Sequence analysis of the L RNA genome segment and predicted encoded L polymerase protein of Crimean-Congo hemorrhagic fever (CCHF) virus (genus Nairovirus, family Bunyaviridae) demonstrates that they are approximately twice the size of those found in viruses of other bunyavirus genera. The CCHF virus L segment and encoded protein (12,164 nucleotides and 3944 amino acids, respectively) are similar in size and sequence to those of the nairovirus Dugbe virus (12,255/62% and 4036/62% nucleotide and amino acid length/identity, respectively). The identification of an ovarian tumor (OTU)-like protease motif in the L protein amino termini of the nairoviruses Dugbe, CCHF, and Nairobi sheep disease (NSD) indicates these proteins are members of the recently described OTU-like protease family and suggests that these large proteins may be polyproteins that are autoproteolytically cleaved or involved in deubiquitination.
BackgroundThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. AimTo investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples.MethodsWe performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study.ResultsDirect metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. ConclusionsThis work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.
The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negativesense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5 terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5-terminal 32 and 3-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 g of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5 terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.Bunyamwera virus (BUN) is the prototype of the genus Bunyavirus and the family Bunyaviridae and possesses a singlestranded negative-sense tripartite RNA genome. The three RNA segments, termed L (large), M (medium), and S (small), encode six proteins. The L segment codes for the L protein, the viral RNA-dependent RNA polymerase, which is responsible for both transcribing and replicating the genome RNAs. The M segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, as a polyprotein which is probably cotranslationally cleaved by host proteases. The S segment encodes the nucleocapsid (N) protein and, in an overlapping reading frame, a second nonstructural protein called NSs (reviewed in reference 8).In common with other negative-sense RNA viruses, the bunyavirus genome RNA segments are replicated via full-length cRNAs termed antigenomes. Both the negative-sense genome and positive-sense antigenome RNAs are encapsidated by the N protein and are associated with the viral polymerase in ribonucleoprotein complexes called nucleocapsids. It is only within the nucleocapsid that the RNA is transcriptionally active. Bunyavirus genome and antigenome RNAs contain highly conserved, complementary terminal sequences that may form panhandle structures in vivo and are probably responsible for the circular appearance of isolated nucleocapsids (19,20,22,26,28).Full-length genome and antigenome segments are usually the only RNAs that are encapsidated in the infected cell. Viral mRNAs, which are not encapsidated, are truncated at the 3Ј end and contain a nontemplated capped primer on the 5Ј terminus (2, 4, 9, 14, 21). It is therefore likely that the terminal sequences of the genome and antigenome RNAs are involved in the encapsidation process. This theory is supported by the observation that an antisense chloramphenicol acetyltransferas...
The genus Nairovirus (family Bunyaviridae) contains seven serogroups consisting of 34 predominantly tick-borne viruses, including several associated with severe human and livestock diseases [e.g., Crimean Congo hemorrhagic fever (CCHF) and Nairobi sheep disease (NSD), respectively]. Before this report, no comparative genetic studies or molecular detection assays had been developed for this virus genus. To characterize at least one representative from each of the seven serogroups, reverse transcriptase-polymerase chain reaction (RT-PCR) primers targeting the L polymerase-encoding region of the RNA genome of these viruses were successfully designed based on conserved amino acid motifs present in the predicted catalytic core region. Sequence analysis showed the nairoviruses to be a highly diverse group, exhibiting up to 39.4% and 46.0% nucleotide and amino acid identity differences, respectively. Virus genetic relationships correlated well with serologic groupings and with tick host associations. Hosts of these viruses include both the hard (family Ixodidae) and soft (family Argasidae) ticks. Virus phylogenetic analysis reveals two major monophyletic groups: hard tick and soft tick-vectored viruses. In addition, viruses vectored by Ornithodoros, Carios, and Argas genera ticks also form three separate monophyletic lineages. The striking similarities between tick and nairovirus phylogenies are consistent with possible coevolution of the viruses and their tick hosts. Fossil and phylogenetic data placing the hard tick-soft tick divergence between 120 and 92 million years ago suggest an ancient origin for viruses of the genus Nairovirus.
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