Molecular Characterization of Human Pathogenic Bunyaviruses of the Nyando and Bwamba/Pongola Virus Groups Leads to the Genetic Identification of Mojuí dos Campos and Kaeng Khoi Virus

<div><p>Background</p><p>Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease.</p><p>Methodology/Principal Findings</p><p>In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types.</p><p>Conclusions/Significance</p><p>On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated.</p></div

Phylogenetic relationships among BWAV/PGAV and NDV/MDCV/KKV group viruses.

<p>Maximum likelihood trees were constructed based on the nucleotide sequences of the S segment, M segment and L segment, as indicated. Bootstrap values based on 1,000 replicates are also indicated. Viruses lineages added based on sequences determined as a part of this study are indicated in color: Bwamba virus (orange), Pongola virus (red), Nyando virus (blue), Mojuí dos Campos virus (purple), Kaeng Khoi virus (pink).</p

Virus strains used in this study.

<p>Virus strains used in this study.</p

Comparison of virus growth in various cell lines.

<p>Cell lines derived from bats (i.e. Tb 1 Lu and RE05), mosquito (i.e. C6/36) or non-human primates (i.e. VeroE6) were infected with the indicated viruses at a multiplicity of infection of 0.1. Supernatants were harvested either immediately after infection (0 h) or after 72 h incubation and titres were determined using plaque assay.</p

Comparison of virus genome structures.

<p>The genomes of the various virus groups sequenced in this study are shown to scale. The non-coding regions are shown in grey while the major open reading frames encoded by each segment [S segment: N (ORF), M segment: GPC (ORF) and L segment: LORF)] are shown in colored boxes. The NSs protein, which is produced from a downstream ATG in the S segment, is shown in a lighter shade of the corresponding color for each virus. The total genome length of each segment is indicated at right.</p

Geographic distribution of viruses used in this study.

<p>Distribution of (A) Bwamba virus, (B) Pongola virus, (C) Nyando virus, (D) Mojuí dos Campos virus, and (E) Kaeng Khoi virus. Countries in which these viruses have been isolated (dark colors) and/or where specific antibodies to these viruses have been detected (light colors) are shown in orange (BWAV), red (PGAV), blue (NDV), purple (MDCV), and pink (KKV), respectively. The geographic location from which the virus strains used in the present study were isolated, are indicated with black dots. The strain names for these isolates are also indicated.</p