Equine herpesvirus type 2: cell-virus relationship during persistent cell-associated viremia

Some aspects of the biology of equine herpesvirus type 2 (EHV-2) were investigated by examination of the persistent cell-associated viremia stage of the infection. The EHV-2 infection of leukocytes was latent, because free virus was not retrieved without first cultivating harvested leukocytes in vitro. A virus infective center (IC) assay was developed to enumerate latently infected cells in the leukocyte population. This assay proved to be simple and reproducible and revealed a linear relationship between IC plaques formed and the number of cells inoculated, except where large numbers of cells (greater than 4 X 10(6)) were inoculated per 10 cm2 dish. This reduction at high cell densities of IC/10(6) cells inoculated was dependent on cells obtained from an EHV-2-infected horse. There was considerable variation in the numbers of IC/10(6) leukocytes harvested from different horses, but little variation in the harvests from the same horse at different times. There seemed to be a direct relationship between serum-neutralization titers and IC numbers. Transfer of viable infected leukocytes to 2 fetuses failed to establish EHV-2 infection. Infection of equine fetal kidney cells with EHV-2 virus failed to produce detectable Fc receptors on the cell surface.     

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.     

H3N8亚型马流感病毒单克隆抗体的制备及鉴定

以灭活马流感病毒(EIV)A/Equine/Jilin/1/1989(H3N8)为免疫原,免疫Balb/c小鼠,经常规细胞融合后,用血凝抑制试验(H1)和间接ELISA方法筛选获得3株(3C2、5G10和5A10)能稳定分泌H3N8亚型马流感病毒单克隆抗体(mAb)的杂交瘤细胞株.其中3C2和5G10为IgG2α,5A...     

Antibody Responses Against Equine Influenza Virus Induced by Concurrent and by Consecutive Use of an Inactivated Equine Influenza Virus Vaccine and a Modified Live Equine Herpesvirus Type 1 Vaccine in Thoroughbred Racehorses

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1–2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naïve horses or horses whose vaccination history is incomplete.     

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 10² plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 10⁶ pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 10⁴ pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.     

Application of a radioimmunoassay for detection of the major internal antigen (p24) of bovine leukemia virus from cultured lymphocytes of cattle

A sensitive and specific radioimmunoassay for the major internal antigen of bovine leukemia virus has been applied to detecting this protein in cultured lymphocytes of infected cattle. The specificity inherent in this assay offers obvious advantages over a previously described syncytium induction assay for infectious bovine leukemia virus, because false positive reactions due to other viruses such as bovine syncytial virus are avoided. Investigations of various culture conditions indicated that maximal amounts of antigen had been produced after incubation for 72 hr at 37°C. Lymphocyte concentrations of 10⁶?5×10⁷ cells/ml gave satisfactory results. Tests of cultured lymphocytes from bovine leukemia virus infected or bovine leukemia virus-free cattle indicated a comparable sensitivity between the radioimmunoassay and syncytium induction assay in the detection of bovine leukemia virus infections.     

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。     

North American Strain of Viral Hemorrhagic Septicemia Virus is Highly Pathogenic for Laboratory-Reared Pacific Herring

Abstract

Specific-pathogen-free Pacific herring Clupea pallasi were reared in the laboratory from eggs and then challenged at 5, 9, and 13 months of age by waterborne exposure to low (10^1.5–2.5 plaque-forming units [PFU] per milliliter), medium (10^3.5–4.5 PFU/mL), or high (10^5.5–6.5 PFU/mL) levels of a North American isolate of viral hemorrhagic septicemia virus (VHSV). The fish were extremely susceptible to the virus, showing clinical disease, mortality approaching 100%, and only a limited increase in resistance with age. Mortality began 4–6 d after exposure and peaked at approximately day 7 in fish exposed to high levels of virus. Whereas the mean time to death showed a significant dose response (P < 0.001), the percent mortality and virus titers in dead fish were generally high in all groups regardless of initial challenge dose. External signs of disease were usually limited to 1–2-mm hemorrhagic areas on the lower jaw and isthmus and around the eye, but 2 of 130 infected fish exhibited extensive cutaneous hemorrhaging. Histopathologic examination of tissues from moribund fish sampled at 2–8 d after exposure revealed multifocal coagulative necrosis of hepatocytes, diffuse necrosis of interstitial hematopoietic tissues in the kidney, diffuse necrosis of the spleen, epidermis, and subcutis, and occasional necrosis of pancreatic acinar cells. Virus titers in tissues of experimentally infected herring were first detected 48 h after exposure and peaked 6-8 d after exposure at 10^7.7 PFU/g. Fish began shedding virus at 48 h after exposure with titers in the flow-through aquaria reaching 10^2.5 PFU/mL at 4–5 d after exposure, just before peak mortality. When the water flow was turned off for 3 h, titers in the water rose to 10^3.5 PFU/mL, and the amount of virus shed by infected fish (on average, greater than 10^6.5 PFU/h per fish) appeared sufficient to sustain a natural epizootic among schooling herring. Taken together, these data suggest that VHSV could be a significant limiting factor for populations of Pacific herring.     

Laboratory Studies on the Transmission of Western Equine Encephalitis Virus by Saskatchewan Mosquitoes I. Culex tarsalis

A Saskatchewan strain of the mosquito Culex tarsalis, transmitted a local strain of western equine encephalitis virus from chick to chick, between four and 44 days after an infective blood meal. At incubation temperatures of 69 and 75°F, 120 transmissions occurred out of a possible 141, and all but seven of these were by single infected mosquitoes. At 75°F virus titers in individual mosquitoes were more uniform and transmission was more efficient, than at 69°F, although infection rates were similar at both temperatures. The minimum concentration of virus required to infect 50% of C.tarsalis was 10^2.5 intracerebral three-week old mouse LD₅₀ per 0.03 ml of donor blood. These findings provide direct evidence that C. tarsalis of Saskatchewan is a highly efficient vector of western equine encephalitis virus.     

First Report on Molecular Detection of Equine Upper Respiratory Infectious Viruses in Republic of Korea

The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.     

马传贫驴白细胞弱毒疫苗株基质蛋白基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码基质蛋白（p15)的基因，并在大肠杆菌中进行了表达，所表达的蛋白是一种可溶性的融合蛋白，其氨基端带有6个组氨酸的标签，因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化，在间接ELISA和免疫印迹试验中，重组的基质蛋白可与马传贫阳性血清样品发生反应，而与健康马血清无任何反应，这表明该重组蛋白具有良好的抗原性和特异性，可用于马传贫弱毒疫苗株在体内外复制及在接种马体人免疫应答的研究中。     

Comparison of the Virucidal Effects of Disinfectant Agents Against Equine Influenza A Virus

Disinfection is one of the most important biosecurity measures to minimize disease spread during outbreaks of equine influenza. Although many disinfectants are commercially available, information about their effects against equine influenza A virus (EIV) is limited. This report describes an evaluation of the effects of six disinfectants against EIV (∼10^4.7 egg infectious dose 50/200 μL) under different conditions (reaction time [10 and/or 30 minutes], temperature [4°C–25°C], and the absence and/or presence of uninfected allantoic fluid that served as our best choice of “organic matter” equivalent). Although the efficacy of didecyldimethylammonium chloride decreased with decreasing reaction temperature, the compound showed the highest efficacy of the three quaternary ammonium compounds tested in this study. The effects of sodium dichloroisocyanurate and nonoxynol iodine were not affected by reaction time or temperature, but they were affected by the presence of organic matter. Antec Virkon S containing potassium peroxymonosulfate and sodium chloride consistently inactivated EIV regardless of reaction time, temperature, and the presence of organic matter. These findings will help us to take rational biosecurity measures during outbreaks of equine influenza.     

Equine herpesvirus type 1: detection of viral DNA sequences in aborted fetuses with the polymerase chain reaction

Primers and probes were selected from the gene encoding glycoprotein 13 (gp 13) of equine herpesvirus 1 (EHV-1). The polymerase chain reaction (PCR) was run on infected and noninfected cultured cells and on 63 specimens from 29 aborted equine fetuses. The results were evaluated by electrophoresis and dot-blot hybridization using an oligonucleotide probe labeled with biotin. In the infected samples electrophoresis showed a PCR product of about 280 base pairs. The dot-blot hybridization confirmed that this product contained EHV-1 DNA sequences. PCR took 4 h and hybridization another 14 h; the results were thus achieved within 24 h and were highly specific for EHV-1. Close concordance was found between the results of PCR and virus isolation.     

Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

Whole-Blood Validation of a New Point-of-care Equine Serum Amyloid A Assay

Serum amyloid A (SAA) is considered a major acute phase protein (APP) in horses. Serum amyloid A stall-side assays are commercially available to assess the inflammatory response of patients with various infectious and noninfectious conditions. The objective of this study was to determine the analytical performance of a new point-of-care (POC) assay for the measurement of SAA in whole blood and plasma of horses. One hundred and sixty blood samples were collected from 60 horses at various time points after immunization with an equine core vaccine. Analytical validation of the SAA POC assay included the measurement of SAA in whole blood and plasma, assessment of linearity and precision, and comparison of the SAA POC results with those obtained with a validated turbidimetric immunoassay (TIA). The SAA POC assay yielded similar results in whole blood and plasma (P > .05), and the results were positively correlated with the TIA (R² = 0.964). The assay displayed solid linearity throughout the detection range of ≤ 20 to 3,000 μg/mL (R² = 0.984) with inter-assay and intra-assay coefficients of variation ranging from 7.8% to 13.3% and 5.7% to 12.0%, respectively. The new SAA POC assay was able to reliably measure SAA in both whole blood and plasma. Similar to previously validated assays, the new SAA POC assay is a valuable tool to investigate the inflammatory response in various clinical diseases of horses.     

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.     

Cloning of the Genomes of Equine Herpesvirus Type 1 (EHV‐1) Strains KyA and RacL11 as Bacterial Artificial Chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV‐1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Escherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co‐transfection of viral DNA and recombinant plasmid pΔ71‐pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high‐molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full‐length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacL11 and pKyA, respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP‐expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full‐length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2‐negative progeny reconstituted from pRacL11 and pKyA (L11Δgp2 and KyAΔgp2) exhibited different growth properties. Whereas both L11Δgp2 and KyAΔgp2 extracellular titres were reduced by 15‐ to 32‐fold, plaque diameters were only markedly (50%) reduced in the case of KyAΔgp2.     

Role of the suppressor of cytokine signaling 2 (SOCS2) during meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5)

The role of suppressors of cytokine signaling (SOCS) in meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5) was evaluated by intracranial infection in C57BL/6 wild-type mice (WT) and SOCS2 deficient mice (SOCS2^−/−). Both infected groups presented weight loss, ruffled fur and hunched posture. Additionally, infected SOCS2^−/− mice showed swollen chamfer and progressive depression. Infected WT animals developed mild meningitis, characterized by infiltration of mononuclear cells. Moreover, viral DNA was detected in liver and lung from infected WT group. This group also showed elevated brain levels of IFN-γ, IL-10, CXCL1 and CCL5, when compared with non-infected WT animals. Brain inflammation was exacerbated in infected SOCS2^−/− mice with widespread distribution of the virus and increased brain levels of TNF-α, IFN-γ, IL-10, IL-12, CXCL1 and CCL5, when compared with WT infected mice. Moreover, infected SOCS2 deficient mice exhibited reduced brain mRNA expression of IFNα and IFNβ and increased expression of mRNA of SOCS1, compared with infected WT mice. Taken together, our study provides an insight into the role of SOCS2 in modulating the immune response to BoHV-5 infection.