African Horse-Sickness Killed-Virus Tissue Culture Vaccine

Formalized African horse-sickness (AHS) type 9 virus cultivated in monkey kidney stable (MS) cell cultures was experimentally used for immunizing horses. Inactivated vaccines prepared either from viscerotropic or neurotropic type 9 AHS virus produced antibodies in vaccinated horses. Immunity developed in all horses vaccinated with various amounts of the vaccine, and protected them from infection, when challenged 5 weeks after vaccination.     

Studies on Bovine Virus Diarrhea: Serum Neutralization, Complement-fixation and Immunofluorescence

The complement-fixation, the serum neutralization tests and the fluorescent-antibody technique were the serological methods applied in this laboratory for the detection of antigens for bovine virus diarrhea (BVD). As observed previously, the modified direct complement-fixation (MDCF) test was required to demonstrate antibodies against virus infections of cattle.

At a certain stage of infection, the MDCF test was found to be as accurate and less time-consuming than the serum neutralization test for the detection of antibodies in bovine sera. The modified direct complement-fixing antibodies were detectable in the serum from approximately three weeks up to a few months after infection as compared to several years for the serum neutralization test. Thus, as in most other viral diseases, the MDCF test was of value for detecting recent infections while the serum neutralization test detects both recent and long-standing infections.

The fluorescent antibody technique was of value to detect viral antigens of both cytopathogenic and noncytopathogenic strains of BVD in primary fetal kidney cell cultures inoculated with field specimens. In addition, the virus was detected in six of 220 fetuses collected at a local slaughter house for the preparation of primary cell cultures. The length of time required for the detection and identification of specific viral antigens by immunofluorescence was considerably reduced over that of the serum neutralization and virus interference tests.

     

Factors affecting the assay of bovine type I interferon on bovine embryonic lung cells

One preparation of interferon (IF) on 7-day-old bovine embryonic lung cultures free of bovine viral diarrhea virus had titers ranging from 20 to 10,240 in plaque-reduction tests, using bovine vesicular stomatitis virus. Factors believed to contribute to this variation were investigated. Although titers differed on different cell strains, different passages, and on cultures of different ages, variations between the two assays definitely could not be established as a function of these factors. However, IF titers on low passage cultures were usually lower than were titers on subsequent passages of the same cell strain. Calf serum in the diluent for IF reduced the titer one or two twofold dilutions. Over the range of 7 to 24 hours contact of IF with bovine embryonic lung cultures, and titer decreased steadily and was one twofold dilution less at 24 hours than it was at 7 hours. Latent viruses were not found in cultures by electron microscopy. Treatment of cultures with a noncytopathogenic strain of bovine viral diarrhea virus almost eliminated the effect of IF. Naturally occurring production of IF was not a major problem. While IF was on the cells, the pH of IF had a pronounced effect on the titer and may have been responsible for the vatiation observed with different passages, different cell strains, use of cultures of different ages, and use of calf serum in the diluent. Interferon at a low pH had a titer three or four twofold dilutions less than did the IF at a high pH.     

Studies with Bovine Parainfluenza — 3 Virus in U.A.R. (Egypt)

A bovine strain of myxovirus parainfluenza-3 (MP3) virus, designated S virus, was isolated from lung tissue collected from cattle with respiratory illness in 1963. The virus agglutinates mammalian and avian erythrocytes, and is sensitive to ether, sodium desoxycholate and trypsin. It grows in primary calf kidney, buffalo kidney, dog kidney, camel kidney and MS cell cultures. The S virus forms well-defined plaques in buffalo and calf kidney cells on the 5th or 6th day after inoculation. Examination of cell cultures following inoculation with S virus revealed giant cell formation, and introcytoplasmic and intranuclear inclusions. At 37°C the virus titer dropped from 10^10.4 to 10^2.6 in 3 days. Virus was completely inactivated at 56°C within 15 minutes. Growth-curve studies in tissue culture monolayer cells revealed a latent period of 10 hours. The intracellular virus titer was slightly lower than that of extracellular virus. The isolate was identified as MP3 virus by serum neutralization and hemagglutination-inhibition tests. Antibodies (HI) to S virus were shown to be present in a significant proportion of Egyptian cattle. The epidemiological significance of MP3 (bovine strain) virus in U.A.R. is discussed.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V.

Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.

     

Immunofluorescence Plaque Assay for African Swine Fever Virus

Suitably diluted cell culture adapted African swine fever virus preparations were inoculated on VERO cell monolayers and grown on coverslips. Gum tragacanth was used as an overlay. After three days incubation at 37°C the infected cultures were fixed with acetone and stained with fluorescent antibody conjugate. Fluorescing plaques consisted of 20-30 infected cells.

Three statistical criteria for a quantitatively reliable assay were met: the Poisson distribution for plaque counts, linearity of the relationship between the concentration of virus and the plaque count and reproducibility of replicate titrations. The method is suitable for counts up to at least 70 plaques per 5 cm² coverslip and computed titers are reproducible within 0.16 log units with a total of 300 plaques enumerated.

     

Variation of virus replication in primary bovine kidney cell cultures derived from 68 three-day-old calves

Primary kidney cell cultures were prepared from 68 three-day-old calves. Complete monolayers of these cultures were infected separately with viral diarrhea, infectious bovine rhinotracheitis, parainfluenza, and adenovirus 7 viruses. The yield of virus from all infected cultures was calculated by plaque titer assay after 2 to 4 days' incubation. The variation of virus yield was substantial between individual cultures.     

浙江地区传染性法氏囊病病毒野毒株的血清亚型分析

７个浙江地区的ＩＢＤＶ野毒株、１个四川地区的ＩＢＤＶ野毒株和２个疫苗毒株经病毒血清交叉中和试验获得了抗原性不同的五个亚型毒株或变异株。根据交叉中和试验所得的Ｒ值，应用聚类分析法分析了各亚型毒株之间的亲缘关系，显示传染性法氏囊病病毒的变异存在地域性差异。     

Infectivity assay and neutralization test for equine influenza virus in microplate cell cultures

Several cell lines including Vero and MDCK cells were tested for susceptibility to the Miami and Prague strains of equine influenza virus to find cell cultures suited for study of these viruses. The viruses readily multiplied with cytopathic effects in these cell cultures. Of the cells tested, ESK cells derived from swine embryo kidney were the most susceptible to the viruses. Based on these results an infectivity assay of the viruses was worked out using ESK cell cultures prepared in microplates. The method is not only simple enough for routine use, but is also practically as sensitive as the egg inoculation method. The method was further adapted to a neutralization test.     

Studies on Transmissible Gastroenteritis of Swine I. The Isolation and Identification of a Cytopathogenic Virus of Transmissible Gastroenteritis in Primary Swine Kidney Cell Cultures

Two virus isolates from transmissible gastroenteritis (TGE) of swine were adapted to grow in primary swine kidney cells. Growth of the virus was indicated by the resistance of the infected cells to the cytopathic effect of a virus diarrhea virus of cattle, and by the development of large round cells on the cell sheet.

Evidence that these virus isolates were TGE was obtained by the development of signs of the disease followed by death of exposed SPF pigs, or the resistance of the recovered pigs to further signs of disease when they were exposed to virulent TGE contained in virus bearing intestinal tissue.

The in vitro and in vivo serum neutralization tests, along with staining of infected cells by fluorescein conjugated TGE antiserum, gave further indication of the specific nature of the virus growing in the cell cultures.

     

赤羽病毒单克隆抗体的研制及鉴定

用纯化的赤羽病毒（akabane virus,AKAV）免疫Balb/c小鼠,取小鼠脾细胞和骨髓瘤细胞SP2/0融合,经间接ELISA筛选和3次有限稀释法克隆,得到2株能稳定分泌抗赤羽病毒单克隆抗体（McAb）的杂交瘤细胞株,分别命名为AKAV McAb 3A株和2C株。ELISA试验和中和试验结果表明,本研究制备的2株McAb均具有良好的特异性,为AKAV阳性,杂交瘤细胞培养上清液抗体的效价分别为1∶640和1∶320,腹水的效价分别为1∶256000和1∶128000,亲和常数（Ka）分别为1.16×10^-9和6.31×10^-8 mol/L,3A株的相对亲和力大于2 C株,具有病毒中和活性,中和效价分别为1∶64和1∶32,其IgG亚类为IgG1,轻链的亚型均为kappa型,2株细胞冻存3次复苏后仍能稳定分泌抗体,表明AKAV McAb制备成功,为赤羽病快速诊断方法的研究奠定了基础。     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

Heterogeneity of Foot-and-mouth Disease Virus: Further Studies on Plaque Formation by Two Plaque-size Variants

Plaque production by a small-plaque (SP) and large-plaque (LP) variant of foot-and-mouth disease virus, type A, strain 119 (FMDV, A119), was influenced by a number of environmental factors. The SP variant produced plaques on cells of the IB-RS-2 cell line from swine kidney and to a lesser degree on primary cultures of swine kidney cells, but plaque formation was inhibited on primary cultures of bovine kidney (BK) cells unless diethylaminoethyl (DEAE) dextran was added to agar overlays. When DEAE dextran-treated agar overlay was used, the LP variant formed larger plaques on BK cells but not on IB-RS-2 cells. Concentrations of DEAE dextran from 0 to 100 µg/ml greatly enhanced the formation of SP virus plaques on BK cells but had little or no effect on the average size of plaques produced by the LP variant. Higher concentrations of polycation enlarged the plaques formed by both variants. Plaque sizes of the SP and LP variants increased as the concentration of agar or agarose in the overlays decreased. Reducing the concentration of agar to 0.75% facilitated the formation of SP virus plaques, but better plaque production occurred under agarose overlays.

The original parent virus consisted predominantly of virus particles that formed small plaques. The rate of neutralization of the parent virus by guinea pig antiserum prepared against the parent virus was faster than antiserum inactivation of a low-passage virus of the same serotype and strain.

     

鸡传染性支气管炎病毒广西分离株血清型的分析

应用病毒感染的鸡胚材料免疫新西兰兔的方法制备抗鸡传染性支气管炎病毒(IBV)单因子血清,然后在鸡胚气管环培养(Tracheal organ cultures,TOC)上对广西分离的7个IBV代表性毒株和3个常用疫苗株进行交叉病毒中和试验。结果显示,10个毒株被分为6个血清型。根据试验所得的R值,应用聚类分析法分析了各血清型毒株之间的亲缘关系,显示目前在广西流行的IBV野毒株之间以及其与疫苗株间的抗原性存在很大程度的差异,分属不同的血清型。同时还对IBV基因分型和血清分型之间的关系进行了探讨。     

Neutralization of African swine fever virus by sera from African swine fever-resistant pigs

Sera from African swine fever-resistant pigs with infection-inhibitory activity decreased virus replication in infected porcine buffy coat cultures. This same effect was observed even after virus was adsorbed. The infection-inhibition was not reversed by removing the immune serum from the assay cultures. Reduction of African swine fever virus replication by immune sera was demonstrated by fluorescent focus assay on MS cell line cultures. Virus-neutralization tests showed a persistent fraction of non-neutralized virus, which was not demonstrable by infection-inhibition tests. One hypothesis for explaining this difference is proposed.     

Characterization and reactivity of monoclonal antibodies to the Miller strain of transmissible gastroenteritis virus of swine.

Hybridomas secreting monoclonal (MAB) to transmissible gastroenteritis virus (TGEV) were produced by fusion of SP2/0 myeloma cells and splenic lymphocytes of BALB/c mice immunized with the virulent cell-passaged Miller strain of TGEV. The MAB secreted by these hybridomas were partially characterized; 4 of them (MA4, MA5, MH11, MB2) had high-neutralization titer for TGEV. The remaining 7 (MC6, MD9, ME5, MG5, MF2, ME9, MG7) did not neutralize TGEV at 1:25 dilution. All 4 neutralizing and 2 of the nonneutralizing MAB reacted with the E2 protein of TGEV in a radioimmunoprecipitation assay. The remaining 5 MAB reacted with the E1 protein of TGEV. Reactivity of the MAB was tested in an indirect immunofluorescent assay with 3 cell culture-adapted strains of TGEV (Miller, Purdue, and Illinois) and 13 wild-type isolates of TGEV. Neutralizing MAB reacted with all 13 wild-type isolates and the 3 cell culture-adapted strains of TGEV. In contrast, nonneutralizing MAB that reacted with the Miller strain of TGEV varied in their reactivity with the wild-type TGEV isolates. Reactivity of neutralizing MAB was also tested, using plaque-reduction neutralization assays with Miller, Purdue, and Illinois strains and 5 wild-type isolates. All 4 neutralizing MAB neutralized the 8 virus isolates, but the neutralization titer was higher with the homologous virus than with the heterologous virus isolates. However, neutralization titers of the 4 neutralizing MAB were 4 to 16 times higher for the homologous Miller strain of TGEV than for the heterologous Illinois and Purdue strains, and were 4 to 1,000 times higher than for the wild-type isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Terminal Dissemination of Rabies Virus in Selected Rat Tissues

Fifty rats were divided into 3 groups and challenged via the foot pad route with a fixed and 2 different strains of street rabies virus in order to study the dissemination of the virus and the affinity for certain tissues of the rat.

The incubation period for rats inoculated with fixed rabies is shorter than with street virus, being 5 to 7 days compared with 10 to 12 days.

Rats inoculated with the fixed strain were less aggressive and irritable than rats inoculated with street virus.

The fixed strain demonstrated a greater affinity for the tissues studied as compared to the street strains of virus.

Both the fixed and street strains revealed a low affinity for the parotid gland since no virus could be demonstrated in 14 of 20 in the fixed virus group and 27 of 30 in the street virus group.

     

A Strain of Siniperca chuatsi Rhabdovirus Causes High Mortality among Cultured Largemouth Bass in South China

Abstract

In April 2011, 40% mortality of Largemouth Bass Micropterus salmoides juveniles occurred at a farm of Zhongshan City, Guangdong Province, China. Infected fish became lethargic, exhibited corkscrew and irregular swimming, and developed a distended abdomen and crooked body. Fish began to die within 2 d after the appearance of clinical signs. In order to analyze the pathogeny and diagnose the disease earlier, observation of clinical signs, cell infection, titer calculation, electron microscopy, immersion infection assay for fish, and nucleotide sequence analysis were carried out. Fathead minnow (FHM) cell cultures, inoculated with filtrate of liver and spleen homogenates from the diseased fish, developed the obvious cytopathic effect 46 h after inoculation in the primary culture and 24 h at the first passage. Typical rhabdovirus particles, 115–143 nm in length and 62–78 nm in diameter, were observed in infected FHM cells by direct transmission electron microscopy. The isolated virus produced a titer of 10^7.15 TCID50/mL. Immersion-Fish infected with the virus had similar clinical signs and 80% mortality with 10^2.5 LD50/mL. The data indicated that the rhabdovirus was the lethal pathogeny of the current disease. Based on nucleoprotein-gene nucleotide sequence multiple alignment analysis, the newly isolated virus is a strain of Siniperca chuatsi rhabdovirus (SCRV) under family Rhabdoviridae, which was initially isolated from Mandarin Fish Siniperca chuatsi. Up to the present, at least four virus strains have been isolated from diseased Largemouth Bass, which have had different clinical signs. Comparison of the clinical signs can help in an early diagnosis of the disease.

Received October 30, 2012; accepted April 19, 2013     

Swine Interferon I. Induction in Porcine Cell Cultures with Viral and Synthetic Inducers

The production of interferon by porcine kidney (PK₁₅) cell culture in response to viral and synthetic inducers was studied. The inducers used included a synthetic double-stranded polyribonucleotide, polyriboinosinic-polyribocytidylic acid (Poly I:C), swine influenza virus and three strains of pseudorabies virus. Following exposure to these inducers cell culture fluids were examined for interferon by the plaque-reduction method.

The Poly I:C and the swine influenza virus induced production of interferon by PK₁₅ cell cultures, whereas, all three strains of pseudorabies virus at the two concentrations tested failed to induce production of interferon in vitro.

The antiviral substance produced in PK₁₅ cells was identified as an interferon because it was pH stable, non-dialyzable, sensitive to trypsin, non-sedimentable, relatively heat stable, host-species specific and it possessed broad-spectrum antiviral activity. The latter was demonstrated by inhibition of vesicular stomatitis, vaccinia and pseudorabies viruses. Differences in interferon activity against the different viruses were observed.

     

Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV-1)

Bovine viral diarrhea virus (BVDV) has various economic impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes. Both genotypes, namely BVDV-1 and BVDV-2, as well as different subgroups within these genotypes have been reported worldwide. Understanding the serological differences among the BVDV subgroups is important for disease epidemiology and prevention as well as vaccination programs. The aim of this study was to determine the serological relatedness among the subgroups in BVDV-1. For that purpose, sheep hyperimmune sera were collected against representative strains from 6 of the subgroups of BVDV-1 (BVDV-1a, -1b, -1d, -1f, -1h and -1l). The serum samples that gave the peak antibody titer to the homologous strains were used to perform cross neutralization assays. The highest homologous antibody titer (1:5160) was obtained against BVDV-1h. Regarding the cross neutralizing (heterologous) antibodies, the lowest titer (1:20) was produced by the BVDV-1f antiserum against the BVDV-1a and BVDV1-b viruses. The highest cross neutralizing titer (1:2580) achieved by the BVDV-1h antiserum was against the BVDV-1b strain. The cross neutralization results indicated particular serological differences between the recently described subgroup (BVDV-1l) and BVDV-1a/-1b, which are widely used in commercial vaccines. Considering the cross neutralization titers, it is concluded that selected BVDV-1l and BVDV-1h strains can be used for the development of diagnostic and control tools.