Thermostability of duck hepatitis virus.

The comparative thermostability of 4 duck hepatitis (DH) viruses were tested at various temperatures for different times. Titer of duckling-passaged, pathogenic DH virus decreased from 10(4.50) to 10(2.33) and 10(2.20) median infective doses (ID50/0.1 ml, respectively, in 2 tests; titer of chicken embryo-passaged, nonpathogenic, but embryo-lethal, DH virus decreased from 10(6.00) to 10(0.46) and from 10(6.62) to 10(0.63) ID50/0.1 ml, respectively; duck embryo fibroblast culture-passaged and duck embryo liver cell culture-passaged, chicken ebryo-infective, but nonlethal, DH viruses were completely inactivated or nearly so after being kept at 56 C for 30 minutes. Duckling-passaged DH virus was not detected on day 21, whereas 10(0.62) ID50 of chicken embryo-passaged DH virus per 0.1 ml remained on day 32 when being kept at 37 C. Titer of chicken embryo-passaged DH virus decreased from 10(7.00) to 10(1.16) ID50/0.1 ml after being kept at room at room temperature for 150 days, to 10(5.17) ID50/0.1 ml after being kept at 4 C for 70 weeks, to 10(6.17) ID50/0.1 ml after being kept at -20 C for 70 weeks, and to 10(6.38) ID50/0.1 ml after being kept at -60 C for 1 year.     

Intranasal infection of Getah virus in experimental horses.

Aerosol transmission in equine Getah virus (GV) infection was examined by intranasal inoculation with 10(3.0) to 10(7.0) TCID50 of the MI-110 strain in 7 experimental horses. The establishment of intranasal infection of GV was confirmed in all these horses by detecting serum neutralizing antibody against the MI-110 strain. Horses inoculated with more than 10(4.0) TCID50 of the virus manifested mild pyrexia, eruptions, serous nasal discharge, lymphopenia or monocytosis. Viremia ranging from 10(1.0) to 10(3.5) TCID50/0.2 ml occurred in horses inoculated with 10(5.0) TCID50, or more. Virus recovery from the nasal cavity was observed only in horses inoculated with 10(7.0) TCID50, and the viral titers recorded were 10(3.0) TICD50/ml or less. From these results, it is assumed that GV disseminated from the nasal cavity of naturally infected horses, except for intranasal infection with a lot of the virus, is probably very low in titer. So it seems to be rare that GV in natural cycles is spread from horse to horse by aerosol transmission.     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

Immune response to vaccinia virus and recombinant virus products in dogs

A study was undertaken to determine the safety and suitability of vaccinia virus as an eukaryotic expression vector in dogs. Clinical signs were not seen in inoculated dogs, with the exception of small nodules at the site of inoculation. Vaccinia virus did not spread from dogs that were inoculated by SC (n = 5), intradermal (ID; n = 13), or intranasal (n = 3) routes to noninoculated dogs maintained in close contact. Replication of vaccinia virus appeared to be restricted in dogs because greater than or equal to 10(5) plaque-forming units of virus were required to induce an immune response by ID inoculation. Results were better with ID inoculation than with SC or intranasal inoculations. Repeated inoculations enhanced serum antibody titers, and annual reinoculation resulted in boosting of antibody titers. Maternal antibody interfered with virus replication and antibody production. Recombinant virus products induced antibody formation in dogs to human influenza-A virus, herpes simplex virus, and human hepatitis-B virus antigens. It was concluded that vaccinia virus would be safe and suitable as an eukaryotic expression vector in dogs.     

Effect of intratesticular inoculation with Aujeszky's disease virus on genital organs of boars

Ten 8-10-month-old Belgian Landrace boars were intratesticularly inoculated with 500 TCID50 of a virulent Belgian Aujeszky's disease virus (ADV) isolate (75V19) in 0.1 ml volume. One control boar was similarly inoculated with phosphate-buffered saline solution. The genital organs of six inoculated boars were examined by virus isolation and immunofluorescence. In spite of high virus titers, the fluorescence in the testicles remained limited to a few small foci in the interstitial connective tissue and tunica albuginea at or close to the inoculation site. Neither virus replication, necrosis nor inflammatory lesions could be demonstrated in the epithelium of the seminiferous tubules. However, virus replication was regularly demonstrated in the serosa covering testicles, plexus pampiniformis, ductus deferens and tunica vaginalis. Virus was also isolated from the scrotal fluid. It is suggested that the serosa is the primary target tissue for ADV. The other four boars were inoculated to study the effect of ADV on semen. Severe morphologic alteration and lowered sperm cell concentrations were observed during several weeks after inoculation or until slaughter at 47, 53 and 58 days post inoculation. Virus was isolated from semen of only two out of four boars examined at 9 and 10 days post inoculation.     

Tween 80: a marker for differentiation of hog cholera and bovine viral diarrhea viruses.

Markers for differentiating hog cholera and bovine viral diarrhea viruses were studied using Tween 80, chloroform, trichlorotrifluoroethane and tri (n-butyl) phosphate. Attenuated A and virulent Ames strains of hog cholera virus were employed. Moreover, the NADL PK-15 cell culture adopted strain and low cell culture passaged Purdue strain of bovine viral diarrhea virus were used. These viruses were reacted with 2,500 micrograms/ml of Tween 80 for one hour at 37 degrees C. When attenuated A and virulent Ames strains of hog cholera virus with titers greater than 10(6) and 10(5) plaque forming units respectively, were reacted with Tween 80 the titer of each strains was reduced by approximately 10(4) plaque forming units of virus. When either strain of bovine viral diarrhea virus was reacted with Tween 80, virus was not detected.     

Bluetongue virus serotypes 20 and 17 infections in sheep: Comparison of clinical and serological responses

The clinical, virological and serological responses of sheep infected with an Australian bluetongue virus (BTV) isolate (serotype 20) were compared to responses in sheep inoculated with an American bluetongue isolate (serotype 17) with which it had shown cross-reactions in serum neutralization tests. In sheep inoculated with BTV 20, clinical signs were very mild and viremia was first detected by day 5; virus was isolated intermittently for a further 2 to 3 days. Neutralizing and precipitating antibodies were first detected in the serum of the sheep between 2 to 3 weeks following inoculation. In contrast, sheep inoculated with BTV 17 showed pyrexia and severe hyperemia of the nasolabial area and oral mucosa from day 7 to 17. Viremia was first detected on day 3 and extended to day 20, while the appearance and titers of serum antibodies was similar in both groups.After challenge with BTV 17 the sheep in both groups remained clinically normal, and virus was not detected in the blood; however, serum neutralizing antibody titers to both viruses increased 2 weeks after challenge and the mean titer of the two groups ranged from 1:250 to 1:640.     

Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretions

Six calves inoculated intranasally with a vaccinal strain of infectious bovine rhinotracheitis (IBR) virus and 6 control calves were given a placebo. All calves were subsequently challenge exposed (by aerosol) with rhinovirus--3 of the calves from each group at 2 days after they were inoculated with IBR virus or with placebo and the remaining calves at 6 days. Nasal excretion of viruses, interferon (IFN) concentrations in nasal secretions (NS), and neutralizing antibody in sera and NS were determined. All calves given the vaccinal IBR virus subsequently had IFN in their NS. Interferon was detected as early as 1 day, reached maximal titers at 2 to 4 days, and persisted in individual calves for 5 to 10 days after inoculation. Rhinovirus shedding was not detected from IBR virus-inoculated calves whose NS contained both rhinovirus antibody and IFN at the time of challenge exposure; such calves were protected at either 2 or 6 days after IBR virus inoculation. The outcome of rhinovirus challenge exposure of calves whose NS contained IFN, but not rhinovirus antibody, varied with the day of challenge exposure. Rhinovirus excretion was detected from 2 of these calves challenge exposed 2 days after IBR virus inoculation, but was not detected from a calf challenge exposed 6 days after inoculation. However, while IFN was present in NS from the former 2 calves, rhinovirus shedding was markedly reduced as compared with that from control calves without IFN or NS antibody at the time of challenge exposure. Consistent relationship was not observed between the rhinovirus neutralizing antibody titer of calves' sera and NS. The antibody titer of NS more closely correlated with protective immunity to rhinovirus infection than did the serum antibody titer.     

Serologic responses of calves to sequential infections with epizootic hemorrhagic disease virus serotypes

Six calves were inoculated with 1 of 2 North American serotypes of epizootic hemorrhagic disease virus (EHDV) and then inoculated with the second serotype 16 weeks later. One calf did not develop an immune response to EHDV after primary inoculation and was removed from the study. Viremia after primary inoculation was transient. Although each infected calf developed a high serum neutralizing antibody titer to EHDV, at no time after inoculation with one or both viruses was antibody detected that neutralized any US serotypes of bluetongue virus. After exposure to both serotypes of EHDV, 4 of 5 calves developed antibodies that cross-reacted with group-specific bluetongue virus antigens.     

Preparing hemagglutinating antigen from isolates of infectious bronchitis virus

The hemagglutinating (HA) activity of 14 strains of infectious bronchitis virus (IBV) was investigated. The optimal conditions for IBV antigen preparation include inoculation of 10- or 11-day-old specific pathogen-free embryonated eggs and incubation for 30 hours at 37 C. Embryos were inoculated via the allantoic cavity with 0.1 ml of a low embryonic passage of the virus (10(7) to 10(8) EID50/ml). Allantoic fluid was harvested and pooled, and a 100-fold concentration of virus particles was achieved by centrifugation for 3 hours at 30,000 x g. Virus pellets were resuspended in Tris-hydrochloride buffer containing 3 units of phospholipase-C (type-1) enzyme/ml and incubated for 2 hours at 37 C. All IBV strains tested demonstrated positive HA activity with chicken red blood cells. The antigen was stored in liquid state or lyophilized at 4 C.     

伪狂犬病病毒鄂A株gG-/LacZ+突变株在不同细胞中增殖规律的研究

伪狂犬病病毒 (Ｐｓｅｕｄｏｒａｂｉｅｓｖｉｒｕｓ ,ＰｒＶ)属疱疹病毒科α 疱疹病毒亚科 ,能引起多种家畜及野生动物的伪狂犬病 ,尤其是猪的伪狂犬病 ,已成为危害当今养猪业的最严重的传染病之一。根据已成功根除伪狂犬病国家的经验以及伪狂犬病病毒分子生物学研究的新成果 ,种猪的免疫还是以灭活苗为主。但传统的灭活苗由于缺少检测标志 ,无法采用鉴别诊断方法区分疫苗免疫猪和野毒感染猪。而在目前广泛使用的三种基因缺失标志疫苗株 (ｇＧ- 、ｇＥ- 、ｇＣ- )中 ,由于 ｇＧ的缺失不影响免疫原性 ,因此 ,ｇＧ- 灭活苗优于 ｇＥ- 、ｇＣ-…     

高致病性PRRS活疫苗(HuN4-F112株)抗体对Ⅱ型不同亚群PRRSV的中和效果

为研究高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)活疫苗(HuN4-F112株)诱导的抗体对II型不同亚群PRRSV的中和作用,本研究将10头PRRSV抗原、抗体阴性的6周龄仔猪,每头仔猪肌肉注射1头份疫苗(106.0TCID50/mL),每周采血并分离血清,检测该血清对II型PRRSV第1、2、4亚群的代表病毒株勃林格PRRSV活疫苗(VR-2332株)、PRRSV活疫苗(CH-1R株)和HP-PRRSV活疫苗(HuN4-F112株)的中和效价。实验结果显示,仔猪免疫3周后开始产生针对HuN4-F112的中和抗体,11周至22周抗体水平达到高峰,抗体持续至少25周,但只有少数免疫猪在几个时间点的血清对VR-2332和CH-1R疫苗株具有血清交叉中和作用,而且中和效价较低。     

Leukocyte-dependent platelet vasoactive amine release and immune complex deposition in African swine fever

Fifteen pigs were inoculated with African swine fever virus in a study of the pathogenesis of the disease. All pigs surviving the first two weeks developed high circulating antibody titers against African swine fever virus and persistent viremia. Hemolytic complement levels declined to 50 to 70 hemolytic complement 50 (CH50) units/ml from mean preinoculation levels of 120 CH50 units/ml. Immune deposits consisting of African swine fever antigen, host immunoglobulin G, and native C3 were found in the glomeruli of surviving pigs. All pigs surviving two weeks after inoculation developed leukocyte-bound antibody functionally characteristic of immunoglobulin E (IgE). Antigen-specific degranulation of antibody-coated leukocytes produced secondary platelet aggregation and vasoactive amine release. The results suggest that the IgE-basophil-platelet loop acting via amplification by leukocyte-derived platelet-activating factor participates in the immune complex deposition process in African swine fever.     

Effects of two adenoviruses (type 1 and type 8) on functional properties of bovine alveolar macrophages in vitro.

Cultures of bovine alveolar macrophages were inoculated with type-1 and type-8 adenoviruses, initially isolated from calves with respiratory tract disease, and functional properties of the cells were observed over a period of 10 to 11 days. Both viruses replicated in macrophages; viral titers were low (less than 3.75 log10 TCID50/0.1 ml), and intranuclear inclusions were detected by indirect immunofluorescence in 5 to 10% of the cells from 3 days after inoculation. Highest titers were induced by type-1 adenovirus, which also induced the greatest functional changes. Expression of Fc and complement receptors was reduced by both viruses, although the greatest effects were seen with type 1. Phagocytosis of Candida krusei cells was reduced following type 1 infection, whereas phagocytosis in type-8-infected cells was not different from that of noninfected macrophages. Ability to kill ingested Candida cells also was reduced following type-1 infection, whereas type-8-infected macrophages had lower killing ability only at 2 to 4 days after inoculation. Neither virus had substantial effects on the production of neutrophil chemotactic factors by the macrophages.     

Infectivity of PRRS virus in pig manure at different temperatures

PRRSv is an economically important swine pathogen which can be disseminated from infected pig herds via movement of contaminated manure. The process of manure handling and inadequate cleaning of transport vehicles are commonly implicated as sources of PRRSv transmission. Stability of PRRSv in pig manure at different temperatures is unknown. The objective of this study was to determine PRRSv-infectivity half-life in manure and in a cell culture medium at 4, 20, 60 and 80°C. To assure sample consistency across the study, all samples were prepared from common homogenized solutions (MEM and manure) and frozen at -20°C. Samples were thawed, transferred to a water bath set at a specific temperature, inoculated with 100μl of PRRSv at designated time points and then tested for virus infectivity. Regression models were created to estimate PRRSv half-life based on incubation temperature. There was an exponential decrease in PRRSv infectivity with increasing temperature. At every temperature tested, PRRSv had shorter half-life when incubated in manure compared to MEM. PRRSv half-life in MEM and manure was estimated at 112.6 and 120.5h at 4°C, 14.6 and 24.5h at 20°C, 1.6 and 1.7h at 40°C, 2.9 and 8.5min at 60°C, and 0.36-0.59min at 80°C, respectively. Results of this study can be used as basis for developing strategies to inactivate PRRSv present in manure-contaminated environments using heating treatments. For example, these data suggest that submitting transport trailers to temperature of 50°C for 8h would decrease PRRSv from 10(6) TCID(50)/ml to less than 10(1) TCID(50)/ml.     

Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.     

白喉型鸡痘病毒Gibbs株免疫特性研究

白喉型鸡痘病毒Gibbs株能在鸡胚绒毛尿囊膜上良好增殖，接种后１２０小时，绒毛尿囊膜明显水肿，且淡黄色痘斑，其毒价达到１０^5.25-10^5.83EID50/0.2ml。用１０倍稀释的病毒悬液肌肉注射１３日龄雏鸡，无任何不良反应。用１００倍稀释病毒液皮下刺种免疫１４－４０日龄鸡，接种后３－６天，接种局部出现痘痂，接种后１４天，用１０倍稀释的Ｇibbs株病毒液二次皮下刺种或用鸡痘野毒株经腿部毛作擦，免疫未见任何局部反应或其它异常，保护率达１００％。，免疫后１６５天，保护率仍达８０％，试验结果证实该毒株具有良好的免疫原性和安全性。     

Experimental reproduction of viral chorioretinitis in kangaroos

OBJECTIVE: To investigate whether preparations containing Wallal and/or Warrego viruses could cause disease when inoculated subcutaneously into captive kangaroos. DESIGN AND PROCEDURE: Four groups of two kangaroos, seronegative to both Wallal and Warrego virus, were each inoculated with wild Wallal virus, cultured Wallal virus, wild Warrego virus, or wild Warrego virus followed by wild Wallal virus after 3 weeks. A single uninoculated animal served as a control. Animals were monitored weekly under anaesthesia, examined ophthalmoscopically (including fundic photography), and samples collected for haematological and serum biochemical analysis, virus isolation, PCR and serological examination for antibodies against Wallal and Warrego viruses. Animals inoculated with cultured Wallal virus were killed at week 10, and remaining kangaroos were reinoculated with cultured Wallal virus at week 12. RESULTS: Virus was isolated from the blood of two kangaroos 2 weeks after inoculation with Wallal virus preparations, and from a third kangaroo 2 weeks after reinoculation. By 3 weeks after inoculation, all kangaroos given Wallal virus preparations had seroconverted to Wallal virus and one had seroconverted to Warrego virus. Fundic changes were detected in the three viraemic kangaroos 4 or more weeks after inoculation, and lesions were present in the eye and brain typical of those seen in field cases of chorioretinitis. No other kangaroos had lesions. Wallal virus was identified by PCR and immunohistochemical analysis in the retina of one affected animal and orbivirus-like particles were seen by electron microscopy in the remains of retinal cells. CONCLUSION: The condition of chorioretinitis was reproduced in three of eight kangaroos by inoculation with preparations containing Wallal virus.     

鸭坦布苏病毒病活疫苗（FX2010-180P株）毒种保存期的研究

鸭坦布苏病毒病是新发的一种以蛋鸭产蛋下降为主要特征的传染病,目前仍没有疫苗可预防和控制该病。将鸭坦布苏病毒强毒（FX2010株）在鸡胚成纤维细胞（chicken embryo fi broblasts,CEFs）上连续传代培养180代,获得1株致弱毒株（FX2010-180P株）,利用该弱毒株研制了鸭坦布苏病毒病活疫苗。本研究为了测定鸭坦布苏病毒病活疫苗（FX2010-180P株）毒种的保存期,将-80℃条件下保存了16个月的原始种子批和基础种子批各取3支,进行纯净性检验、鉴别检验以及病毒滴度测定。结果表明,各批次毒种在保存16个月后,病毒纯净无污染,病毒滴度没有明显下降。把基础种子在CEFs上增殖后,低剂量接种鸭子,检验毒种的免疫原性。结果显示,用毒种增殖的病毒免疫原性良好,101.5和102.5 TCID50的剂量可以为接种鸭提供90%和100%的保护。