Polymyositis in mice experimentally inoculated with Getah virus

Mice inoculated intracerebrally with parent, large-plaque (LP) and small-plaque (SP) strains of Kanagawa strain of Getah virus showed clinically recumbency and paralysis. The LP strain caused recumbency more rapidly and killed mice more early after inoculation than the parent and SP strains. Microscopically, skeletal muscles of the whole body were involved showing degenerative or inflammatory changes. In mice inoculated with the parent or SP strains, there were degeneration and necrosis of the muscle fibers with inflammatory cell infiltration and regenerative reaction. The lesions were particularly conspicuous in muscles of the hind legs. In mice inoculated with the LP strain, most of the muscle fibers revealed degeneration and necrosis, but reactive changes were poor. In addition, the periosteum and muscular connective tissue were thickened with karyorrhexis. Electron microscopically, virus particles were recognized mainly in cisternae of sarcoplasmic reticulum in skeletal muscle fibers of mice inoculated with the LP strain, while they were rare in those of animals injected with the parent and SP strains. From these finding, it was suggested that Kanagawa strain of Getah virus has the virulence to skeletal muscles of mice.     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

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.

     

Pathogenicity for horses of original Sagiyama virus, a member of the Getah virus group

Sagiyama virus is a member of the Getah virus group. Its pathogenicity for horses was examined. All the horses infected with the original 4 strains of Sagiyama virus (M6/Mag 33, Mag 121, Mag 132 and Mag 258) developed pyrexia ranging from 39.0 to 40.0 degrees C. Other clinical signs, characterized by eruptions, edema in the hind legs, enlargement of the submandibular lymph node and mild leukopenia, were also manifested. Viremia occurred 1-4 days post-inoculation (p.i.). Virus was recovered from spleen, liver, lung and various lymph nodes of a horse autopsied on Day 4 p.i. The maximum titer of virus (10(6.0) TCID50 g-1) was detected in the inguinal lymph node. Seroconversion was demonstrated in all the infected horses on Day 5 p.i. These clinical signs and virological findings were similar to those of horses infected naturally. The results indicate that Sagiyama virus has pathogenicity for horses and is similar to that of Getah virus.     

Getah virus as an equine pathogen.

Getah virus is a member of the genus Alphavirus in the family Togaviridae and has been frequently isolated from mosquitoes. Seroepizootiologic studies indicate that the virus is mosquito-borne and widespread, ranging from Eurasia to southeast and far eastern Asia, the Pacific islands, and Australasia. The natural host animal of the virus was not known until the first recognized occurrence of Getah virus infection among racehorses in two training centers in Japan in 1978. Outbreaks of clinical disease due to Getah virus infection occur infrequently, and only one outbreak has been reported outside Japan; this was in India in 1990. Clinical signs of the disease are mild and nonlife-threatening and are characterized by pyrexia, edema of the hind limbs, swelling of the submandibular lymph nodes, and urticarial rash, as reported in the 1978 epizootic. The morbidity was 37.9% (722 of 1903 horses) in one training center, with 96% of 722 affected horses making a full clinical recovery within a week without any significant sequelae. Antibodies against Getah virus were detected in 61.2% (172 of 281) and 55.8% (254 of 455) of horses at two training centers, respectively. Virus isolation can be attempted in VERO, RK-13, BHK-21, and many other cell lines as well as in suckling mouse brain. Blood plasma collected from suspect cases of infection at the onset of pyrexia is the specimen of choice. A diagnosis of Getah virus infection can also be confirmed serologically based on testing acute and convalescent phase sera by using SN, CF, HI, and ELISA tests. An inactivated vaccine is available for the prevention and control of Getah virus infection in horses in Japan.     

The Effect of Diethylaminoethyl Dextran and Agar Overlay pH on Plaque Formation by Two Plaque-size Variants of Foot-and-Mouth Disease Virus

The effect of diethylaminoethyl (DEAE) dextran and agar overlay medium pH on a small-plaque (SP) and large-plaque (LP) foot-and-mouth disease virus (FMDV), type A, strain 119 (A119) was studied. The SP virus was inhibited under normal agar overlay but the addition of 100, 1,000 and 2,000 µg DEAE dextran/ml of agar overlay permitted plaque development. By using untreated and DEAE dextran-treated agar overlay medium, plaque formation by the SP virus was enhanced when the pH of agar medium was raised to a more alkaline level before overlay. Plaques formed by the LP virus were relatively uninhibited under the regular overlay but were larger in the presence of 1,000 µg DEAE dextran/ml. The enhancement of LP virus plaques occurred at various pH levels and was also inversely related to the hydrogen ion concentration of agar overlays; regular and DEAE dextrantreated alkaline overlays produced larger plaques.     

猪乙型脑炎减毒活疫苗毒株的选育研究

以乙脑弱毒SA14－14－2作为母种,采用代地鼠肾细胞和乳鼠皮下传代方法并结合蚀斑纯化技术,选育获得乙脑减毒活疫苗猪用毒株SA14－14－2VS株。该毒株经细胞传到15－18代,脑内接种12－14g小鼠不引起发病和死亡;皮下接种10－12g小鼠,从脑组织中不能分离出病毒;经3－5日龄乳鼠回传一代后,脑内毒力Log LD50仅为1.32-2.10,皮下接种小鼠无致病力。该毒株细胞18代毒,用乳猪传至第五代,从乳猪血液、脑、肝组织中分离的病毒再经细胞传二代,其滴度与原毒液相近,并仍对小鼠脑内感染不致死;8头不公猪经该毒接种后,未发生睾丸炎,睾丸组织中未回收到病毒;经静脉接种的2头怀孕30d的初产母猪,蝇接种后2d和4d血样中检出病毒,但接种21d剖杀时,胎儿均健尖,胎盘、羊水及胎儿脑组织中均未回收到病毒,该毒株能使豚鼠和猪产生较强的免疫应答,对小白鼠攻击的保护力比灭活疫苗主;纯毒及外源因子污染 结果表明,该毒株是纯将的乙脑病毒,符合兽用生物制品种毒标准。     

新城疫Ⅰ系病毒不同空斑克隆株生物学测定试验

新城疫Ⅰ系种毒稀释接种鸡胚成纤维细胞(CEF)单层后覆盖含中性红的1.6%营养琼脂糖,37℃培养至72h,分别挑取大(φ2～3mm)、中(φ1～2mm)、小(φ1mm)三种空斑,反复冻融后收获病毒,同样方法空斑克隆三代后,通过SPF鸡胚继代增殖,对收获物进行ELD50、MDT、ICPI测定。结果表明,三种空斑克隆株继代培养物中,中斑毒力最强,大斑次之,小斑最弱;大斑克隆株增殖力最强,中斑次之,小斑最差。     

Isolation and characterization of attenuated plaque variants of infectious bursal disease virus

Attenuated plaque variants were obtained from infectious bursal disease virus adapted to chick embryo cell cultures. The large plaque (Lp) clone and the small plaque (Sp) clone formed homogeneous plaques about 5 and 1 mm in diameter, respectively. Neutralization tests indicated that these clones differed little from their parent strain in antigenicity. Sp clones showed a retarded growth rate in chick embryo cell cultures as compared with Lp clones. The clones were significantly less pathogenic for chick embryos than the parent strain, although Lp clones were more pathogenic than Sp clones, and they were much less pathogenic for 1-day-old chicks and 28-day-old chickens. Both clones had immunizing potency in 28-day-old chickens, although the Lp clone had a somewhat higher potency than the Sp clone. These findings suggest the Lp and Sp clones, in particular the Lp clones, to be useful as live virus vaccine strains.     

鸡大肠杆菌traT基因的扩增及TraT-DIG探针制备

以致病性Ｅ．ｃｏｌｉ的质粒为模板,用人工合成引物扩增出致病性相关的ｔｒａＴ基因核心部分;将菌株的致病性与ｔｒａＴ基因的扩增结果进行比较,强致病力菌株均扩增出ｔｒａＴ基因,无致病力菌株未扩增出ｔｒａＴ基因,约大多数中等致病力菌株亦扩增出ｔｒａＴ基因。扩增获得的ｔｒａＴ基因经纯化后标记地高辛（Ｄｉｇｏｘｉｎ,ＤＩＧ）研制出ｔｒａＴ－ＤＩＧ基因探针,该探针与鸡沙门氏菌、鸡白沙门氏菌、副伤寒沙门氏菌不发生     

驯化克隆的CAV-2大斑株的实验免疫研究

对MDCK细胞上驯化克隆产毒量高的大斑株 (LP)的第 5 0代毒和 75代毒进行了实验免疫研究 ,结果表明该毒株具有安全性好 ,通过静脉和口鼻接种CAV易感犬 ,无任何致病反应 ;用10 4 .0 TCID50 以上的该毒免疫CAV易感犬 ,即获得 97.5 %以上的免疫保护 ;将其在CAV易感犬连续传 5代 ,其安全性与原毒相同 ,表明该毒株在 5 0～ 75代之间遗传性稳定 ,免疫原性良好 ;与CPV和CDV弱毒联合免疫 ,结果与各弱毒单独免疫所产生的抗体无差异 ,说明该毒株不但可以单独用于免疫 ,也可与其他弱毒联合免疫。是一株较好的疫苗候选株 ,具有开发应用价值。     

Sensitivity of Suckling Mice to Various Strains of Infectious Bronchitis Virus

Suckling mice are serviceable in many virological experiments because they are sensitive to various virus species. Simpson & Groupé (2) succeeded in adapting the Beaudette strain of infectious bronchitis virus (IBV) to suckling mice, using the intracerebral route of inoculation. A study of the properties of a Finnish strain of infectious bronchitis virus (IBV_F) has been published recently (1). It successfully established the intracerebral adaptation of IBV_F to suckling mice. The suckling mice were 100 % sensitive to this virus strain up to an age of 12 days. Symptoms of the central nervous system with a typical clearly discernible gibbosity in the vertebral column were elicited after an incubation period of 2—3 days, and the animals died 3—4 days after the inoculation. Parallelly with IBV_F, transmission experiments with other IBV strains were carried out. The present paper is a report on the results of these experiments.     

Clinical and virological observations on swine experimentally infected with Getah virus

The pathogenicity of Getah virus for swine was examined. All 8 pigs (4 adults and 4 piglets) inoculated with Strains MIP-99 and MI-110 developed pyrexia ranging from 39.4 to 40.7 degrees C and anorexia. Mild depression and diarrhea were observed in 2 of the 4 piglets. These clinical signs were transient. Viremia occurred 1-2 days post-inoculation (p.i.) and the maximum titer was 10(3.0) TCID50 0.1 ml-1. The virus was recovered from a piglet autopsied on Day 3 p.i. from spleen and various lymph nodes. The maximum titer of virus (10(3.75) TCID50 0.1 g-1) was detected in the inguinal lymph node. Seroconversion was demonstrated in all the pigs on Day 6 p.i. These results suggest that Getah virus is mildly pathogenic for swine, which may play a role as an amplifying host in nature.     

Isolations of Cache Valley virus in Texas, 1981

Two strains of the same virus (isolates AR 168 and 7856), were isolated in 1981 from an apparently healthy cow and a sick sheep in TX, U.S.A. These isolates were shown to be members of the Bunyamwera serogroup (family Bunyaviridae, genus Bunyavirus) by complement-fixation tests. Serum dilution-plaque reduction neutralization test results indicated that the isolates are closely related to Cache Valley virus. The virus isolates were characterized by sensitivity to lipid solvent, size (50-100 nm by filtration and 70 nm by electron microscopy), heat (56 degrees C) and pH 3 lability, cytopathic effects or plaques in cultures of Vero, LLC-MK2, embryonic bovine testicle and PS cells, and pathogenicity for suckling and weaned mice by the intracranial but not the intraperitoneal route. Gnotobiotic and conventional sheep and goats were experimentally infected by inoculation with one of the isolates given either intravenously or intraperitoneally. Elevation of body temperature, depression, tremors, muscle spasms, disorientation, feeding anomalies, convulsions, or other signs of central nervous system disturbances were observed.     

Selection of an infectious bursal disease virus mutant with increased immunogenicity following passage under humoral immune pressure.

It is generally known that the pathogenicity of infectious bursal disease virus (IBDV) strains decreases following passage in cell culture. However, there is no information about the effect of passage under immune pressure on the phenotypic and molecular properties of IBDV. In the present study, a small plaque mutant virus with poor neutralization capability, but showing similar growth characteristics as the parental virus strain, QC2, was isolated after serial passage in Vero cells in presence of IBDV serotype 1 chicken polyclonal antiserum. This mutant virus showed reduced pathogenicity but enhanced immunogenicity compared to the parental virus. Sequence analysis of the non-coding regions of the genome revealed 4 and 3 nucleotide changes in the 3' non-coding regions of segments A and B, respectively, and none in the 5' non-coding regions. Restriction enzyme analysis of selected coding regions of the IBDV genome in both viruses revealed a loss of the PstI site in the VP2 region of the mutant virus. Selection of such mutant viruses by passaging under immune pressure may offer an improved method for developing safer and more effective attenuated vaccine strains against infectious bursal disease of chickens.     

Avirulent ts and thymidine kinase-deficient mutant of Aujeszky's disease virus.

The temperature-sensitive (ts), thymidine kinase-deficient (TK-) mutant designated ZHtsTK- strain, of Aujeszky's disease virus (ADV) was isolated from a virulent strain with the treatments using 5-bromodeoxyuridine and arabinosylthymine. The ZHtsTK- strain was easily distinguished from the other virulent ADV strains by plaque size on HmLu-1 and chicken embryo fibroblast cells and by restriction endonuclease analyses using Bam HI, Sal I and Kpn I. The ZHtsTK- strain was avirulent for mice, guinea pigs and rabbits, and produced neutralizing antibodies to ADV in these animals. The rabbits inoculated with the ZHtsTK- strain did not shed detectable amounts of virus after dexamethasone treatment. The ZHtsTK- strain was also avirulent for 5-day-old piglets and did not cause disease. No virus was detected from the piglets inoculated intramuscularly in the nasal swabs or the tissues examined on postinoculation day 9. These findings presented here suggested that there is a significant correlation between pathogenicity and properties such as ts and TK-, and the combination of ts and TK- properties plays a much larger role in reducing virulence for animals.     

采用蚀斑技术克隆纯化口蹄疫病毒的研究

本研究应用BHK₂₁细胞通过蚀斑试验克隆和再次克隆牛AsiaⅠ型口蹄疫病毒AKT03毒株并对克隆前和第1次、第2次克隆的口蹄疫病毒毒株的细胞半数感染量（TCID₅₀）、乳鼠半数致死量（LD₅₀）进行比较测定,结果表明,通过蚀斑试验成功克隆了牛AsiaⅠ型口蹄疫病毒AKT03毒株,第1次克隆的毒株和第2次克隆的毒株TCID₅₀和LD₅₀效价明显提高。     

African swine fever: microplaque assay by an immunoperoxidase method.

A microplaque assay for Vero cell-adapted Lisborn '60 strain of African swine fever virus (L'60-uncloned) and a large plaque-forming strain cloned from the L'60-uncloned strain was developed by an immunoperoxidase method. The immunoperoxidase method can be used to stain microplaques of 3 days after inoculation, whereas the conventional plaque assay requires 5 to 7 days to develop visible plaques. A linear relationship between viral concentration in the inoculum and plaque numbers was observed. Viral titers obtained by both microplaque assay and conventional plaque assay were comparable, and both methods were reproducible and reliable. The viral titer obtained by either one of the plaque assay methods was approximately 0.9 log10 lower than that obtained by the hemadsorption test.     

Physical, Chemical and Biological Characteristics of Two Bovine Enterovirus Plaque Variants

Large plaque (4LP) and small plaque (4SP) variants were derived from a parent bovine virus strain by serial plaque passage. Both 4LP and 4SP were resistant to chloroform and stabilized at 50°C for one hour by 1.0 M magnesium chloride. Both 4LP and 4SP had buoyant densities in cesium chloride of 1.36 gm/ml. Antigenically, 4LP and 4SP were reciprocally cross neutralizable.

The nucleic acid of 4LP was shown to be ribonucleic acid (RNA) by resistance of its infectivity to deoxynuclease (DNase) but not ribonuclease (RNase) and by increased incorporation of [³H]-uridine into cytoplasmic RNA in cells of virus infected cultures. In growth characteristics, both 4LP and 4SP had maximum adsorption times of 75 to 90 minutes but 4LP had more rapid replication and release rates and yielded nearly twice as many infectious units per cell as 4SP. The differences in growth properties correlated directly with the differential in plaque diameter which was 40-50%.