Report of isolations of unusual lyssaviruses (rabies and Mokola virus) identified retrospectively from Zimbabwe

Rabies isolates that had been stored between 1983 and 1997 were examined with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies. Out of 56 isolates from cats and various wild carnivore species, 1 isolate of Mokola virus and 5 other non-typical rabies viruses were identified. The Mokola virus isolate was diagnosed as rabies in 1993 from a cat. Genetic analysis of this isolate suggests that it falls in a distinct subgroup of the Mokola virus genotype. The 5 non-typical rabies viruses were isolated from honey badgers (Mellivora capensis), African civets (Civettictis civetta) and an unidentified mongoose (Herpestidae). These isolates are representatives of rarely-reported wildlife-associated strains of rabies, probably maintained by the slender mongoose (Galerella sanguinea). These findings indicate that both Mokola virus and the mongoose-associated variant may be more common in Zimbabwe than is apparent from routine surveillance.     

New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: comparative efficacy

Attempts were made, through selection of optimum viral strains, to develop improved vaccines against Marek's disease (MD). Seven attenuated serotype 1 strains and 22 avirulent serotype 2 strains, both alone and in combination with the FC126 strain of serotype 3, were screened for protective efficacy against challenge with virulent and very virulent MD viral strains. The three viruses selected as most promising were evaluated alone and in various combinations and compared with commercially available vaccines, including FC126, bivalent (FC126 + SB-1), and CV1988/C, in 12 separate assays. Two of these new viruses--301B/1 (serotype 2) and Md11/75C/R2 (serotype 1)--were exceptionally protective compared with prototype vaccine strains. Four new monovalent and polyvalent vaccines based on these two isolates protected chickens better than FC126 alone or CV1988/C alone. Three of these new vaccines provided better protection than the bivalent (FC126 + SB-1) vaccine. Protective synergism was noted commonly between viruses of serotypes 2 and 3 but only sporadically between serotypes 1 and 2 or between serotypes 1 and 3. Strain CVI988/C was protective but was no better than FC126 alone, and it was less effective than bivalent (FC126 + SB-1) vaccine, even when used as a bivalent vaccine with FC126 or SB-1.     

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.     

Restriction endonuclease analysis of Marek's disease virus DNA: differentiation of viral strains and determination of passage history.

The restriction endonuclease (RE) patterns of DNA from serotype 1 Marek's disease viruses (MDVs) are unique to serotype 1 viruses and can also be used to differentiate between low and high cell-culture-passaged viruses. We compared the RE patterns of DNA from seven serotype 2 and 3 MDVs before and after serial in vitro passage. Passage of four serotype 2 strains resulted in a variety of changes in the RE pattern. Individual strains within a serotype exhibited unique restriction patterns that allowed individual isolates to be differentiated. In a similar manner, the serotype 3 virus strains displayed RE pattern variations that were unique to each strain, as well as differences between low and high cell-culture passage. Our findings, together with earlier reports, suggest that the RE patterns of MDV DNA provide a simple and accurate method to: 1) differentiate between the three MDV serotypes, 2) differentiate between virus strains within a serotype, and 3) determine whether the viruses have been passaged extensively in cell culture.     

The rabies viruses of bats

In the 1930s rabies was shown to affect blood-, insect- and fruit-eating bats. We have prepared anti-nucleocapsid monoclonal antibodies (MAbs) using Mokola and bat (Lagos, Duvenhage and Denmark) rabies viruses as immunogens. With these MAbs we have examined rabies viruses from vampire, insectivorous and frugivorous bats from the Americas, Africa, Europe and the Soviet Union and have compared them with isolates from terrestrial species including man. As well as confirming the findings of others with viruses of African and American bat origin, the results revealed the presence of a second biotype in European bats and demonstrated the presence of serotype 1 as well as serotype 4 viruses in bats of the Soviet Union.     

Identification of optimal regions for phylogenetic studies on VP1 gene of foot-and-mouth disease virus: analysis of types A and O Argentinean viruses

An analysis of the informative content of sequence stretches on the foot-and-mouth disease virus (FMDV) VPI gene was applied to two important viral serotypes: A and O. Several sequence regions were identified to allow the reconstruction of phylogenetic trees equivalent to those derived from the whole VPI gene. The optimal informative regions for sequence windows of 150 to 250 nt were predicted between positions 250 and 550 of the gene. The sequences spanning the 250 nt of the 3' end (positions 400 to 650), extensively used for FMDV phylogenetic analyses, showed a lower informative content. In spite of this, the use of sequences from this region allowed the derivation of phylogenetic trees for type A and type O FMDVs which showed topologies similar to those previously reported for the whole VP1 gene. When the sequences determined for viruses isolated in Argentina, between 1990 and 1993, were included in these analyses, the results obtained revealed features of the circulation of type A and type O viruses in the field, in the months that preceded the eradication of the disease in this country. Type A viruses were closely related to an Argentinean vaccine strain, and defined an independent cluster within this serotype. Among the type O viruses analysed, two groups were distinguished; one was closely related to the South American vaccine strains, while the other was grouped with viruses of the O3 subtype. In addition, a detailed phylogeny for type A FMDV is presented.     

Control of rabies: epidemiology of rabies in Asia and development of new-generation vaccines for rabies

Rabies is an enzootic viral disease widespread throughout the world. Although it is a vaccine-preventable disease, the annual number of human deaths caused by rabies is estimated to be 32,000 in Asia. Phylogenetic analysis based on sequence data of the partial N gene of rabies viruses in Asia has shown that the viruses are divided into five genogroups, distributed in Middle East, South Asia, South East Asia, Malay, and Arctic regions. The genetic relationships among these rabies viruses agree basically with the results of previous studies. Meanwhile, new types of vaccines are being developed by applying gene manipulation techniques to rabies virus in order to overcome the disadvantages of current vaccines. This article reviews the molecular epidemiology of rabies in Asia and progress made in the development of new-generation rabies vaccines with the goal of elimination or control of rabies in Asia.     

Nucleic acid and structural proteins of infectious bursal disease virus isolates belonging to serotypes I and II

The nucleic acid and structural proteins of infectious bursal disease virus serotype I (six isolates) were compared with those of serotype II (two isolates). Five of the serotype I isolates originated from chickens, whereas both serotype II isolates were from turkeys. The growth curves of representative isolates of both serotypes were similar, but the latent period and virus yield were different. The seven isolates tested had two segments of double-stranded genomic RNA. The RNA migration patterns of viruses belonging to each serotype were similar, but differences were noticed between the two serotypes. There were differences in the molecular weights of viral proteins (VP) 3 and 4 from the two serotypes, and serotype II isolates lacked VP-2.     

In ovo vaccination of specific-pathogen-free chickens with vaccines containing multiple agents.

We used in ovo technology to protect chickens against multiple diseases by inoculating vaccines containing mixtures of live viral agents. A single in ovo injection of a vaccine containing serotypes 1, 2, and 3 of Marek's disease virus (MDV), a vaccine strain of serotype 1 infectious bursal disease virus (IBDV), and recombinant fowl pox vaccine with HN and F genes of Newcastle disease virus (rFP-NDV) induced protection against virulent MDV, IBDV, Newcastle disease virus, and fowl poxvirus. The multiple-agent vaccine induced specific antibodies against the viral agents present in the mixture and did not adversely affect the survival of hatched chickens. Inoculation of a vaccine containing serotypes 1, 2, and 3 of MDV and IBDV did not affect hatchability of eggs, although the addition of rFP-NDV to the mixture reduced hatchability by 23%-26%. In ovo vaccination with a vaccine containing MDV and IBDV vaccine viruses did not exacerbate the inhibitory effect of individual viral agents on humoral and cellular immune competence.     

Immunogenicity of infectious bursal disease viruses in chickens.

Cross-protective properties of infectious bursal disease viruses (IBDVs) were studied. Viruses represented different subtypes of serotype 1, including recently isolated viruses (variants), and a serotype 2 virus. Chickens were vaccinated at 3 weeks of age with inactivated vaccines containing 10(5), 10(6), 10(7), or 10(8) mean tissue-culture infectious dose of a given virus and challenged 2 weeks later using either 10(2) or 10(3.5) mean embryo infectious dose (EID50) of either a standard virus or a variant serotype 1 virus. Protection was evaluated at 5 and 10 days post-challenge, based on gross and microscopic lesions, body weight, and bursa/body-weight ratios. The serotype 2 virus did not confer protection on birds challenged with the serotype 1 viruses. Vaccines made of variant viruses at the low doses protected chickens challenged with the high or low doses of either the standard or the variant viruses. Vaccines made of the standard or variant strains at low doses protected against high or low challenge doses of the standard strain. Vaccines made of the high dose of any of the standard strains protected chickens against the variant virus when the low challenge dose (10(2) EID50) was used, but not when the high challenge dose (10(3.5) EID50) was used. The lowest dose of the standard viruses vaccines required to confer protection against the variant virus varied depending on the strain. Results indicated that protection depended on the strain and dose of both the vaccine and challenge viruses and that the variant strains and standard strains share a common protective antigen(s).     

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

两株狂犬病病毒强毒株糖蛋白基因的克隆及潜在抗原表位分析

通过RT-PCR分别获得了狂犬病病毒强毒CVS株、DRV82株糖蛋白基因,进行克隆及测序,并推导出氨基酸序列,与犬用疫苗弱毒株ERA、SRV9、犬源性街毒株CGX及人用疫苗株PG的糖蛋白序列进行比较。结果表明,以上狂犬病病毒毒株间的核苷酸同源性为83.1%～99.2%,氨基酸序列同源性为87.0%～98.5%。经Jameson-Wolf抗原表位优势图分析,CVS株与其他各株相比发现在304位、372位抗原表位优势升高;而DRV82株与其它各株差异不明显。抗原优势变化可能导致狂犬病病毒糖蛋白出现新的潜在抗原位点,为下一步构建不同毒株的狂犬病病毒糖蛋白重组疫苗奠定了基础。     

Detection and differentiation of bovine group A rotavirus serotypes using polymerase chain reaction-generated probes to the VP7 gene.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.     

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.     

吉林鹿源狂犬病病毒野毒株磷蛋白基因的测序及分析比较

通过对狂犬病病毒鹿源野毒株(DRV)总RNA的提取,用RT-PCR方法扩增磷(N S)蛋白基因并与T载体连接、克隆,将其转化到大肠杆菌JM109细胞中,测定N S蛋白基因的序列,与其他已经发表的国际标准株、疫苗株等进行比较,分析其氨基酸的同源性和N S蛋白的分子生物学功能,为狂犬病病毒野毒株全基因序列的测定奠定基础。     

Foot-and-mouth disease: past,present and future

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals including cattle, pigs, sheep and many wildlife species. It can cause enormous economic losses when incursions occur into countries which are normally disease free. In addition, it has long-term effects within countries where the disease is endemic due to reduced animal productivity and the restrictions on international trade in animal products. The disease is caused by infection with foot-and-mouth disease virus (FMDV), a picornavirus. Seven different serotypes (and numerous variants) of FMDV have been identified. Some serotypes have a restricted geographical distribution, e.g. Asia-1, whereas others, notably serotype O, occur in many different regions. There is no cross-protection between serotypes and sometimes protection conferred by vaccines even of the same serotype can be limited. Thus it is important to characterize the viruses that are circulating if vaccination is being used for disease control. This review describes current methods for the detection and characterization of FMDVs. Sequence information is increasingly being used for identifying the source of outbreaks. In addition such information can be used to understand antigenic change within virus strains. The challenges and opportunities for improving the control of the disease within endemic settings, with a focus on Eurasia, are discussed, including the role of the FAO/EuFMD/OIE Progressive Control Pathway. Better control of the disease in endemic areas reduces the risk of incursions into disease-free regions.     

Complete nucleotide sequences of S1 and N genes of infectious bronchitis virus isolated in Japan and Taiwan

The complete nucleotide sequences of the S1 and N genes of three Japanese and one Taiwanese field strains of IBV are reported. These Japanese strains were found to have S1 sequences most similar to those of Australian strains and N sequences most similar to those of North American strains. This result suggested that these Japanese strains might all be recombinant viruses derived from recombination of Australia- and North America-related viruses. Moreover, the S1 proteins of all these Japanese and Taiwanese strains exhibit only a limited sequence homology to strains of Massachusetts and Connecticut serotypes that have been commonly used as vaccine strains. This result high lightens the importance of development of vaccines based on the local strains of IBV.     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.