Sequence analysis of the small (s) RNA segment of viruses in the genus Orthobunyavirus
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Viruses in the genus Orthobunyavirus (family Bunyaviridae) are classified serologically into 18 serogroups. The viruses have a tripartite genome of negative sense RNA composed of large (L), medium (M) and small (S) segments. The L segment encodes the polymerase protein, the M segment encodes two glycoproteins, Gc and Gn, and a non-structural protein (NSm), and the S segment encodes nucleocapsid (N) and NSs proteins, in overlapping reading frames (ORF). The NSs proteins of Bunyamwera and California serogroup viruses have been shown to play a role in inhibiting host cell protein synthesis and preventing induction of interferon in infected cells. To-date, viruses in only 4 serogroups: Bunyamwera, California, Group C and Simbu, have been studied intensively. Therefore, this study was conducted with the aim to sequence the S RNA segments of representative viruses in the other 14 orthobunyavirus serogroups, to analyse virus-encoded proteins synthesised in infected cells, and to investigate their ability to cause shutoff of host protein synthesis. S RNA segment sequences were obtained from cloned RT-PCR products. They were compared with the available sequences and each other. Complete S RNA sequences of Anopheles A (ANAV) and Tacaiuma virus (TCMV) [Anopheles A serogroup], Anopheles B (ANBV) and Boraceia virus (BORV) [Anopheles B serogroup], Eretmapodites (E147V) and Nyando virus (NDV)[Nyando serogroup], Bwamba virus (BWAV) [Bwamba serogroup], Mâ Poko virus (MPOV) [Turlock serogroup], Tete (TETEV) and Batama virus (BMAV) [Tete serogroup], and Gamboa (GAMV) and San Juan 2441 virus (SJ244V) [Gamboa serogroup], and partial sequences of Patois virus (PATV) [Patois serogroup], Guama (GMAV) and Bertioga virus (BERV) [Guama serogroup], Capim virus (CAPV) [Capim serogroup] and Palestina virus (PLSV) [Minatitlan serogroup] were obtained. Complete S segment sequences revealed that viruses in the same serogroup have same length of N and NSs proteins, except for the viruses in Gamboa serogroup which were found to have two lengths of NSs protein. Viruses in 4 serogroups (Anopheles A, Anopheles B, Tete and Capim) were found not to encode an NSs ORF, presenting the first report of naturally isolated orthobunyaviruses without an NSs protein. Most of these viruses were found to have longer N proteins compared to those with NSs protein, with the largest N protein observed to date in TETEV and BMAV (258 amino acids). Other viruses 3 (EREV, NDV, GAMV, SJ2441V, BWAV and MPOV) were found to encode both N and NSs proteins in their S segment with the largest and smallest NSs protein detected to date in SJ2441V (137 amino acids) and MPOV (70 amino acids) respectively. The conserved CA rich motif in 5â non coding region (NCR) of Bunyamwera and California serogroups viruses was absent in BWAV and MPOV, while ANBV and BORV were found to have two copies of this motif. Repeated sequences, as observed previously in the 5â NCR of genomic-sense RNA of Lumbo virus (LUMV), were also detected in BWAV and TCMV S RNA segments. Sequence comparisons and phylogenetic analyses of the sequences determined in this study were in agreement with previous serological classification of the viruses, except for BERV and TCMV. BERV, in the Guama serogroup, was found to have a closer relationship with CAPV compared to GMAV. However high sequence identities (>70%) were observed between these 3 viruses, suggesting that they are derived from the same ancestor. N protein and nucleotide sequence identities of TCMV with ANAV were only 53% and 59% respectively. However, Neighbour-Joining (NJ) plot based on complete N amino acid sequence and Maximum Parsimony (MP) plot based on partial N sequence supported previous serological classification which placed this virus in the same clade as ANAV. This study first reports on the proteins synthesised by Bakau, Bwamba, Koongol, Gamboa, Minatitlan, Olifantsvlei and Tete serogroup viruses. Analysis of radio-labelled cell extracts revealed similar protein migration patterns for all the studied viruses compared with other viruses in the genus Orthobunyavirus. Shutoff of host cell protein synthesis, similar to that seen in Bunyamwera virus (BUNV)-infected cells was only observed in ACAV, BAKV, BWAV, CAPV, PAHV, PATV and WONV-infected cells. However, this shutoff was found not related to the presence of NSs protein. In general, viruses in the same serogroup were found to have almost same size of plaque and plaque-size did not correlate with the presence of NSs protein and the virulence of the virus in the mice. In vitro transcription and translation (TnT) using rabbit reticulocyte and wheat germ lysate expression systems further confirmed the sequencing results that no NSs protein was expressed from S cDNA clones of ANAV, TCMV, ANBV, BORV, BMAV and TETEV. S RNA segments shutoff almost similar to BUNV-infected cells was observed in A549 cells infected with TCMV, suggesting that TCMV might use a different mechanism to induce shutoff. No significant shutoff was observed in Hep2, Hep2/V and C6/36 cells infected with any of the viruses. RT-PCR specific for IFN- Ã mRNA in 293 infected cells and IFN reporter gene assays revealed that TCMV was capable of counteracting IFN production similar to wt BUNV, whereas the other NSs minus viruses (ANAV, ANBV, BORV, TETEV and BMAV) were found to be capable of inducing IFN in infected cells. However, only low level of IFN- Ã mRNA and weak activation of the IFN- Ã promoter was detected in ANAV and BMAV- infected cells.
Thesis, PhD Doctor of Philosophy
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