帕利亚姆血清群病毒一步法RT-PCR检测技术的建立

帕利亚姆血清群病毒(PALV)在我国广泛流行,给我国牛羊养殖业健康发展构成了严重威胁,目前国内尚无PALV核酸检测方法的报道。本研究根据流行于我国的中山病毒(CHUV)、Bunyip Creek virus(BCV)和D’Aguilar virus(DAV)3种血清型PALV的Seg-7保守基因序列,设计针对PALV的群特异性RT-PCR扩增引物,通过反应条件优化,建立了PALV的一步法RT-PCR检测方法,其最佳引物浓度为0.8μmol/L;最佳退火温度为56℃。对该方法进行特异性试验,结果显示该方法可特异性的检测出CHUV、BCV和DAV核酸,而对环状病毒属的蓝舌病病毒、流行性出血病病毒、非洲马瘟病毒和广西环状病毒的检测结果均为阴性;敏感性试验结果显示,该方法对RNA标准品的最低检测限为6.9×10~1拷贝/μL。利用该方法检测血液样品,结果显示该方法检测结果与病毒分离结果基本一致。本研究建立的PALV一步法RT-PCR方法具有特异性强、敏感度高等优点,为开展PALV感染疫病诊断与流行病学研究提供了技术保障。     

Expression and antigenic characterization of the major core protein VP7 of Chuzan virus, a member of the Palyam serogroup orbiviruses

The Palyam serogroup-specific antigen, VP7, of Chuzan virus strain K-47 was expressed in insect cells by a recombinant baculovirus. The expressed protein appeared as a single band of 38kDa corresponding to the predicted molecular mass of Chuzan virus VP7 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In immunoprecipitation analysis, the recombinant VP7 was not only recognized by all polyclonal antibodies against the Palyam serogroup viruses (PALV) tested in this study, but also by antisera to bluetongue virus (BTV) serotype 1, epizootic haemorrhagic disease virus (EHDV) serotypes 1 and 2. However, in Western immunoblot assay, no positive signals were observed between this protein and these antisera, even in the homologous reaction using antiserum to Chuzan virus. These findings demonstrate that the common antigenic determinants on the VP7 proteins of Chuzan virus and the other PALV serotypes are mainly conformational and that the proteins share some epitopes with those of BTV and EHDV beyond the serogroup. No cross-reactivities were detected between Chuzan virus VP7 and antisera to BTV and EHDV in agar gel immunodiffusion (AGID) and indirect ELISA tests, indicating that the recombinant VP7 is useful as a diagnostic reagent for serological tests of congenital abnormalities of cattle caused by PALV.     

Serologic evidence for the etiologic role of Chuzan virus in an epizootic of congenital abnormalities with hydranencephaly-cerebellar hypoplasia syndrome of calves in Japan

An epizootic of congenital abnormalities of calves was observed in the Kyushu district of Japan from November 1985 through April 1986. The main clinical signs of the disease were impairment of mobility and signs of impairment of the nervous system. Opisthotonos was pronounced, and almost all calves were unable to suckle by themselves. The main macroscopic pathologic changes were hydranencephaly and cerebellar hypoplasia. Although an etiologic agent was not isolated from the calves, serotest results of precolostral serum samples indicated that 128 of 139 (92%) abnormal calves had antibody for Chuzan virus, a new virus belonging to the Palyam subgroup of the Orbivirus genus; 34 healthy calves in the epizootiologic area did not have antibody for the virus. The presence of Chuzan virus in Kyushu in 1985 was confirmed serologically.     

A monoclonal antibody blocking ELISA detects antibodies specific for epizootic haemorrhagic disease virus.

The isolation of a monoclonal antibody (1G9/C9) with specificity for the epizootic haemorrhagic disease (EHD) serogroup has enabled the development of a highly sensitive and specific blocking ELISA (B-ELISA) for the detection of serum antibodies to EHD viruses. The assay was sensitive to blocking antibodies present in hyperimmune reference antisera to all six EHD serotypes tested but was unaffected by reference antisera to 19 South African and eight Australian serotypes of the related orbivirus bluetongue virus (BTV). The sensitivity of the EHD B-ELISA exceeded that of an indirect ELISA (I-ELISA) for EHD-specific antibody detection. Serum antibody titres to BTV and EHD in experimental and field sera, including a sentinel herd from which virus isolations were made, were examined in both the BTV and EHD B-ELISA tests. These results showed the B-ELISA was only sensitive to antibodies specific for the homologous serogroup in each case, even where sequential and mixed infections with each virus type occurred.     

The orbivirus genus. Diversity, structure, replication and phylogenetic relationships

The general properties of the orbiviruses have been examined at the physical, structural and molecular level. At the structural level, the orbiviruses (with the exception of the Kemerovo serogroup) appear similar. The replicative events are also similar, however differences in the ultrastructure of virus-specific structures and their association with components of the host cell have been observed. Further research in this area may be used to differentiate between the serogroups and even some serotypes, of orbiviruses. At the molecular level the properties of the genome can be used to determine relationships between members of the orbivirus genus. These relationships are revealed using a variety of techniques including serology and gene sequence analysis. Not only are the different serological responses to gene products present in the mature virus particle used for differential diagnosis, but the gene sequences themselves can also be utilized. Understanding of the relationships between these viruses is progressing to the point that insights into orbivirus molecular epidemiology is now possible.     

Epizootics of sudden death in tammar wallabies (Macropus eugenii) associated with an orbivirus infection

Epizootics of sudden death in tammar wallabies (Macropus eugenii) occurred at six research facilities and zoological gardens in New South Wales, Australia, in late 1998 and at one Queensland research facility in March 1999. There were 120 confirmed tammar wallaby deaths during this period; however, population censuses indicated that up to 230 tammar wallabies may have died. The majority of animals died without premonitory signs. A small proportion of wallabies exhibited increased respiratory rate, sat with a lowered head shortly before death or were discovered in lateral recumbency, moribund and with muscle fasciculations. Gross postmortem findings consistently included massive pulmonary congestion, mottled hepatic parenchyma and subcutaneous oedema throughout the hindlimbs and inguinal region. Approximately 30% of the animals examined also had extensive haemorrhage within the fascial planes and skeletal muscle of the hindlimb adductors, inguinal region, ventral thorax, dorsal cervical region and perirenal retroperitoneal area. The tissues of affected animals became autolytic within a short period after death. Bacteriological examination of tissues from 14 animals did not provide any significant findings. Toxicological examination of the gastric and colonic contents of four animals did not reveal evidence of brodifacoume or other rodenticides. Viruses from the Eubenangee serogroup of the Orbivirus genus were isolated from the cerebral cortex of nine, and the myocardium of two, tammar wallabies and the liver and intestine of another tammar wallaby. A similar orbivirus was also isolated from the cerebrospinal fluid of another tammar wallaby that died suddenly. The disease agent appears to be a previously unrecognised orbivirus in the Eubenangee serogroup. This is the first report of epizootics of sudden deaths in tammar wallabies apparently associated with an orbivirus infection.     

Isolation of Akabane virus from the biting midge Culicoides oxystoma in Japan

Akabane virus was isolated from the biting midge, Culicoides oxystoma, collected in a cowshed in Kagoshima on Kyushu Island of Japan. This is the first report on the isolation of Akabane virus from biting midges of the genus Culicoides in Japan. Two calves kept as bait in the cowshed seroconverted to Akabane virus. These results strongly suggest that C. oxystoma may be a vector of Akabane virus.     

Investigations of bluetongue and other arboviruses in the blood and semen of naturally infected bulls

Small groups of bulls were exposed to natural infection with arboviruses. The bulls were bled and ejaculated regularly and the blood and semen were processed for virus isolation. Over a 5-year observation period, virus isolation and serology indicated that the 29 exposed bulls had experienced 79 viraemic episodes with the viruses of the bluetongue, epizootic haemorrhagic disease, Palyam and Simbu serogroups and an incompletely characterised rhabdovirus. In no instance was there unequivocal evidence of bluetongue virus contamination of semen, despite 18 infections in the study period.     

Genomic characterization and distribution of bovine foamy virus in Japan

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.     

Isolation of Chuzan virus, a new member of the Palyam subgroup of the genus Orbivirus, from cattle and Culicoides oxystoma in Japan

Five virus strains with identical antigenic properties were isolated from 3 RBC suspensions obtained from 2 healthy sentinel calves and from 2 pools of Culicoides oxystoma in cultures of a hamster lung cell line (HmLu-1). The virus was tentatively named Chuzan virus. The Chuzan virus was classified as a new member of the Palyam subgroup of the genus Orbivirus on the basis of its physicochemical, morphologic, and antigenic properties.     

Encephalitis of cattle caused by Iriki isolate, a new strain belonging to Akabane virus

A disease characterized by nervous signs was found in 10 calves in two districts in Kagoshima Prefecture, Japan, from October to November, 1984. Histopathological changes of nonpurulent encephalitis were found in every case. An agent, named Iriki isolate, was isolated from the cerebellum of a calf in HmLu-1 cell cultures. All of the affected calves possessed neutralizing antibody to the virus. A high seropositive rate to the virus in cohabiting cattle and cattle kept in the epizootic area, and seroconversion to the virus in 1984, were disclosed. Experimental infection of calves with Iriki isolate produced severe nervous signs and histopathological changes similar to those of the natural infection. These seroepidemiological findings and animal experiments established that Iriki isolate is the causative agent of the disease. Iriki isolate was considered as a variant of Akabane virus since the virus showed cross reaction with Akabane virus in virus neutralization tests.     

The use of chicken IgY in a double antibody sandwich ELISA for detecting African horsesickness virus.

An indirect sandwich ELISA that can detect as little as 8 ng of African horsesickness virus (AHSV) was developed. Viral antigen was captured from suspension using an immobilized monoclonal antibody specific for an epitope on VP7, a protein that is a major constituent of the virus core. Egg-yolk derived chicken IgY directed against AHSV (serotype 3) was used as the secondary antibody. Since IgY and mouse IgG do not cross-react serologically, the secondary antibody was not labelled, but was instead detected with enzyme-coupled sheep antibodies directed against avian immunoglobulins. The assay recognized all nine AHSV serotypes, but not the Cascara isolate of equine encephalosis virus, a related orbivirus that also infects horses. In addition to being able to detect and quantify whole AHSV, the ELISA could show the presence of VP7 produced by recombinant baculoviruses.     

Isolation and characterisation of an H3N8 equine influenza virus in Australia, 2007

Before 2007, equine influenza had never been diagnosed in Australia. On 22 August 2007, infection was confirmed in horses at Eastern Creek Animal Quarantine Station near Sydney. The virus subsequently isolated (A/equine/Sydney/2888-8/2007) was confirmed by sequence analysis of the haemagglutinin (HA) gene as an H3 virus of the variant American Florida lineage that is now referred to as Clade 1. The HA sequence of the virus was identical to that of a virus isolated from a contemporaneous outbreak in Japan and showed high homology to viruses circulating in North America.     

In vivo and in vitro propagation and transmission of Toggenburg orbivirus

The Toggenburg orbivirus (TOV), a recently discovered virus related to bluetongue virus (BTV), has been identified in goats in Switzerland, Italy and Germany. Isolation of TOV in vitro has not yet been achieved and the transmission mechanisms are still unknown. In the experimental infection of pregnant goats described here, TOV could not be detected in secretion/excretion samples or fetal blood. Material from the goat experiment was used as inoculum for propagating the virus in vitro. To enhance the infectivity of TOV several modified protocols, e.g. pretreatment of the virus with trypsin, polyethylene glycol-mediated infection and lipofection were applied. Isolation of TOV, attempts to infect Culicoides nubeculosus by feeding TOV-positive blood and intracerebral inoculation of newborn mice were unsuccessful. The results of these studies suggest that TOV requires specific but different factors than other BTVs for infection and replication outside of its natural caprine host.     

In vivo and in vitro propagation and transmission of Toggenburg orbivirus

The Toggenburg orbivirus (TOV), a recently discovered virus related to bluetongue virus (BTV), has been identified in goats in Switzerland, Italy and Germany. Isolation of TOV in vitro has not yet been achieved and the transmission mechanisms are still unknown. In the experimental infection of pregnant goats described here, TOV could not be detected in secretion/excretion samples or fetal blood. Material from the goat experiment was used as inoculum for propagating the virus in vitro. To enhance the infectivity of TOV several modified protocols, e.g. pretreatment of the virus with trypsin, polyethylene glycol-mediated infection and lipofection were applied. Isolation of TOV, attempts to infect Culicoides nubeculosus by feeding TOV-positive blood and intracerebral inoculation of newborn mice were unsuccessful. The results of these studies suggest that TOV requires specific but different factors than other BTVs for infection and replication outside of its natural caprine host.     

Viruses isolated from Culicoides midges in South Africa during unsuccessful attempts to isolate bovine ephemeral fever virus

Five viruses, unrelated to bovine ephemeral fever virus (BEFV), were isolated from Culicoides biting-midges collected during the summer months of the years 1968-69 and 1969-70 near a cattle herd in which cases of BEF occurred and at an open horse stable at Onderstepoort. These viruses were investigated by means of serological, electron-microscopical and physicochemical tests. It was established that 2 isolates, Cul. 1/69 and Cul. 2/69, were related to each other and belonged to the Palyam subgroup of the genus Orbivirus, that isolate Cul. 3/69 belonged to the equine encephalosis subgroup of the genus Orbivirus, while Cul. 1/70 was related to Akabane virus, which belongs to the Simbu subgroup of the family Bunyaviridae. One isolate, Cul. 5/69, though prevalent in the cattle population, could not be identified at this point. A brief serological survey indicated that the cattle in the nearby herd possessed antibodies against all the isolates except Cul. 3/69. BEFV could not be isolated in mice or in cultured cells from the wild-caught Culicoides.     

A case of stranded Indo-Pacific bottlenose dolphin (Tursiops aduncus) with lobomycosis-like skin lesions in Kinko-wan,Kagoshima, Japan

Lobomycosis is a chronic fungal disease caused by the etiologic agent, Lacazia loboi, in the skin and subcutaneous tissues in humans and dolphins in tropical and transitional tropical climates. An Indo-Pacific bottlenose dolphin (Tursiops aduncus) stranded in Kagoshima, Japan, had severe skin lesions characterized by granulomatous reactions and hyperkeratosis that were similar to those of the lobomycosis, but no fungal organism was observed in the skin lesion. In this paper, we report a stranded Indo-Pacific bottlenose dolphin with lobomycosis-like lesions based on pathological examinations in Japan.     

Arboviruses recovered from sentinel cattle using several virus isolation methods

A group of 20 sentinel steers was bled weekly for 5 months in 1986 and the blood samples were examined for arboviruses by inoculation firstly into embryonated chicken eggs (ECE), baby mice, Aedes albopictus cells and BHK21 monolayers. A second group of cattle was similarly examined for virus in 1987, except that baby mice were not used. Viruses were recovered from 26% of the 878 weekly bleeds. The viruses identified consisted of 14 types belonging to the bluetongue, epizootic haemorrhagic disease (EHD), Palyam and Simbu groups with a single isolation of bovine ephemeral fever virus. The ECE system was found to be the best for isolating bluetongue and Simbu viruses, though the eggs were not usually killed by the inoculum. The ECE and A. albopictus systems were equally sensitive for recovering EHD viruses, while Palyam group viruses were most efficiently isolated in BHK21 monolayers.     

A sentinel herd system for the study of arbovirus infections in Australia and Papua-New Guinea

The establishment, and development between 1969 and 1978, of a system of sentinel cattle in herds located in many areas of Australia and in Papua New Guinea is described. Though the system was established for the study of the epidemiology of a variety of viruses infecting cattle, the study has been limited since 1974 to arboviruses. By means of serology, it was established that bovine ephemeral fever virus was present in Australia in subclinical form between major epidemics but was not detected in Papua-New Guinea. The development of antibody to bovine ephemeral fever virus in individual cattle before they developed clinical signs in epidemics was clearly demonstrated. The sentinel technique was used to demonstrate that subclinical Akabane virus infection in cattle occurred at the time that virus was present in its suspected vector,Culicoides brevitarsis which had been collected nearby. The epidemiology of other Simbu group viruses, D'Aguilar virus, and bluetongue virus, (serotype 20) was also studied. A limited programme of arbovirus isolation in tissue cultures produced 0.8% of isolates from 2090 of the blood clots which accompanied sentinel herd serum samples.The most valuable aspect of the sentinel herd scheme has been the accumulation of a well documented representative set of serum samples for retrospective serology by the use of newly isolated or imported antigens.