野马鼻肺炎病毒的分离鉴定

从新疆昌吉州吉木萨尔野马饲料养繁殖中心送检的野马病料中分离到１株病毒,我们对其进行了鉴定。该病毒株在ＢＨＫ－２１细胞上连传６代出现典型的细胞病变,用适应ＢＨＫ－２１细胞的病毒接种鸡胚成纤维细胞,乳豚鼠肾原代细胞,豚鼠睾丸细胞均出现程度不同的细胞病变。     

STUDIES ON EQUINE HERPESVIRUSES 5. Isolation and Characterisation of Slowly Cytopathic Equine Herpesviruses in Queensland

The isolation and characterization of 71 strains of slowly cytopathic herpesviruses (EHV2) from horses in Queensland is described. Specimens from aged horses at slaughter yielded 7 isolates from leucocytes and 4 from tonsils, but EHV2 strains could not be detected in salivary glands, spleen, kidney or intestinal wall. A further EHV2 strain (EK550) was isolated from cell cultures prepared from the kidney of an adult horse. Repeated sampling yielded 39 nasal, ocular and genital EHV2 strains from a group of 16 mares and their young foals, and 20 strains from a group of 6 yearlings.
Laboratory examination of these EHV2 strains demonstrated variations in plaque size and growth in non-host cell cultures, and at least two serological groups were detected. EHV2 infection of foals transplacentally was not detected, but neonatal infection of foals from chronically infected mares appeared probable.     

2. Persistence of Equine Herpesviruses in Experimentally Infected Horses and the Experimental Induction of Abortion

An 8-month-old filly (No. 2) developed an acute vulvo-vaginitis and respiratory disease following inoculation of equine herpesvirus (EH virus) type 1 (EH 39 virus; equine rhinopneumonitis virus) into the vestibule of the vagina. The same virus produced acute respiratory disease but not balanoposthitis following intranasal, intravenous and intrapreputial inoculation of a 12-month-old colt (No. 3). A second 8-month-old filly (No. 1) developed a mild respiratory disease but not vulvo-vaginitis following intravestibular inoculation of EH 39 virus. EH viruses that were slowly cytopathic for equine foetal kidney cell cultures and serologically unrelated to the inoculated EH 39 virus were isolated from the buffy coat cells at 3 days and from the nasal cavity at 6 days after inoculation of horse No. 1. EH virus that was slowly cytopathic and serologically unrelated to EH 39 virus was isolated at 16 days from the vagina of the filly (No. 2) that developed acute vulvovaginitis and was frequently isolated from the nasal cavities of 2 of the 3 horses for 83 days and from the nasal cavity of the third horse for 57 days under conditions that precluded reinfection from other equidae except from each other. EH viruses were recovered from the 3 horses for a further 58 days under conditions where contact with other equidae may, although was not known to, have occurred between 83 and 141 days postinoculation. It was concluded that these viruses represented a single virus type that was present in the nasal cavity (designated EH 1–6 virus) perhaps also the blood stream of filly No. 1 at the time the 3 horses were purchased and that this virus was subsequently transmitted to the vagina of 1 and the nasal cavities of the other 2 horses. Accordingly a carrier state for EH 39 virus was not shown to occur. These findings are discussed in relation to the natural history of EH virus infections. Attempts to reactivate the EH viruses to cause clinical respiratory disease, by a series of injections of adrenalin and cortisone, were inconclusive. The 3 horses showed no clinical evidence of respiratory disease when they were reinfected intranasally with EH 39 virus 360 days (1 horse) and 412 days (2 horses) after the initial infection with this virus. Abortion was produced when EH 39 virus was inoculated directly into the allantoic or amniotic cavity of a pregnant mare although naturally occurring EH virus abortion remains unrecognised in Australia.     

Isolation of Herpesvirus from Equine Leukocytes: Comparison with Equine Rhinopneumonitis Virus

An agent which possessed the properties of herpesviruses was isolated from the leukocytes of 71 out of 80 (88.7%) apparently normal Iowa horses. It was ether- and heat-sensitive, DNA type, and produced type-A intranuclear inclusion bodies in cell cultures. Electron micrographs revealed a virion of typical herpesvirus structure. Leukocyte isolate virus could be differentiated from equine rhinopneumonitis virus (ERV) by serum neutralization, by growth differences in rabbit kidney cells, and by fluorescent antibody staining. Specific neutralizing antibody against this agent was found in a pooled serum sample from normal horses and in the serums of herpesvirus carrier horses. Serum from a mare inhibited growth of both ERV and leukocyte viral isolates. Normal sheep, calf, and rabbit serum did not neutralize either virus.     

Lack of Detectable Equine Herpesviruses 1 and 2 in Paraffin-Embedded Specimens of Equine Sarcoidosis

Background: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2).
Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis.
Animals: Eight horses with sarcoidosis, reported previously.
Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2.
Results: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses.
Conclusion: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.     

Infectious Center Assay of Intracellular Virus and Infective Virus Titer for Equine Mononuclear Cells Infected in vivo and in vitro with Equine Herpesviruses

A novel, simple method of infectious center assay was developed to detect and quantitate the intracellular existence of equine herpesvirus 1 and equine herpesvirus 2 in peripheral blood mononuclear cells infected in vivo and in vitro with the viruses by cocultivation of these cells with a permissive equine cell culture. The infectious center titers were correlated with the infectious virus titers. In vivo equine herpesvirus 1-infected mononuclear cells obtained from ponies experimentally infected with the virus and equine herpesvirus 2-infected mononuclear cells obtained from selected naturally infected ponies with the virus gave by infectious center assay a mean value of 67 infectious center/2 x 10⁶ cells as a peak titer on day 4 postinfection and 26 infectious center/2 x 10⁶ cells for equine herpesvirus 1 and equine herpesvirus 2 respectively. The mononuclear cells, in both cases, did not contain detectable infectious virus, but the infectious virus was detected from the respective cells when they were cultured in the presence of mitogen. The equine herpesvirus 1 infected mononuclear cells in culture gave a mean count of 8.05 x 10² infectious center/2 x 10⁶ cells/mL and contained 1.08 x 10⁴ plague assay/mL of infectious virus. Similarly the equine herpesvirus 2 infected mononuclear cells in culture gave a mean count of 7.1 x 10¹ infectious center/2 x 10⁶ cells/mL and contained <10¹ tissue culture infective dose₅₀/mL of infectious virus. Mononuclear cells infected in vitro with equine herpesvirus 1 gave a mean count of 9.3 x 10⁴ infectious center/2 x 10⁶ cells/mL and contained 5.75 x 10³ plaque assay/mL of infectius virus. Culturing these cells in the presence of mitogen gave a mean count of 5.5 x 10³ infectious center/2 x 10⁶ cells/mL and contained 9 x 10³ plague assay/mL of infectious virus. A correlation between infectious center assay and infectious virus assay is discussed.     

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.     

马疱疹病毒1型LAMP检测方法的建立

本试验旨在建立一种检测马疱疹病毒1型（EHV-1）的快速、灵敏、特异的环介导等温扩增技术（LAMP）,同时评价该方法的可靠性。根据马鼻肺炎糖蛋白B（gB）基因特异保守序列设计多对LAMP引物,利用LAMP Real Time Turbidimeter LA-320仪监测反应进程,进行引物筛选和反应条件的优化,建立能特异性扩增EHV-1 DNA的LAMP检测方法,并加入SYBR GreenⅠ通过肉眼判断结果。该方法在65 ℃恒温下作用50 min,使EHV-1 DNA获得了高效率的特异性扩增,与其他马易感病毒如马疱疹病毒4型（EHV-4）等无交叉反应;且具有极高的灵敏性,可检测到10^-4稀释的目标病毒,比普通PCR的灵敏度高10倍;反应结束后加入SYBR GreenⅠ肉眼观察的结果与LAMP Real Time Turbidimeter LA-320仪监测结果一致。通过将4份临床样品的LAMP检测结果与已得到验证的PCR结果进行比对,结果显示符合率为100%。本研究建立的LAMP检测方法具有快速、特异、灵敏、简单易操作且设备要求低等特点,具有实地检测EHV-1的前景。     

Time-related Pathological Changes in Horses Experimentally Inoculated with Equine Influenza A Virus

To investigate the pathology of equine influenza, necropsy of 7 horses experimentally infected with equine influenza A virus (EIV) subtype H3N8 was conducted on post-infection days (PID) 2, 3, 7, and 14. Histopathologically, rhinitis or tracheitis including epithelial degeneration or necrosis with loss of ciliated epithelia and a reduction in goblet cell numbers, was observed in the respiratory tracts on PIDs 2 and 3. Epithelial hyperplasia or squamous metaplasia and suppurative bronchopneumonia with proliferation of type II pneumocytes were observed on PIDs 7 and 14. Viral antigen was detected immunohistochemically in the epithelia of the nasal mucosa, trachea, and bronchi on PIDs 2 and 3. The sodA gene of Streptococcus equi subsp. zooepidemicus, a suspected cause of suppurative bronchopneumonia, was detected in paraffin-embedded lung tissue sections, but only on PIDs 7 and 14. These findings suggest that damage caused to ciliated epithelia and goblet cells by EIV infection results in secondary bacterial bronchopneumonia due to a reduction in mucociliary clearance.     

检测马疱疹病毒4型抗体间接ELISA方法的建立及应用简

In order to establish an indirect ELISA method for detection of equine herpesviruses type 4 （EHV4） antibody, the glycoprotein G （gG） protein with specific epitope worked as a detection antigen. After optimizing conditions, the indirect ELISA method was developed successfully,specificity and repeatability tests were determined. The result was that the EHV4 gG only reacted with antibodies against EHV4;but not with antibodies against EHV1; both the intro-batch and inter-batch variation coefficiencies were lower than 10%.Concordance of the indirect ELISA relative to commercial EHV1/4 antibody kit was above 90%.The results indicated that the indirect ELISA method could be used for the detection and epidemiological surveys of EHV4 infection.     

Identification of Equine Herpesvirus 5 in Horses with Lymphoma

Equine multinodular pulmonary fibrosis, equine herpesvirus 5 (EHV-5), and multicentric lymphoma were discovered in one patient. Review of gamma herpesvirus activity in humans revealed a propensity for lymphoproliferative disorders associated with infection. The objective was to determine the frequency of EHV-5 in lymphoma tissues and compare with the frequency found in the lymph nodes of clinically normal horses. Case control investigation of lymphoma-positive tissues and analysis via polymerase chain reaction (PCR) for EHV-5 was performed on 12 horses. Prospective collection and PCR analysis of lymph nodes (mesenteric or submandibular) for EHV-5 was performed on 21 control horses. Thirteen samples of lymphoma-positive tissues and fluid were submitted for PCR analysis for EHV-5. Of these, 67% was positive. In the control horse population, 14% was positive for EHV-5 (P = .004). Neoplastic samples positive for EHV-5 were classified as T-cell rich B-cell lymphoma (three), T-cell lymphoma (one), one was nondifferentiated, and two were not stained. Gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as Kaposi sarcoma and Burkitt lymphoma. This study reveals an increased frequency of EHV-5 (gamma herpesvirus) in horses diagnosed with lymphoma compared with healthy control horses. Although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia is unknown, EHV-5 may be an etiologic agent associated with the development of some types of equine lymphoma.     

猪痘病毒江西株(SWPV-JXO1株)的分离鉴定

本试验利用PK15细胞从江西省某猪场疑似猪痘的保育猪皮肤痘痂中分离到1株病毒,该分离毒株在PK15单层细胞上能连续盲传培养,不产生明显的细胞病变,通过PCR扩增猪痘病毒P35基因,扩增序列与猪痘病毒参考株(AF410153.1)P35基因的核苷酸同源性为97.9％,表明该分离病毒为猪痘病毒,并命名为SWPV-JX01.将SWPV-JX01株F5代细胞培养液经肌肉和皮下注射感染兔和小鼠,人工感染80 h后,通过PCR检测不同组织的猪痘病毒P35基因,从兔的肾脏、脾脏、皮肤、心肌、淋巴结均能扩增到P35基因,且扩增序列与SWPV-JX01株同源性为100％.试验结果表明,SWPV-JX01株能在兔体内增殖,但不能在昆明鼠体内增殖.     

Herpesviruses of carnivores.

This review focuses on felid herpesvirus 1 (FHV-1), the most studied of the carnivore herpesviruses. Canid herpesvirus (CHV-1) and phocid (seal) herpesvirus 1 (PhHV-1) are also included where information is available. FHV-1 is a member of the Varicellovirus genus of the Alphaherpesvirinae, which appears to be closely related phylogenetically to both CHV-1 and PhHV-1. FHV-1 infects both domestic and some wild Felidae, such as cheetahs, and is predominantly a respiratory pathogen of cats. As in other herpesviruses, infection with FHV-1 is characterised by a latent carrier state, during which intermittent shedding of infectious virus may occur. Typical of an alphaherpesvirus, the primary site of FHV-1 latency is neurological tissue (trigeminal ganglion), though recent studies using the polymerase chain reaction have suggested that some latency may occur in non-neurological sites. Latently infected carriers are epidemiologically important as sources of infection for susceptible animals. Though conventional modified live and inactivated vaccines have been available for a number of years, they do not protect against infection nor the development of latency. Recently, work has focused on molecular characterisation of FHV-1, detecting genes such as glycoproteins or regulatory genes. Such work will enable better understanding of the interaction of FHV-1 with the natural host. Deletion mutants of some of these genes may also have potential as vaccine strains.     

Isolation of Western Equine Encephalomyelitis (Wee) Virus From Mosquitoes In Saskatchewan, 1962

Saskatchewan, in the summer of 1962, was the scene of an extensive outbreak of western equine encephalomyelitis (WEE) in horses. The results of mosquito survey work showed Culiseta inornata and Culex tarsalis respectively to be the two most abundant mosquito species during midsummer. These species are those reported to be most commonly associated with outbreaks of WEE. Five hundred and sixty-four pools of mosquitoes were examined for the presence of WEE virus. Six pools, three of C. tarsalis and one each of C. inornata, Aedes flavescens and Aedes dorsalis, yielded WEE virus. Positive mosquitoes were from St. Walburg (C. inornata), Saskatoon (C. tarsalis — two, A. dorsalis — one), Outlook (C. tarsalis) and Kisbey (A. flavescens).     

Isolation of Nicoletella semolina from Equine Tracheal Washes

The aim of the present study was to describe the prevalence of Nicoletella semolina in equine airways and its relationships with cytological evaluation of tracheal wash (TW). Samples were collected in the framework of routine bacteriological diagnostics of equine TW between May 2010 and June 2011. N semolina has been isolated, along with either common pathogens or contaminants, from 19 (1.8%) of the 1,054 TW samples. Median TW neutrophil counts (87.0%) in specimens from N semolina-positive horses were significantly different from those from N semolina-“negative” horses (52.0%). The data presented in this report may suggest considering such bacteria in horses clinically suffering from airway inflammation.