Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro.

Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.

Akabane virus in Israel: a new virus lineage.

This report describes the first molecular characterization of Akabane virus (AKAV) in Israel. The virus was recognized by real-time RT-PCR in extracts from Culicoides imicola insects trapped at the Volcani Center located in the center of Israel. This is also the first report on the use of real-time RT-PCR to identify the virus. The quantitative capability of this technique was applied, and it was calculated that the insect extract contains 1.5 x 10(5) copies of the genome segment S. Following amplification of the small (S) genome segment, its nucleotide sequence was determined to have 93.4% identity or greater with the S segment of other AKAV isolates. The deduced amino acid (aa) sequence of the combined nucleocapsid and the non-structural protein showed more than 96.6% identity. Phylogentic trees constructed using the combined deduced nucleocapsid and the non-structural protein aa sequences showed that the Israeli isolate forms a fourth cluster of AKAV, indicating a separate virus lineage. Attempts to isolate the virus by inoculation to Vero cells and by intracerebral inoculation to mice were unsuccessful.

Nucleotide sequence and deduced amino acid sequence of the medium RNA segment of Oropouche, a Simbu serogroup virus: comparison with the middle RNA of Bunyamwera and California serogroup viruses.

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50-52%) and amino acid (30-32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743-748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain.

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.