Genetic diversity and population structure of wild and cultivated Crotalaria species based on genotyping-by-sequencing.

Crotalaria is a plant genus that is found all over the world, with over 700 species of herbs and shrubs. The species are potential alternative food and industrial crops due to their adaptability to different environments. Currently, information on the genetic diversity and population structure of these species is scanty. Genotyping-by-sequencing (GBS) is a cost-effective high-throughput technique in diversity evaluation of plant species that have not been fully sequenced. In the current study, de novo GBS was used to characterize 80 Crotalaria accessions from five geographical regions in Kenya. A total of 9820 single nucleotide polymorphism (SNP) markers were obtained after thinning and filtering, which were then used for the analysis of genetic diversity and population structure in Crotalaria. The proportion of SNPs with a minor allele frequency (maf) > = 0.05 was 45.08%, while the Guanine-Cytosine (GC) content was 0.45, from an average sequence depth of 455,909 reads per base. The transition vs transversion ratio was 1.81 and Heterozygosity (He) ranged between 0.01-0.07 in all the sites and 0.04 to 0.52 in the segregating sites. The mean Tajima's D value for the population was -0.094, suggesting an excess of rare alleles. The fixation index (Fst) between the different populations based on the Wright Fst (1943) ranged from 0.0119 to 0.066 for the Eastern-Western and Nairobi-Western populations. Model based techniques of population structure analysis including structure, k-means and cross-entropy depicted eight clusters in the study accessions. Non-model based techniques especially DAPC depicted poor population stratification. Correspondence Analysis (CA), Principal coordinate analyses (PCoA) and phylogenetic analysis identified a moderate level of population stratification. Results from this study will help conservationists and breeders understand the genetic diversity of Crotalaria. The study also provides valuable information for genetic improvement of domesticated species.

A comparative genome analysis of Rift Valley Fever virus isolates from foci of the disease outbreak in South Africa in 2008-2010.

Rift Valley fever (RVF) is a re-emerging zoonotic disease responsible for major losses in livestock production, with negative impact on the livelihoods of both commercial and resource-poor farmers in sub-Sahara African countries. The disease remains a threat in countries where its mosquito vector thrives. Outbreaks of RVF usually follow weather conditions which favour increase in mosquito populations. Such outbreaks are usually cyclical, occurring every 10-15 years. Recent outbreaks of the disease in South Africa have occurred unpredictably and with increased frequency. In 2008, outbreaks were reported in Mpumalanga, Limpopo and Gauteng provinces, followed by 2009 outbreaks in KwaZulu-Natal, Mpumalanga and Northern Cape provinces and in 2010 in the Eastern Cape, Northern Cape, Western Cape, North West, Free State and Mpumalanga provinces. By August 2010, 232 confirmed infections had been reported in humans, with 26 confirmed deaths.To investigate the evolutionary dynamics of RVF viruses (RVFVs) circulating in South Africa, we undertook complete genome sequence analysis of isolates from animals at discrete foci of the 2008-2010 outbreaks. The genome sequences of these viruses were compared with those of the viruses from earlier outbreaks in South Africa and in other countries. The data indicate that one 2009 and all the 2008 isolates from South Africa and Madagascar (M49/08) cluster in Lineage C or Kenya-1. The remaining of the 2009 and 2010 isolates cluster within Lineage H, except isolate M259_RSA_09, which is a probable segment M reassortant. This information will be useful to agencies involved in the control and management of Rift Valley fever in South Africa and the neighbouring countries.

Viral metagenomics of aphids present in bean and maize plots on mixed-use farms in Kenya reveals the presence of three dicistroviruses including a novel Big Sioux River virus-like dicistrovirus.

BACKGROUND: Aphids are major vectors of plant viruses. Common bean (Phaseolus vulgaris L.) and maize (Zea mays L.) are important crops that are vulnerable to aphid herbivory and aphid-transmitted viruses. In East and Central Africa, common bean is frequently intercropped by smallholder farmers to provide fixed nitrogen for cultivation of starch crops such as maize. We used a PCR-based technique to identify aphids prevalent in smallholder bean farms and next generation sequencing shotgun metagenomics to examine the diversity of viruses present in aphids and in maize leaf samples. Samples were collected from farms in Kenya in a range of agro-ecological zones. RESULTS: Cytochrome oxidase 1 (CO1) gene sequencing showed that Aphis fabae was the sole aphid species present in bean plots in the farms visited. Sequencing of total RNA from aphids using the Illumina platform detected three dicistroviruses. Maize leaf RNA was also analysed. Identification of Aphid lethal paralysis virus (ALPV), Rhopalosiphum padi virus (RhPV), and a novel Big Sioux River virus (BSRV)-like dicistrovirus in aphid and maize samples was confirmed using reverse transcription-polymerase chain reactions and sequencing of amplified DNA products. Phylogenetic, nucleotide and protein sequence analyses of eight ALPV genomes revealed evidence of intra-species recombination, with the data suggesting there may be two ALPV lineages. Analysis of BSRV-like virus genomic RNA sequences revealed features that are consistent with other dicistroviruses and that it is phylogenetically closely related to dicistroviruses of the genus Cripavirus. CONCLUSIONS: The discovery of ALPV and RhPV in aphids and maize further demonstrates the broad occurrence of these dicistroviruses. Dicistroviruses are remarkable in that they use plants as reservoirs that facilitate infection of their insect replicative hosts, such as aphids. This is the first report of these viruses being isolated from either organism. The BSRV-like sequences represent a potentially novel dicistrovirus infecting A. fabae.

Detection of West Nile virus in wild birds in Tana River and Garissa Counties, Kenya.

BACKGROUND: West Nile fever virus is a zoonotic arboviral infection maintained in a sylvatic cycle involving mosquito vectors and birds. It is one the arboviruses whose geographical range is expanding because of climate and land use changes that enhance the densities of mosquitoes and promote mosquito-bird-human interactions. We carried out a survey to determine the reservoirs of WNV among wild birds in Tana River and Garissa counties, Kenya. METHODS: Blood samples were obtained from 361 randomly trapped wild birds. Using real-time polymerase chain reaction (PCR), all samples were screened for WNV using gene specific primer sets amplifying a portion of the E region of the genome encoding the envelope protein. RESULTS: Sixty five (65) out of 361 birds screened tested positive for WNV on real-time PCR assay. Sequencing of the selected positive samples reveals that the isolated WNV were most closely related to strains isolated from China (2011). A regression analysis indicated that sampling location influenced the occurrence of WNV while species, age, weight and sex of the birds did not have any effect. CONCLUSIONS: This study provides baseline information on the existing circulation of WNV in this region among wild bird reservoirs that could spill over to the human population and points to the need for implementation of surveillance programs to map the distribution of the virus among reservoirs. Awareness creation about West Nile fever in this region is important to improve its detection and management.

COMPLETE GENOMIC SEQUENCE OF VIRULENT PIGEON PARAMYXOVIRUS IN LAUGHING DOVES (STREPTOPELIA SENEGALENSIS) IN KENYA.

Following mass deaths of Laughing Doves (Streptopelia senegalensis) in different localities throughout Kenya, internal organs obtained during necropsy of two moribund birds were sampled and analyzed by next generation sequencing. We isolated the virulent strain of pigeon paramyxovirus type-1 (PPMV-1), PPMV1/Laughing Dove/Kenya/Isiolo/B2/2012, which had a characteristic fusion gene motif (110)GGRRQKRF(117). We obtained a partial full genome of 15,114 nucleotides. The phylogenetic relationship based on the fusion gene and genomic sequence grouped our isolate as class II genotype VI, a group of viruses commonly isolated from wild birds but potentially lethal to Chickens ( Gallus gallus domesticus ). The fusion gene isolate clustered with PPMV-I strains from pigeons (Columbidae) in Nigeria. The complete genome showed a basal and highly divergent lineage to American, European, and Asian strains, indicating a divergent evolutionary pathway. The isolated strain is highly virulent and apparently species-specific to Laughing Doves in Kenya. Risk of transmission of such a strain to poultry is potentially high whereas the cyclic epizootic in doves is a threat to conservation of wild Columbidae in Kenya.

Delineation of the population genetic structure of Culicoides imicola in East and South Africa.

BACKGROUND: Culicoides imicola Kieffer, 1913 is the main vector of bluetongue virus (BTV) and African horse sickness virus (AHSV) in Sub-Saharan Africa. Understanding the population genetic structure of this midge and the nature of barriers to gene flow will lead to a deeper understanding of bluetongue epidemiology and more effective vector control in this region. METHODS: A panel of 12 DNA microsatellite markers isolated de novo and mitochondrial DNA were utilized in a study of C. imicola populations from Africa and an outlier population from the Balearic Islands. The DNA microsatellite markers and mitochondrial DNA were also used to examine a population of closely related C. bolitinos Meiswinkel midges. RESULTS: The microsatellite data suggest gene flow between Kenya and south-west Indian Ocean Islands exist while a restricted gene flow between Kenya and South Africa C. imicola populations occurs. Genetic distance correlated with geographic distance by Mantel test. The mitochondrial DNA analysis results imply that the C. imicola populations from Kenya and south-west Indian Ocean Islands (Madagascar and Mauritius) shared haplotypes while C. imicola population from South Africa possessed private haplotypes and the highest nucleotide diversity among the African populations. The Bayesian skyline plot suggested a population growth. CONCLUSIONS: The gene flow demonstrated by this study indicates a potential risk of introduction of new BTV serotypes by wind-borne infected Culicoides into the Islands. Genetic similarity between Mauritius and South Africa may be due to translocation as a result of human-induced activities; this could impact negatively on the livestock industry. The microsatellite markers isolated in this study may be utilised to study C. bolitinos, an important vector of BTV and AHSV in Africa and identify sources of future incursions.

Genetic divergence of Chikungunya virus plaque variants from the Comoros Island (2005).

Chikungunya virus (CHIKV) from a human sample collected during the 2005 Chikungunya outbreak in the Comoros Island, showed distinct and reproducible large (L2) and small (S7) plaques which were characterized in this study. The parent strain and plaque variants were analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. In vitro growth kinetic assays showed similar growth patterns of both plaque variants in Vero cells but higher viral titres of S7 compared to L2 in C6/36 cells. Amino acids (AA) alignments of the CHIKV plaque variants and S27 African prototype strain, showed 30 AA changes in the non-structural proteins (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, only two AAs differences were observed. A missense substitution (C642Y) of L2 in the nsP2, involving a conservative AA substitution and a nonsense substitution (R524X) of S7 in the nsP3, which has been shown to enhance O'nyong-nyong virus infectivity and dissemination in Anopheles mosquitoes. The phenotypic difference observed in plaque size could be attributed to one of these AA substitutions. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains indicating circulation of isolates with close evolutionary relatedness in the same outbreak. These observations pave way for important functional studies to understand the significance of the identified genetic changes in virulence and viral transmission in mosquito and mammalian hosts.

Whole genome phylogenetic investigation of a West Nile virus strain isolated from a tick sampled from livestock in north eastern Kenya.

BACKGROUND: West Nile virus (WNV) has a wide geographical distribution and has been associated to cause neurological disease in humans and horses. Mosquitoes are the traditional vectors for WNV; however, the virus has also been isolated from tick species in North Africa and Europe which could be a means of introduction and spread of the virus over long distances through migratory birds. Although WNV has been isolated in mosquitoes in Kenya, paucity of genetic and pathogenicity data exists. We previously reported the isolation of WNV from ticks collected from livestock and wildlife in Ijara District of Kenya, a hotspot for arbovirus activity. Here we report the full genome sequence and phylogenetic investigation of their origin and relation to strains from other regions. METHODS: A total of 10,488 ticks were sampled from animal hosts, classified to species and processed in pools of up to eight ticks per pool. Virus screening was performed by cell culture, RT-PCR and sequencing. Phylogenetic analysis was carried out to determine the evolutionary relationships of our isolate. RESULTS: Among other viruses, WNV was isolated from a pool of Rhipicephalus pulchellus sampled from cattle, sequenced and submitted to GenBank (Accession number: KC243146). Comparative analysis with 27 different strains revealed that our isolate belongs to lineage 1 and clustered relatively closely to isolates from North Africa and Europe, Russia and the United States. Overall, Bayesian analysis based on nucleotide sequences showed that lineage 1 strains including the Kenyan strain had diverged 200 years ago from lineage 2 strains of southern Africa. Ijara strain collected from a tick sampled on livestock was closest to another Kenyan strain and had diverged 20 years ago from strains detected in Morocco and Europe and 30 years ago from strains identified in the USA. CONCLUSION: To our knowledge, this is the first characterized WNV strain isolated from R. pulchellus. The epidemiological role of this tick in WNV transmission and dissemination remains equivocal but presents tick verses mosquito virus transmission has been neglected. Genetic data of this strain suggest that lineage 1 strains from Africa could be dispersed through tick vectors by wild migratory birds to Europe and beyond.

Isolation of tick and mosquito-borne arboviruses from ticks sampled from livestock and wild animal hosts in Ijara District, Kenya.

Tick-borne viruses infect humans through the bite of infected ticks during opportunistic feeding or through crushing of ticks by hand and, in some instances, through contact with infected viremic animals. The Ijara District, an arid to semiarid region in northern Kenya, is home to a pastoralist community for whom livestock keeping is a way of life. Part of the Ijara District lies within the boundaries of a Kenya Wildlife Service-protected conservation area. Arbovirus activity among mosquitoes, animals, and humans is reported in the region, mainly because prevailing conditions necessitate that people continuously move their animals in search of pasture, bringing them in contact with ongoing arbovirus transmission cycles. To identify the tick-borne viruses circulating among these communities, we analyzed ticks sampled from diverse animal hosts. A total of 10,488 ticks were sampled from both wildlife and livestock hosts and processed in 1520 pools of up to eight ticks per pool. The sampled ticks were classified to species, processed for virus screening by cell culture using Vero cells and RT-PCR (in the case of Hyalomma species), followed by amplicon sequencing. The tick species sampled included Rhipicephalus pulchellus (76.12%), Hyalomma truncatum (8.68%), Amblyomma gemma (5.00%), Amblyomma lepidum (4.34%), and others (5.86%). We isolated and identified Bunyamwera (44), Dugbe (5), Ndumu (2), Semliki forest (25), Thogoto (3), and West Nile (3) virus strains. This observation constitutes a previously unreported detection of mosquito-borne Semliki forest and Bunyamwera viruses in ticks, and association of West Nile virus with A. gemma and Rh. pulchellus ticks. These findings provide additional evidence on the potential role of ticks and associated animals in the circulation of diverse arboviruses in northeastern Kenya, including viruses previously known to be essentially mosquito borne.

Viral metagenomics demonstrates that domestic pigs are a potential reservoir for Ndumu virus.

BACKGROUND: The rising demand for pork has resulted in a massive expansion of pig production in Uganda. This has resulted in increased contact between humans and pigs. Pigs can act as reservoirs for emerging infectious diseases. Therefore identification of potential zoonotic pathogens is important for public health surveillance. In this study, during a routine general surveillance for African swine fever, domestic pigs from Uganda were screened for the presence of RNA and DNA viruses using a high-throughput pyrosequencing method. FINDINGS: Serum samples from 16 domestic pigs were collected from five regions in Uganda and pooled accordingly. Genomic DNA and RNA were extracted and sequenced on the 454 GS-FLX platform. Among the sequences assigned to a taxon, 53% mapped to the domestic pig (Sus scrofa). African swine fever virus, Torque teno viruses (TTVs), and porcine endogenous retroviruses were identified. Interestingly, two pools (B and C) of RNA origin had sequences that showed 98% sequence identity to Ndumu virus (NDUV). None of the reads had identity to the class Insecta indicating that these sequences were unlikely to result from contamination with mosquito nucleic acids. CONCLUSIONS: This is the first report of the domestic pig as a vertebrate host for Ndumu virus. NDUV had been previously isolated only from culicine mosquitoes. NDUV therefore represents a potential zoonotic pathogen, particularly given the increasing risk of human-livestock-mosquito contact.