Brain lesions and transmission of experimental equine herpesvirus type 9 in pigs

We demonstrated that pigs are susceptible to acute infection by equine herpesvirus type 9 (EHV-9). Six 8-week-old SPF pigs were inoculated intranasally and four were inoculated orally with different doses of EHV-9, and observed for 6 days. Although neurological signs did not develop in any of the infected pigs, the six intranasally infected pigs and one of the orally infected pigs developed lesions of encephalitis consisting of neuronal necrosis, neuronophagia, and intranuclear inclusion bodies, distributed mainly in the rhinencephalon. EHV-9 antigen was localized in the necrotic neuronal cells and was closely associated with the presence of inclusion bodies. These findings clearly demonstrate that pigs are fully susceptible to EHV-9 infection following intranasal inoculation (but less so following oral inoculation), and that EHV-9 in pigs has a highly neurotropic nature.     

Neuropathogenicity of Equine Herpesvirus 9 in Cattle

The pathogenicity of equine herpesvirus 9 (EHV-9), a neurotropic equine herpesvirus isolated from a herd of Gazella thomsoni, was studied in cattle. Seven calves were inoculated intranasally with 10⁵ and 10⁷ plaque-forming units of the EHV-9 P19. Three animals showed brain lesions consisting of glial reactions and perivascular cuffings in the olfactory bulb and the frontal and temporal lobes. Additionally, the animal that was inoculated with 10⁷ plaque-forming units showed neuronal degeneration and loss, as well as nuclear inclusions compatible with herpesvirus. EHV-9 was isolated from the brain of calf 6 and the lungs of calves 1 and 2. The results suggested that cattle are susceptible to experimental infection with EHV-9 and at risk from natural infection from reservoir hosts.     

Pathogenicity of a new neurotropic equine herpesvirus 9 (gazelle herpesvirus 1) in horses

Pathogenicity of equine herpesvirus 9 (EHV-9), a new type of equine herpesvirus isolated from Gazella thomsoni, in horses was investigated by intranasal inoculation of EHV-9 (10(7) pfu) to two conventionally reared 8-months old half-bred weanling horses. Fever higher than 39 degrees C was recorded. Virus was recovered from nasal swabs and peripheral blood mononuclear cells. Both horses developed neutralizing antibody to EHV-9. Perivascular infiltration of mononuclear cells and glial reaction were found in the olfactory and limbic systems. The results suggested that EHV-9 has a pathogenicity in horses.     

Equine Multinodular Pulmonary Fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report

A standardbred gelding with a history of 10 days pyrexia and lethargy was referred to the Equine Hospital at the Swedish University of Agricultural Sciences in Uppsala, Sweden.The horse had tachypnea with increased respiratory effort and was in thin body condition. Laboratory findings included leukocytosis, hyperfibrinogenemia and hypoxemia. Thoracic radiographs showed signs of pneumonia with a multifocal nodular pattern, which in combination with lung biopsy findings indicated Equine Multinodular Pulmonary Fibrosis (EMPF). EMPF is a recently described disease in adult horses with clinical signs of fever, weight loss and respiratory problems. The pathological findings include loss of functional pulmonary parenchyma due to extensive nodular interstitial fibrosis which has been related to infection with the equine herpesvirus type 5 (EHV-5). In this case, lung biopsy and tracheal wash samples tested positive for both asinine herpesvirus type 5 (AHV-5) and EHV-5 using PCR assays. The horse failed to respond to treatment and was euthanized for humane reasons. Postmortem examination confirmed the diagnosis of EMPF. This case suggests that not only EHV-5 alone should be considered in association with the development of this disease.     

EHV-1 and EHV-4 infection in vaccinated mares and their foals

A silent cycle of equine herpesvirus 1 infection was described following epidemiological studies of unvaccinated mares and foals on a Hunter Valley stud farm. Following the introduction of routine vaccination with an inactivated whole virus equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) vaccine in 1997, a subsequent study identified excretion of EHV-1 and EHV-4 in nasal swab samples tested by PCR from vaccinated mares and their unweaned, unvaccinated foals. The current sero-epidemiological investigation of vaccinated mares and their young foals found serological evidence of EHV-1 and EHV-4 infection in mares and foals in the first 5 weeks of life. The results further support that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned foals and confirms the continuation of the cycle of EHV-1 and EHV-4 infection.     

Induction of a Th-1-biased IgG subclass response against equine herpesvirus type 1 in horses previously infected with type 4 virus

An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were na?ve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses na?ve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.     

Equine herpesvirus type-1 modulates CCL2, CCL3, CCL5, CXCL9, and CXCL10 chemokine expression

Equine herpesvirus type 1 (EHV-1) is highly prevalent in horses and causes rhinopneumonitis, abortion, and encephalopathy. Studies on the related human herpes simplex virus and of murine models of EHV-1 suggest that chemokines play important roles in coordinating of innate and adaptive immune responses, and thus effective control of herpesvirus infection and prevention of severe clinical disease. Here, equine peripheral blood mononuclear cells (PBMC) were infected with one of three EHV-1 strains, which differ in pathogenicity (RacL11, NY03=abortogenic, Ab4=neurogenic). Changes in CCL2, CCL3, CCL5, CXCL9 and CXCL10 chemokine gene expression relative to non-infected PBMC were measured by real-time PCR. CXCL9 and CXCL10 gene expression was up-regulated 10h post infection and decreased to the level of non-infected cells after 24h. CCL2 and CCL3 were significantly down-regulated 24h post infection with NY03 and Ab4. CCL5 was up-regulated 24h after infection with RacL11. Ab4 infected PBMC had significantly lower expression of all chemokines except CCL2 24h post infection then RacL11 infected cells. While there was not a significant difference between NY03 and the other strains, there was a trend with each chemokine toward NY03 inducing less expression then RacL11 but more then Ab4. The data suggested that EHV-1 infection of PBMC induced up-regulation of inflammatory chemokines CCL5, CXCL9 and CXCL10, and down-regulation of chemotactic CCL2 and CCL3. The data also implies that different EHV-1 strains have varying effects on all five chemokines, with the nuropathogenic strain, Ab4, having the greatest suppressive potential.     

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Molecular data of UL24 homolog gene (ORF37) from Brazilian isolates of equine herpesvirus type 1

Equine herpesvirus type 1 (EHV-1) is associated with abortions, respiratory distress, and neurological disturbances in horses. The ORF37 of EHV-1 encodes a protein homolog to UL24 gene product of human herpesvirus that has been associated with neurovirulence. In the present work, ORF37 PCR fragments derived from two Brazilian EHV-1 isolates, a German isolate and an American reference strain were sequenced and characterized by molecular phylogenetic analysis. This genomic region is highly conserved an allowed to infer genetic distances between EHV-1 strains and other animal herpesvirus.     

Respiratory disease in foals and the epizootiology of equine herpesvirus type 2 infection

The epizootiology of equine herpesvirus type 2 (EHV-2) infection was investigated in Thoroughbred foals on a stud farm which in previous years had suffered economic loss due to respiratory disease. Sixteen pairs of foals and their dams were selected for this study and all of the foals became infected with EHV-2 by two to four months of age. These animals responded serologically to the virus infection as detected by an enzyme-linked immunosorbent assay (ELISA). EHV-2 infection persisted in these foals for two to six months with constant or intermittent virus recovery. This persistent infection stimulated continuous production of antibodies against EHV-2. As soon as the antibody levels reached their peak at five to six months, the isolation rate of EHV-2 from the nasal cavity of these animals decreased, and eventually by nine months of age virus could no longer be recovered. Respiratory disease was observed in ten of the 16 foals; and two severely affected animals died at two months of age. EHV-2 was isolated from both foals at ante and/or post mortem examination. It is postulated that EHV-2, either as an initiating agent or by means of immnunosuppression, caused the respiratory disease observed in these foals.     

In vitro cytotoxicity of serum and peripheral blood leukocytes for equine herpesvirus type 1-infected target cells.

The immune response in horses following experimental infection with equine herpesvirus type 1 (EHV-1) was assessed by measuring cytotoxicity for EHV-1-infected target cells. A technique was developed, using [125I]5-iodo-2'-deoxyuridine ([125I]IUDR)-labeled equine fetal kidney cells infected with EHV-1 as the target cells. It was shown that peripheral blood leukocytes from a recovered horse were capable of lysing target cells, as measured by the loss of radio-active label. Following the experimental infection of specific-pathogen-free ponies with EHV-1, cytotoxicity was obtained with fresh autologous serum, peripheral blood leukocytes in autologous serum, and washed peripheral blood leukocytes. Cytotoxicity of the serum and peripheral blood leukocytes was detected as early as one day after infection. It is suggested that cytotoxic antibodies or cells could play an important part in restricting virus spread after infection of the horse with EHV-1.     

Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals

Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by PCR and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the pcr for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to EHV-1 and/or EHV-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.     

An outbreak of Equid herpesvirus abortion in New South Wales.

Thirty-three of the 44 mares on a Thoroughbred stud in New South Wales aborted or lost foals within one day of birth. Gross pathological and histological changes were in keeping with Equid herpesvirus I (EHV-1) abortion. In the six foals that underwent virological examination, EHV was isolated and typed as EHV-1 by restriction endonuclease analysis. EHV-1 abortion had not occurred previously on this stud and the source of the infection was not identified.     

Prevalence of equine herpesvirus-1 and equine herpesvirus-4 infections in equidae species in Turkey as determined by ELISA and multiplex nested PCR

In this report we examined the presence of specific antibodies against equine herpesvirus type 1 (EHV-1), and equine herpesvirus type 4 (EHV-4) in several equidae, including mules, donkeys, horses. The presence of EHV-1 and EHV-4 in respiratory diseases of equids, and ability of multiplex nested polymerase chain reaction (PCR) screening in simultaneous diagnosis of horses acutely infected by EHV-1 and EHV-4 were also investigated. Sera from 504 horses, mules and donkeys sampled were tested for the presence of EHV-1 and EHV-4 specific antibodies. Blood samples taken from 21 symptomatic horses and nasal swabs taken from 40 symptomatic horses were tested for the presence of EHV-1 and EHV-4 by a multiplex nested PCR. A total of 14.3% (3/21) of buffy coat samples and 32.5% (13/40) nasal swab samples were found to contain EHV-1 DNA, while 19% (4/21) buffy coat samples and 22.5% (9/40) nasal swab samples were found to be positive for EHV-4 DNA. By species, 14.5% of horses, 37.2% of mules and 24.2% of donkeys tested were EHV-1 seropositive. EHV-4 specific antibodies were detected in 237 (81.7%) of 290 horse sera tested. Results from this investigation demonstrate that EHV-1 and EHV-4 are prevalent throughout the equid population, and that donkeys and mules might also represent an important source of infection for other equids. We also showed that the multiplex nested PCR assay might be useful for diagnosis of mixed respiratory infections in horses due to EHV-1 and EHV-4.     

马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。     

马疱疹病毒1型感染马疾病模型的建立及评价

试验旨在建立马疱疹病毒1型（Equine herpesvirus type1,EHV-1）人工发病模型,确定EHV-1感染马的半数感染量（ID₅₀）及感染发病的判定标准,为该病的预防与治疗药物的研发奠定基础。以新疆伊犁地区某发病马场流产胎儿中分离的EHV-1 XJ2015株为研究对象,设立4组不同病毒剂量感染组及对照组,经鼻内喷雾感染马,5 mL/匹,每天观察试验马的临床症状和发病情况,14 d后进行剖检,观察各组织脏器病理变化并应用实时荧光定量PCR方法检测鼻腔排毒及病毒分布情况。结果显示,EHV-1 XJ2015株感染马的ID₅₀为10^-6.67/5 mL,其病毒含量为10^4.33 TCID₅₀/mL。与对照组相比,1×10⁶和1×10⁵ TCID₅₀/mL感染组马临床评分显著升高,主要表现为体温升高（高达39.5 ℃,一般持续2~6 d）、食欲不振、流浆液性鼻液和下颌淋巴结肿大;且1×10⁶和1×10⁵ TCID₅₀/mL感染组试验马均表现出不同程度的排毒,肺脏及脑组织中可检测出大量病毒,与对照组相比极显著或显著升高（P<0.01;P<0.05）;病理学检查发现,患马脑组织出现非化脓性脑炎及神经元水肿,肺脏组织出现间质性肺炎、嗜中性粒细胞、炎性细胞浸润、出血和肺泡间隔增厚。以上结果表明,EHV-1 XJ2015株对马具有较强的致病性,患病马临床症状典型,病毒主要随鼻液排出,并富集在肺脏及脑组织,通过上述指标确定EHV-1感染马发病的判定标准,本试验成功建立EHV-1感染本体动物疾病模型。     

Isolation and comparative restriction endonuclease DNA fingerprinting of equine herpesvirus-1 from cattle

Deoxyribonucleic acid fingerprinting analyses with 4 restriction endonucleases (EcoRI, BamHI, BglII, and HindIII) and serotest results have definitively indicated that 5 herpesviruses isolated from 1974 to 1986 from aborted bovine fetuses and from bovine tissues and nasal secretions were abortigenic subtypes of equine herpesvirus type 1 (EHV-1). The herpesviruses, designated BH1247, 3M20-3, G118, H1753, and 9BSV4, were neutralized by EHV-1-specific antiserum and could be propagated in cultures of either bovine or equine cells. Only minor differences in restriction endonuclease patterns were detected from the pattern of an Army 183 isolate of EHV-1 subtype 1 that had been passaged only in equine cells and from that of an attenuated EHV-1 subtype 1 (RQ) strain that had been passaged several hundred times in non-equine cells. The individual differences in the restriction endonuclease fragments of the 5 bovine isolates and the Army 183 and RQ strains mainly were attributable to alterations in the terminally repeated and the unique short nucleotide sequences of the EHV-1 genomes, which are known to be hot spots for deletions and tandem repeats. The BamHI restriction endonuclease pattern of the 1977 bovine isolate H1753 was identical to that of EHV-1 subtype-1 strains responsible for most of the virus abortions in vaccinated horses since 1981. Abortigenic EHV-1 strains have the ability to infect cattle and cause disease under natural conditions.     

An immunoperoxidase method applied to the diagnosis of equine herpesvirus abortion, using conventional and rapid microwave techniques.

An indirect immunoperoxidase (IMP) technique was applied to cryostat and paraffin sections of liver from ten aborted equine foetuses. Equid herpesvirus type 1 (EHV-1) had been isolated from seven of them and EHV-4 from one: the remaining two were virologically negative and were not used as controls. In the eight virus-infected cases the immunostaining revealed foci of cells exhibiting a distinct brown cytoplasmic and inclusion body pigmentation. No specific signal was present in the non-infected controls. The method also was adapted for incubation in a microwave oven, which allowed the total length of the procedure to be reduced to a minimum of 35 mins for cryostat sections, without significant loss of signal.     

Detection of equine herpesviruses in aborted foetuses by consensus PCR

The major role of EHV-1 in equine abortion is widely reported in the literature but the contribution of EHV-2, EHV-3, EHV-4 or EHV-5 remains less well documented. The objective of this study is to evaluate the contribution of these five different EHVs to equine abortion in a variety of biological tissues using a consensus polymerase chain reaction (PCR). The test was validated for specificity and sensitivity in horses before screening specimens from 407 foetuses, stillbirths and premature foals collected over a 2.5-year interval. Positive results obtained with this assay were compared to other EHV type-specific PCR or by sequencing. EHV-1 was identified as the major cause of abortion in French mares (59/407 cases). However, there was evidence to suggest some variation in the potential of EHV-1 strains to induce abortion. Indeed, DNA samples from EHV-2 (in three cases) and EHV-5 (in one case) inferred a role of these viruses in abortion. The presence of viral DNA from EHV-3 or EHV-4 strains was not detected in the specimens studied. The data obtained suggest that the consensus herpesvirus PCR is an efficient screening tool. In association with a specific PCR, the test provides a rapid identification of the type of herpesvirus involved in abortion and is useful for routine diagnostic tests as it allows the identification of herpesviruses other than the EHV-1 strain.