Ingwavuma virus (Simbu group) from Culex and Mansonia mosquitoes (Diptera: Culicidae) in Indonesia.
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Two Australian members of the Simbu group, Douglas and Tinaroo viruses, were found to be distinct, by virus-neutralization tests, from three previously known Simbu group viruses isolated in Australia, namely Akabane, Aino and Peaton viruses. A low-titre, two-way, cross-reaction was noted between Akabane and Tinaroo viruses. Antibody to Tinaroo and Douglas viruses was detected in serum from cattle, buffalo, sheep, goats and deer but not in humans, pigs, kangaroos and wallabies. The results for horses were inconclusive. The distribution of antibodies to each virus falls mainly within the geographical distribution of the biting midge Culicoides brevitarsis, an insect from which each virus has been isolated.
The antigenicity and RNA genome structures of five Simbu serogroup bunyaviruses isolated in Japan and Australia were analyzed using monoclonal antibodies (Mabs) raised to Akabane (AKA) virus and oligonucleotide fingerprinting. The virion surface glycoprotein (G1) and the nucleocapsid (N) protein of heterologous viruses showed no reactivity to the Mabs, while the AKA-derived anti-G1 Mab (2F1) reacted with Peaton virus and all three AKA anti-N Mabs reacted with Tinaroo (TIN) virus at almost the same antibody titers as the homologous virus. Oligonucleotide fingerprinting analyses indicated that the three RNA species of all the viruses were unique and distinguishable. However, AKA and TIN viruses exhibited very similar S RNA oligonucleotide fingerprints, while the L and M RNA fingerprints were quite different. The S RNA sequence of TIN virus has been determined and compared with that of AKA and Aino viruses. The results revealed 95.1% S sequence homology between the AKA and TIN viruses. The antigenic and genetic comparisons of AKA and TIN viruses suggest that the two viruses may represent naturally occurring reassortant viruses.
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Antigenic relationships among 24 bunyaviruses of the Simbu serogroup were determined by complement-fixation (CF), serum dilution-plaque reduction neutralization (N) and, where possible, hemagglutination-inhibition (HI) tests. By CF, three distinct complexes of closely related viruses were identified within the serogroup. Nola and Thimiri viruses, which showed little relationship with other members of the serogroup, may represent two additional complexes. N tests in Vero cells showed that individual viruses generally were distinguishable with little difficulty. Aino and Kaikalur viruses were indistinguishable by CF or N. Seven viruses showed hemagglutination activity, and antigenic relationships among these viruses by HI paralleled those established by N tests. A classification scheme, based on both CF and N test results, for the viruses of the Simbu serogroup is proposed.
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The complete nucleotide sequence has been determined for the S RNA of Aino virus, a member of the Simbu serogroup (Bunyavirus genus, family Bunyaviridae). The S RNA is 850 nucleotides long (2.76 X 10(5) daltons) and in the viral complementary sequence has a short 5' non-coding region of 34 nucleotides and a more extensive 3' non-coding region of 117 nucleotides. The 3'-5' complementarity of the Aino S RNA is about 25 residues long, depending on the arrangement. The Aino sequence predicts that, like snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses (Bishop, D.H.L., et al. (1982) Nucleic Acids Res., 10, 3703-3713; Akashi, H. and Bishop, D.H.L. (1983) J. Virol. 45, 1155-1158), there are two S coded gene products, a nucleoprotein N, and a non-structural protein, NSS, that are read from overlapping reading frames in the viral complementary sequence. The Aino N primary gene product is composed of 233 amino acids (26.2 X 10(3) daltons) and is 45% homologous in sequence with that of LAC virus. The NSS protein of Aino virus is composed of 91 amino acids (10.5 X 10(3) daltons) and is 35% homologous in sequence with the LAC NSS protein. Unlike those viruses there are no uridylate tracts longer than 4 residues in the 5' non-coding region of the S viral RNA that could function as a template for polyadenylation of Aino S mRNA species.
A new member of the Simbu group of arboviruses, for which the name Peaton virus is proposed, has been isolated from midges and cattle in Australia. Nine isolates were obtained from 101 pools of the biting midge Culicoides brevitarsis collected at Peachester, Qld, (26.51 degrees S., 152.53 degrees E.) between 30 November and 8 December 1976. Three isolations of the same virus were made from the blood of sentinel cattle collected at Grafton and Tamworth, N.S.W., on 20 January and 13 April 1977, respectively. Peaton virus was shown to be a member of the Simbu group of arboviruses by complement-fixation tests using antisera prepared against Australian strains of Akabane and Aino viruses. It was readily distinguishable from these viruses in cross-neutralization tests in tissue cultures and mice. A serological survey of sentinel cattle showed that neutralizing antibody was detectable only in cattle within the recorded limits of the suspected vector C. brevitarsis. Neutralizing antibody in blood serum was detected in 22 of 157 sheep, 21 of 137 horses, 7 of 18 buffaloes, 7 of 20 goats and 3 of 62 pigs, but not in 22 camels, 34 dogs, 3 cats, 76 human beings, 240 marsupials, 19 reptiles or 31 wild birds. The pathogenecity of Peaton virus has yet to be determined. The Yale Arbovirus Research Unit and the Center for Disease Control, Fort Collins, U.S.A., found that Peaton virus was distinguishable from all other Simbu group viruses and thus is a new virus.
The antigenic properties of 21 Japanese field isolates and two Australian strains of Akabane (AKA) virus (Simbu serogroup, bunyavirus) isolated from 1959 to 1990 were compared by enzyme-linked immunosorbent assay (ELISA), plaque-reduction neutralization (PRNT) and hemagglutination inhibition (HI) tests using monoclonal antibodies (Mabs) to the OBE-1 strain of AKA virus. Sixteen Mabs were established by fusing P3X63Ag8U1 mouse myeloma cells and spleen cells from BALB/c mice immunized with the OBE-1 strain. Of the 16 clones, 13 produced immunoglobulin (Ig) which precipitated glycoprotein G1 and three produced Ig which precipitated nucleoprotein (N). Twelve out of 13 Mabs had both NT and HI activities to not only the homologous OBE-1 strain but also the other isolates. By the competitive binding assay, at least five antigenic regions for G1, and two for N were defined. Some of the anti-G1 Mabs which reacted to the same antigenic region had unique reactivity while anti-N Mabs recognizing the same epitope reacted with almost the same degree to all of the isolates. Finally, nine epitopes of the G1 protein in five different antigenic regions have been identified. There was no striking correlation between isolation date and place of the isolates and their reactivity to Mabs. A most interesting result is that three isolates collected in the same place over a three week period had different reactivity patterns detected by ELISA, showing great antigenic variation of the virus. AKA virus may be a single gene pool consisting of different genotypes in the field.
A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.
Heparin inhibited the hemagglutinin activity of Akabane and Aino viruses. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of Akabane and Aino viruses was 10 U/ml. Goose erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, goose erythrocytes treated with heparinase had greatly reduced agglutinability by Akabane virus. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.
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The Bunyavirus genus, belonging to the Bunyaviridae family, is comprised of a large group of antigenically and geographically disparate arthropod-borne viruses of medical and veterinary significance. In Australia, viruses belonging to the Simbu serogroup of the Bunyavirus genus, Akabane, Tinaroo, Peaton, Aino, Douglas, Thimiri and Facey's Paddock have been isolated. In this communication we describe two indirect ELISAs, referred to as the Simbu serogroup ELISA (SG-ELISA), and the Simbu typing ELISA (ST-ELISA), for the identification of these Simbu serogroup viruses. Infected cell lysate antigens prepared from Simbu serogroup virus isolates were assessed in the SG-ELISA for reactivity with a mouse monoclonal antibody (4H9/B11/F1). The monoclonal antibody reacted strongly with all Australian members of Simbu serogroup reference viruses and is proposed for use as a serogrouping reagent for Simbu viruses. Furthermore, the ST-ELISA enabled specific identification of viruses from within this group by recognition of characteristic reaction patterns between infected cell lysate antigens and a panel of polyclonal antisera raised to Simbu serogroup viruses.
Type 9 African horse sickness virus and type 4 bluetongue virus multiplied to a high titre in an Israeli strain of Culicoides puncticollis after intrathoracic inoculation. Akabane virus persisted for at least 10 days in this midge after intrathoracic inoculation but with little evidence of virus multiplication. All 3 viruses failed to multiply in C. puncticollis after ingestion by the oral route and all were inactivated by 4 days post infection. Five other species of Israeli Culicoides supported multiplication of bluetongue virus after intrathoracic inoculation.
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