, and we propose a standardization based on genomic genetic distance. A recent study reported no recombination between sequenced FHV-1 strains, however in the present study, both splitstree, bootscan, and PHI analysis indicated recombination. We also found that the recently sequenced Brazilian CHV-1 strain BTU-1 may contain a genetic signal in the UL50 gene from an unknown varicellovirus. CONCLUSION: Together, the data contribute to a greater understanding of varicellovirus genomics, and we also suggest a new clade nomenclature scheme based on genetic distances.


Assuntos
Varicellovirus/classificação , Varicellovirus/genética , Composição de Bases , Códon , Herpesvirus Bovino 1/classificação , Herpesvirus Bovino 1/genética , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/classificação , Herpesvirus Equídeo 4/genética , Mutação , Filogenia , Recombinação Genética
6.
Anal Chem ; 89(21): 11219-11226, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-28819973

RESUMO

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a hand-held "cradle" that interfaces the phone with a silicon-based microfluidic chip embedded within a credit-card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to utilize a single 15 µL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 min. Our approach utilizes loop-mediated isothermal amplification (LAMP) reagents predeposited into distinct lanes of the microfluidic chip, which when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that when excited by appropriately selected light emitting diodes (LEDs), are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with the information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results.


Assuntos
DNA Bacteriano/análise , DNA Viral/análise , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Smartphone , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Dispositivos Lab-On-A-Chip , Limite de Detecção , Pneumopatias/diagnóstico , Pneumopatias/veterinária , Técnicas Analíticas Microfluídicas/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Streptococcus equi/genética
7.
J Vet Med Sci ; 79(1): 206-212, 2017 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-27840393

RESUMO

Equine herpesvirus type 4 (EHV-4) is one of the most important pathogens in horses. To clarify the key genes of the EHV-4 genome that cause abortion in female horses, we determined the whole genome sequences of a laboratory strain and 7 Japanese EHV-4 isolates that were isolated from 2 aborted fetuses and nasal swabs of 5 horses with respiratory disease. The full genome sequences and predicted amino acid sequences of each gene of these isolates were compared with of the reference EHV-4 strain NS80567 and Australian isolates that were reported in 2015. The EHV-4 isolates clustered in 2 groups which did not reflect their pathogenicity. A comparison of the predicted amino acid sequences of the genes did not reveal any genes that were associated with EHV-4-induced abortion.


Assuntos
Genoma Viral/genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/virologia , Animais , Feminino , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 4/isolamento & purificação , Doenças dos Cavalos/epidemiologia , Cavalos/virologia , Japão/epidemiologia , Análise de Sequência de DNA/veterinária
8.
J Gen Virol ; 97(3): 747-755, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26691326

RESUMO

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/virologia , Recombinação Genética , Animais , Sequência de Bases , Genoma Viral , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 4/classificação , Herpesvirus Equídeo 4/isolamento & purificação , Cavalos , Dados de Sequência Molecular , Nova Zelândia , Filogenia
9.
Biomed Res Int ; 2015: 917854, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26421307

RESUMO

This study was undertaken to investigate molecularly the occurrence of EHV-1 and EHV-4 infection among equine population in regions, Iran. Blood samples from 53 and 37 randomly selected horses settled in Isfahan and Shahrekord, Iran, respectively, were collected. Detection of EHV-1 and EHV-4 genes in the blood samples was done using polymerase chain reaction (PCR). Out of 53 and 37 samples from Isfahan and Shahrekord, 4 (18.18%) and 3 (8.10%) were positive for PCR of EHV-1, respectively. Nine (16.98%) and 6 (16.21%) were positive for PCR of EHV-4, while 6 (11.32%) and 3 (8.10%) were positive for PCR of both EHV-1 and EHV-4, in Isfahan and Shahrekord, respectively. Of the 7 blood samples positive for EHV-1, 4 (16.66%) and 3 (8.10%) were from horses >3 years old while 2 (18.18%) and 1 (16.66%) were from 2-3 years old horses, in Isfahan and Shahrekord, respectively. Out of the 7 and 3 samples positive for PCR of EHV-1 in Isfahan and Shahrekord, 4 (22.2%) and 1 (7.69%) were Standardbred, while 3 (14.28%) and 2 (13.33%) were Thoroughbreds, respectively. EHV-4 was detected in blood of 4 (22.22%) and 2 (15.83%) Standardbreds and from 4 (19.04%) and 4 (26.66%) Thoroughbred horses in Isfahan and Shahrekord, respectively. This study has shown that horses settled in Isfahan central and Shahrekord southwest regions, Iran, are infected by EHV-1 and EHV-4 and thus serve as potential reservoirs and disseminators of the viruses.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/virologia , Cavalos/virologia , Envelhecimento , Animais , Cruzamento , Eletroforese em Gel de Ágar , Genes Virais , Infecções por Herpesviridae/virologia , Irã (Geográfico)
10.
Vet Microbiol ; 176(3-4): 219-28, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25666453

RESUMO

Infection with equid herpesvirus 1 (EHV-1) may be asymptomatic, or may result in respiratory disease, abortion, neonatal death, or neurological disease. The aim of this study was to estimate the prevalence of EHV-1 infection, including differentiation between genotypes with aspartic acid (D) and asparagine (N) at position 752 of the DNA polymerase sequence, within a selected population of New Zealand horses. The second aim was to determine the predictive value of serology for detection of latently infected horses. Retropharyngeal lymph nodes (RLN) and trigeminal ganglia (TG) were dissected from 52 horses at slaughter and tested for the presence of EHV-1 DNA using magnetic bead, sequence-capture enrichment followed by nested PCR. Sera were tested for EHV-1 antibody using type-specific glycoprotein G ELISA. Overall, 17/52 horses tested positive for EHV-1 DNA. All but one positive PCR results were obtained from RLN samples. Fifteen of the EHV-1 positive horses harboured EHV-1 with N752 genotype, one of which was additionally infected with the D752 genotypes of the virus. Our data comprise the first detection of EHV-1 with D752 genotype in New Zealand and suggest that the "neurovirulent" variant of EHV-1 had been present in New Zealand for at least two years before the first reported outbreak of EHM. All sampled horses tested positive for EHV-4 antibody, and 11/52 tested positive for EHV-1 antibody. The strength of agreement between results of EHV-1 PCR and EHV-1 serology was "fair" (Kappa 0.259, 95% CI: -0.022-0.539), which was likely a reflection of low levels of both EHV-1 antibody in sera and EHV-1 DNA in tissues tested.


Assuntos
Anticorpos Antivirais/sangue , Surtos de Doenças/veterinária , Infecções por Herpesviridae/epidemiologia , Herpesvirus Equídeo 1/imunologia , Herpesvirus Equídeo 4/imunologia , Doenças dos Cavalos/epidemiologia , Animais , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Genótipo , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/isolamento & purificação , Doenças dos Cavalos/virologia , Cavalos , Nova Zelândia/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Gravidez , Prevalência
11.
Vet Microbiol ; 169(1-2): 50-7, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24418044

RESUMO

A single nucleotide polymorphism (SNP) has been previously associated with EHV-1 neurological disease in several countries around the world. This disease is very uncommon in Australia and little information is available about the presence of this SNP in Australian EHV-1 isolates. The ORF30 sequence of 66 Australian EHV-1 isolates was determined and the genotype was compared to the disease manifestation of the case from which the virus was isolated. Of the 66 isolates, 61 were from cases of abortion and 5 were cases associated with equine herpesvirus myeloencephalopathy (EHM). There was no association between pathotype and genotype in these isolates. In total, 64 of the 66 isolates encoded N752, including 4 isolates from EHM cases. The ORF30 sequence was also determined for 14 EHV-4 isolates, including 2 isolates from confirmed EHV-4 abortion cases. All 14 EHV-4 isolates had aspartic acid at the position equivalent to EHV-1 AA752. Aspartic acid was also confirmed in this position for the single isolate of AHV-3 sequenced in this study. The nucleotide sequence of ORF68 was also determined and showed considerable genetic heterogeneity in the EHV-1 isolates, however, this ORF was highly conserved among the 14 EHV-4 isolates sequenced, with only one SNP identified among 7 isolates. These results confirm that the EHV1 ORF30 N752 is unique and that the D752 sequence is most likely to be the true parent strain of this virus. We suggest that the abortigenic form of EHV-1 should be considered to be the more recently emerged mutant.


Assuntos
Variação Genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/virologia , Fases de Leitura Aberta , Varicellovirus/genética , Aborto Animal/virologia , Sequência de Aminoácidos , Animais , Austrália , Sequência de Bases , Feminino , Genes Virais , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 4/isolamento & purificação , Cavalos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Gravidez , Análise de Sequência de DNA , Varicellovirus/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/genética
12.
Biochem Pharmacol ; 87(3): 435-44, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24316433

RESUMO

The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.


Assuntos
Ganciclovir/farmacologia , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Equídeo 4/enzimologia , Timidina Quinase/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proliferação de Células , Desenho de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Herpesvirus Equídeo 4/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Timidina Quinase/genética , Proteínas Virais/genética
13.
Vet Microbiol ; 167(1-2): 123-34, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-23890672

RESUMO

The equine herpesviruses type 1 (EHV-1) and 4 (EHV-4) are ubiquitous pathogens that affect horse populations on all continents. Despite widespread vaccination, EHV-1 and EHV-4 infections remain a permanent risk. While the two viruses share a high degree of genetic and antigenic similarity, they differ significantly in host range and pathogenicity. Compared to EHV-4, which mainly infects horses and causes respiratory disease, EHV-1 has a broader host range and can result in respiratory disease, abortions, neonatal death, and equine herpesvirusmyeloencephalopathy (EHM). Recent studies have elucidated a number of mechanisms that may, at least partly, explain the differential pathogenic potential of the two viruses. While both EHV-1 and EHV-4 can escape host immune responses and establish latent infection, there are differences with respect to virus entry and their ability to interfere with the innate immune response. Understanding the virus' repertoire of immunomodulatory mechanisms may lead the way to develop more efficient vaccines.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/fisiologia , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/virologia , Animais , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/classificação , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Herpesvirus Equídeo 4/classificação , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/imunologia , Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/patologia , Doenças dos Cavalos/prevenção & controle , Cavalos , Internalização do Vírus
14.
Viruses ; 4(8): 1258-63, 2012 08.
Artigo em Inglês | MEDLINE | ID: mdl-23012623

RESUMO

Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. Glycoprotein K (gK) homologues have been identified in several alphaherpesviruses as a major player in virus entry, replication, and spread. In the present study, EHV-4 gK-deletion mutant has been generated by using bacterial artificial chromosome technology and Red mutagenesis to investigate the role of gK in EHV-4 replication. Our findings reported here show that gK is essential for virus replication in vitro and that the gK-negative strain was not able to be reconstituted in equine cells. It is noteworthy that these findings agree with the previously published study describing gK deletion in other alphaherpesviruses.


Assuntos
Glicoproteínas/metabolismo , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/virologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Glicoproteínas/genética , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 4/genética , Cavalos , Humanos , Deleção de Sequência , Proteínas Virais/genética
15.
Virus Res ; 169(1): 203-11, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22902480

RESUMO

Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) are important pathogens of horses worldwide. Infection with EHV-4 usually remains restricted to the upper respiratory tract, whereas infection with EHV-1 can generalize after leukocyte-associated viremia. Here we examined whether differences in the immunomodulatory glycoprotein G (gG) between the two viruses determine EHV-1's ability to cause systemic infection. To this end, mutant viruses were constructed based on the neurovirulent EHV-1 strain OH-03, in which the entire gG gene or parts thereof were exchanged with EHV-4 gG sequences. In vitro chemotaxis assays showed that supernatants of cells infected with the various gG mutant viruses interfered to variable degrees with neutrophil migration. More specifically, supernatants of cells infected with the gG deletion virus (vOH-ΔgG1) or OH-03 expressing EHV-4 gG (vOH-gG4) were unable to interfere with chemotaxis. Re-insertion of the predicted chemokine-binding region of EHV-1 gG in the vOH-gG4 mutant (vOH-gG4hyp1) did not completely restore the ability to inhibit neutrophil migration, whereas insertion of the hypervariable region of EHV-4 gG into vOH-03 (vOH-gG1hyp4) did not lead to a complete loss of chemokine-binding function. Very similar results were obtained in an in vivo study where the amount of neutrophils present in bronchioalveolar lavages (BALs) of mice infected with the different mutants was analyzed by flow cytometry. Taken together, our results show that, in a virus background, the hypervariable region is not solely responsible for the immunomodulatory potential of EHV-1 gG.


Assuntos
Herpesvirus Equídeo 1/patogenicidade , Herpesvirus Equídeo 4/patogenicidade , Tolerância Imunológica , Proteínas do Envelope Viral/imunologia , Fatores de Virulência/imunologia , Animais , Feminino , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/imunologia , Doenças do Sistema Imunitário , Transtornos Leucocíticos , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Recombinação Genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
Vet Res ; 43: 61, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22909178

RESUMO

Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to α4ß1 or α4ß7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that α4ß1 and α4ß7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/fisiologia , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/virologia , Integrinas/metabolismo , Proteínas do Envelope Viral/genética , Animais , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/crescimento & desenvolvimento , Cavalos , Especificidade de Hospedeiro , Proteínas do Envelope Viral/metabolismo , Ensaio de Placa Viral/veterinária , Replicação Viral
17.
J Virol ; 86(15): 8059-71, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22623773

RESUMO

Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.


Assuntos
Apresentação de Antígeno/imunologia , Regulação para Baixo/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Equídeo 4/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Apresentação de Antígeno/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Cães , Regulação para Baixo/genética , Células HEK293 , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/metabolismo , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Cavalos , Humanos , Camundongos , Transporte Proteico/genética , Transporte Proteico/imunologia , Células Vero , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
18.
Res Vet Sci ; 93(3): 1504-7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22401978

RESUMO

The aim of the present study was to investigate abortion storms that occurred in the Marmara region of Turkey in 2008-2009 using a real-time PCR. Two aborted foetuses were necropsied and histo-pathological findings reported herein. Ten lungs, 3 brains and one nasal swab from 10 aborted foetuses, 6 nasal swabs and 3 vaginal swabs from aborting mares were included in this study. EHV-1 was isolated from the lung, liver and brain of 1 aborted foetus. EHV-1 DNA was detected in the lungs, livers and spleens of 2 necropsied foetuses and in 3 lungs from 10 foetuses submitted for diagnosis. A brain from one of the aborted foetuses was also positive for EHV-1 DNA. EHV-4 DNA was detected only in a nasal swab of one of the tested foetuses. Neither EHV-1 nor EHV-4 DNA was detected in the swabs of aborting mares. Sequence analysis of the glycoprotein B of the strains was performed and a phylogenetic tree was generated. The results indicated that 4 of the 5 Turkish EHV-1 strains (TR02, TR03, TR04 and TR05) clustered together; the fifth strain (TR01) was slightly removed from the group and clustered with other EHV-1 from various origins. Single nucleotide polyporphism (SNP in ORF30) associated with neuropathogenesis was not detected in any of the strains. At necropsy, sub-milier focal necrosis in the liver and spleen was observed. Microscopically, focal coagulation necrosis and marked eosinophilic intranuclear and intracytoplasmic inclusion bodies in the hepatocytes localised around the necrotic areas in the liver. Severe coagulation necrosis in white pulp of the spleen was also observed.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1 , Herpesvirus Equídeo 4 , Doenças dos Cavalos/virologia , Animais , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/patologia , Cavalos , Filogenia , Turquia/epidemiologia
19.
Virus Genes ; 44(1): 109-11, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21960433

RESUMO

Equine herpesvirus 4 (EHV-4) is an important pathogen that causes respiratory tract disease in horse populations worldwide. Glycoprotein G (gG) homologs have been identified in several alphaherpesviruses as minor non-essential membrane-anchored glycoproteins. In this study, EHV-4 gG deletion mutant has been generated by using bacterial artificial chromosome technology to investigate the role of gG in viral pathogenesis. Our findings reported here revealed no significant difference between parental EHV-4 and gG-negative strain in their replication cycle in cell culture. Furthermore, virus titers and plaque formation were comparable in both viruses. It is noteworthy that these findings disagree with the previously published study describing gG deletion in another EHV-4 strain.


Assuntos
Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/virologia , Deleção de Sequência , Proteínas do Envelope Viral/genética , Animais , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 4/patogenicidade , Herpesvirus Equídeo 4/fisiologia , Cavalos , Proteínas do Envelope Viral/metabolismo , Virulência , Replicação Viral
20.
J Virol ; 86(4): 2031-44, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22171258

RESUMO

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVß3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/fisiologia , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/metabolismo , Integrinas/metabolismo , Proteínas do Envelope Viral/metabolismo , Tropismo Viral , Animais , Linhagem Celular , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Doenças dos Cavalos/virologia , Cavalos , Especificidade de Hospedeiro , Integrinas/genética , Proteínas do Envelope Viral/genética
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