Pangolin Genomes Offer Key Insights and Resources for the World's Most Trafficked Wild Mammals.

Pangolins form a group of scaly mammals that are trafficked at record numbers for their meat and purported medicinal properties. Despite their conservation concern, knowledge of their evolution is limited by a paucity of genomic data. We aim to produce exhaustive genomic resources that include 3,238 orthologous genes and whole-genome polymorphisms to assess the evolution of all eight extant pangolin species. Robust orthologous gene-based phylogenies recovered the monophyly of the three genera and highlighted the existence of an undescribed species closely related to Southeast Asian pangolins. Signatures of middle Miocene admixture between an extinct, possibly European, lineage and the ancestor of Southeast Asian pangolins, provide new insights into the early evolutionary history of the group. Demographic trajectories and genome-wide heterozygosity estimates revealed contrasts between continental versus island populations and species lineages, suggesting that conservation planning should consider intraspecific patterns. With the expected loss of genomic diversity from recent, extensive trafficking not yet realized in pangolins, we recommend that populations be genetically surveyed to anticipate any deleterious impact of the illegal trade. Finally, we produce a complete set of genomic resources that will be integral for future conservation management and forensic endeavors for pangolins, including tracing their illegal trade. These comprise the completion of whole-genomes for pangolins through the hybrid assembly of the first reference genome for the giant pangolin (Smutsia gigantea) and new draft genomes (∼43x-77x) for four additional species, as well as a database of orthologous genes with over 3.4 million polymorphic sites.

Molecular screening reveals non-uniform malaria transmission in western Kenya and absence of Rickettsia africae and selected arboviruses in hospital patients.

BACKGROUND: In sub-Saharan Africa, malaria is the common diagnosis for febrile illness and related clinical features, resulting in the under-diagnosis of other aetiologies, such as arboviruses and Rickettsia. While these may not be significant causes of mortality in malaria-endemic areas, they affect the daily life and performance of affected individuals. It is, therefore, important to have a clear picture of these other aetiologies to institute correct diagnoses at hospitals and improve patient outcomes. METHODS: Blood samples were collected from patients with fever and other clinical features associated with febrile illness at selected hospitals in the malaria-endemic counties of Busia, Bungoma, and Kakamega, and screened for Crimean-Congo haemorrhagic fever, Sindbis, dengue and chikungunya viruses, Rickettsia africae, and Plasmodium spp. using high-throughput real-time PCR techniques. A logistic regression was performed on the results to explore the effect of demographic and socio-economic independent variables on malaria infection. RESULTS: A total of 336 blood samples collected from hospital patients between January 2018 and February 2019 were screened, of which 17.6% (59/336) were positive for Plasmodium falciparum and 1.5% (5/336) for Plasmodium malariae. Two patients had dual P. falciparum/P. malariae infections. The most common clinical features reported by the patients who tested positive for malaria were fever and headache. None of the patients were positive for the arboviruses of interest or R. africae. Patients living in Busia (OR 5.2; 95% CI 2.46-11.79; p < 0.001) and Bungoma counties (OR 2.7; 95% CI 1.27-6.16; p = 0.013) had higher odds of being infected with malaria, compared to those living in Kakamega County. CONCLUSIONS: The reported malaria prevalence is in line with previous studies. The absence of arboviral and R. africae cases in this study may have been due to the limited number of samples screened, low-level circulation of arboviruses during inter-epidemic periods, and/or the use of PCR alone as a detection method. Other sero-surveys confirming their circulation in the area indicate that further investigations are warranted.

Molecular prevalence and risk factors associated with tick-borne pathogens in cattle in western Kenya.

BACKGROUND: Tick-borne pathogens (TBPs) are of global importance, especially in sub-Saharan Africa where they represent a major constraint to livestock production. Their association with human disease is also increasingly recognized, signalling their zoonotic importance. It is therefore crucial to investigate TBPs prevalence in livestock populations and the factors associated with their presence. We set out to identify TBPs present in cattle and to determine associated risk factors in western Kenya, where smallholder livestock production is important for subsistence and market-driven income. RESULTS: Tick-borne pathogen infections in blood samples collected from cattle at livestock markets and slaughterhouses between May 2017 and January 2019 were identified by high-resolution melting analysis and sequencing of PCR products of genus-specific primers. Of the 422 cattle sampled, 30.1% (127/422) were infected with at least one TBP, while 8.8% (37/422) had dual infections. Anaplasma spp. (19.7%) were the most prevalent, followed by Theileria (12.3%), Ehrlichia (6.6%), and Babesia (0.2%) spp. Sequence analysis of the TBPs revealed them to be Anaplasma platys-like organisms (13.5%), Theileria velifera (7.4%), Anaplasma marginale (4.9%), Theileria mutans (3.1%), Theileria parva (1.6%), and Babesia bigemina (0.2%). Ehrlichia ruminantium, Rickettsia spp., and arboviruses were not detected. Exotic breeds of cattle were more likely to be infected with A. marginale compared to local breeds (OR: 7.99, 95% CI: 3.04-22.02, p <  0.001). Presence of ticks was a significant predictor for Anaplasma spp. (OR: 2.18, 95% CI: 1.32-3.69, p = 0.003) and Ehrlichia spp. (OR: 2.79, 95% CI: 1.22-7.23, p = 0.022) infection. Cattle sampled at slaughterhouses were more likely to be positive for Anaplasma spp. (OR: 1.64, 95% CI: 1.01-2.70, p = 0.048) and A. marginale (OR: 3.84, 95% CI: 1.43-12.21, p = 0.012), compared to those sampled at livestock markets. CONCLUSION: This study reports TBP prevalence and associated risk factors in western Kenya, factors which are key to informing surveillance and control measures.

Prevalence and Diversity of the Streptobacillus Rat-bite Fever Agent, in Three Invasive, Commensal Rattus Species from South Africa.

Rat-bite fever is an over-looked, global zoonotic disease that has a mortality rate of up to 13%, if untreated. Historically, this rat-borne disease has been attributed to one of two causative agents, Streptobacillus moniliformis or Spirillum minus. Given the confirmed presence of multiple invasive Rattus host species, high rat densities in urban, informal human settlements and increasing reports of rat bites in South Africa, we undertook a retrospective assessment of Streptobacillus in rats sampled from 16 urban sites, in Gauteng, the smallest but most populous Province in South Africa. Using a multi-gene PCR-sequencing approach, we confirmed Streptobacillus presence in 50.9% of oral swabs from three rat species and the presence of two Streptobacillus species, viz.S. moniliformis and S. notomytis. The two members of the cryptic Rattus rattus species complex (R. rattus and R. tanezumi), which are morphologically indistinguishable from each other, had markedly different colonization rates. Whereas 48.6% of rats from this species complex were Streptobacillus-positive, only 32.3% of Rattus tanezumi were positive compared to 61.5% R. rattus. Rattus norvegicus had an intermediate prevalence of 55.6%. Phylogenetic analysis of four gene regions (16S rRNA, gyrB, groEL, recA) identified two discrete lineages; S. moniliformis occurred exclusively in R.norvegicus, and S. notomytis was restricted to the two members of the R. rattus species complex; this represents the first report of Streptobacillus in R. tanezumi. These results highlight a largely overlooked zoonotic threat posed by invasive rats and confirm the presence of two discrete and potentially host-specific Streptobacillus lineages in South Africa.

Genetic insights into dispersal distance and disperser fitness of African lions (Panthera leo) from the latitudinal extremes of the Kruger National Park, South Africa.

BACKGROUND: Female lions generally do not disperse far beyond their natal range, while males can disperse distances of over 200 km. However, in bush-like ecosystems dispersal distances less than 25 km are reported. Here, we investigate dispersal in lions sampled from the northern and southern extremes of Kruger National Park, a bush-like ecosystem in South Africa where bovine tuberculosis prevalence ranges from low to high across a north-south gradient. RESULTS: A total of 109 individuals sampled from 1998 to 2004 were typed using 11 microsatellite markers, and mitochondrial RS-3 gene sequences were generated for 28 of these individuals. Considerable north-south genetic differentiation was observed in both datasets. Dispersal was male-biased and generally further than 25 km, with long-distance male gene flow (75-200 km, detected for two individuals) confirming that male lions can travel large distances, even in bush-like ecosystems. In contrast, females generally did not disperse further than 20 km, with two distinctive RS-3 gene clusters for northern and southern females indicating no or rare long-distance female dispersal. However, dispersal rate for the predominantly non-territorial females from southern Kruger (fraction dispersers ≥0.68) was higher than previously reported. Of relevance was the below-average body condition of dispersers and their low presence in prides, suggesting low fitness. CONCLUSIONS: Large genetic differences between the two sampling localities, and low relatedness among males and high dispersal rates among females in the south, suggestive of unstable territory structure and high pride turnover, have potential implications for spread of diseases and the management of the Kruger lion population.

Attempted molecular detection of the thermally dimorphic human fungal pathogen Emergomyces africanus in terrestrial small mammals in South Africa.

The ecological niche of Emergomyces africanus (formerly Emmonsia species), a dimorphic fungus that causes an AIDS-related mycosis in South Africa, is unknown. We hypothesized that natural infection with E. africanus occurs in wild small mammals. Using molecular detection with primers specific for E. africanus, we examined 1402 DNA samples from 26 species of mole-rats, rodents, and insectivores trapped in South Africa that included 1324 lung, 37 kidney, and 41 liver specimens. DNA of E. africanus was not detected in any animals. We conclude that natural infection of wild small mammals in South Africa with E. africanus has not been proven.

Virus genome dynamics under different propagation pressures: reconstruction of whole genome haplotypes of West Nile viruses from NGS data.

BACKGROUND: Extensive focus is placed on the comparative analyses of consensus genotypes in the study of West Nile virus (WNV) emergence. Few studies account for genetic change in the underlying WNV quasispecies population variants. These variants are not discernable in the consensus genome at the time of emergence, and the maintenance of mutation-selection equilibria of population variants is greatly underestimated. The emergence of lineage 1 WNV strains has been studied extensively, but recent epidemics caused by lineage 2 WNV strains in Hungary, Austria, Greece and Italy emphasizes the increasing importance of this lineage to public health. In this study we explored the quasispecies dynamics of minority variants that contribute to cell-tropism and host determination, i.e. the ability to infect different cell types or cells from different species from Next Generation Sequencing (NGS) data of a historic lineage 2 WNV strain. RESULTS: Minority variants contributing to host cell membrane association persist in the viral population without contributing to the genetic change in the consensus genome. Minority variants are shown to maintain a stable mutation-selection equilibrium under positive selection, particularly in the capsid gene region. CONCLUSIONS: This study is the first to infer positive selection and the persistence of WNV haplotype variants that contribute to viral fitness without accompanying genetic change in the consensus genotype, documented solely from NGS sequence data. The approach used in this study streamlines the experimental design seeking viral minority variants accurately from NGS data whilst minimizing the influence of associated sequence error.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. Methods: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. Results: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. Conclusions: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Genome Sequencing of Historical Encephalomyocarditis Viruses from South Africa Links the Historical 1993/4 Savanna Elephant (Loxodonta africana) Outbreak to Cryptic Mastomys Rodents.

From 1993 to 1994, 64 free-ranging elephants (Loxodonta africana) succumbed to encephalomyocarditis in the Kruger National Park, South Africa, of which 83% were adult bulls. Mastomys rodents were implicated as the reservoir host of the Encephalomyocarditis virus (EMCV) based on serology and RT-PCR. However, in the absence of sequence-confirmation of both the virus and the rodent host, definitive links between the elephant outbreak strains and rodent reservoir could not be established. In this study, we generate the first reference genome sequences for three historical EMCVs isolated from two Mastomys rodents and one Mastomys-associated mite, Laelaps muricola, in Gauteng Province, South Africa, in 1961. In addition, near-complete genome sequences were generated for two elephant outbreak virus strains, for which data were previously limited to the P1 and 3D genome regions. The consensus sequence of each virus was determined using a PCR-Sanger sequencing approach. Phylogenetic analysis confirmed the three near-identical (99.95-99.97%) Mastomys-associated viruses to be sister to the two near-identical (99.85%) elephant outbreak strains, differing from each other at 6.4% of sites across the ~7400-nucleotide region characterised. This study demonstrates a link between Mastomys-associated viruses and the historical elephant outbreak strains and implicates Mastomys as reservoirs of EMCV in South Africa.

African swine fever virus NAM P1/95 is a mixture of genotype I and genotype VIII viruses.

African swine fever virus causes a lethal hemorrhagic disease of domestic pigs. The NAM P1/1995 isolate was originally described as B646L genotype XVIII; however, full genome sequencing revealed that this assignment was incorrect.

Phytochemical analysis and in-vitro anti-African swine fever virus activity of extracts and fractions of Ancistrocladus uncinatus, Hutch and Dalziel (Ancistrocladaceae).

BACKGROUND: African swine fever (ASF), a highly contagious fatal acute haemorrhagic viral disease of pigs currently has no treatment or vaccination protocol and it threatens the pig industry worldwide. Recent outbreaks were managed by farmers with ethnoveterinary preparations with various claims of effectiveness. RESULTS: We identified 35 compounds using GC-MS protocol and ASF virus (NIG 99) was significantly reduced by some extracts and fractions of the plant. However, the plant was poorly extracted by water and cytotoxicity was found to be a major problem with the use of the plant since its extracts also reduced the primary cells used in the assay. CONCLUSION: It is confirmed that the plant has antiviral potentials against ASF virus and farmers' claims seem to have certain degree of veracity, but finding the best means of exploring the potential of the plant while reducing its cytotoxic effect in-vitro and in-vivo will be necessary.

Phylogenomic Comparison of Seven African Swine Fever Genotype II Outbreak Viruses (1998-2019) Reveals the Likely African Origin of Georgia 2007/1.

Since the initial report of African swine fever (ASF) in Kenya in 1921, the disease has predominantly been confined to Africa. However, in 2007, an ASF genotype II virus of unknown provenance was introduced to Georgia. This was followed by its rampant spread to 73 countries, and the disease is now a global threat to pig production, with limited effective treatment and vaccine options. Here, we investigate the origin of Georgia 2007/1 through genome sequencing of three viruses from outbreaks that predated the genotype II introduction to the Caucasus, namely Madagascar (MAD/01/1998), Mozambique (MOZ/01/2005), and Mauritius (MAU/01/2007). In addition, genome sequences were generated for viruses from East African countries historically affected by genotype II (Malawi (MAL/04/2011) and Tanzania (TAN/01/2011)) and newly invaded southern African countries (Zimbabwe (ZIM/2015) and South Africa (RSA/08/2019). Phylogenomic analyses revealed that MOZ/01/2005, MAL/04/2011, ZIM/2015 and RSA/08/2019 share a recent common ancestor with Georgia 2007/1 and that none contain the large (~550 bp) deletion in the MGT110 4L ORF observed in the MAD/01/1998, MAU/01/2007 and TAN/01/2011 isolates. Furthermore, MOZ/01/2005 and Georgia 2007/1 only differ by a single synonymous SNP in the EP402R ORF, confirming that the closest link to Georgia 2007/1 is a virus that was circulating in Mozambique in 2005.

Retrospective Multi-Locus Sequence Analysis of African Swine Fever Viruses by "PACT" Confirms Co-Circulation of Multiple Outbreak Strains in Uganda.

African swine fever (ASF) is a haemorrhagic fever of swine that severely constrains pig production, globally. In Uganda, at least 388 outbreaks of ASF were documented from 2001 to 2012. We undertook a retrospective serological and molecular survey of ASF virus (ASFV) using banked samples collected from seven districts (Pallisa, Lira, Abim, Nebbi, Kabarole, Kibaale, and Mukono) of Uganda. Six assays (ELISA for antibody detection, diagnostic p72 gene PCR and genomic amplification, and sequencing of four gene regions (p72 [P], p54 [A], CVR of the 9RL-ORF [C], and TK [T]), hereinafter referred to as P-A-C-T (PACT)) were evaluated. Antibodies to ASFV were detected in the Abim district (6/25; 24.0%), and the remainder of the serum samples were negative (187/193; 96.9%). For the tissue samples, ASFV detection by assay was 8.47% for P, 6.78% for A, 8.47% for C, and 16.95% for T. The diagnostic PCR (p72 gene) detected seven positive animals from four districts, whereas the TK assay detected ten positives from all seven districts. In addition to the superior detection capability of TK, two virus variants were discernible, whereas CVR recovered three variants, and p72 and p54 sequencing each identified a single variant belonging to genotype IX. Our results indicate that dependence on serology alone underestimates ASF positivity in any infected region, that multi-locus sequence analysis provides better estimates of outbreak strain diversity, and that the TK assay is superior to the WOAH-prescribed conventional p72 diagnostic PCR, and warrants further investigation.

Experimental Infection of Domestic Pigs (Sus scrofa) with Rift Valley Fever Virus.

Rift valley fever (RVF), caused by the RVF virus (RVFV), is a vector-borne zoonotic disease that primarily affects domestic ruminants. Abortion storms and neonatal deaths characterise the disease in animals. Humans develop flu-like symptoms, which can progress to severe disease. The susceptibility of domestic pigs (Sus scrofa domesticus) to RVFV remains unresolved due to conflicting experimental infection results. To address this, we infected two groups of pregnant sows, neonates and weaners, each with a different RVFV isolate, and a third group of weaners with a mixture of the two viruses. Serum, blood and oral, nasal and rectal swabs were collected periodically, and two neonates and a weaner from group 1 and 2 euthanised from 2 days post infection (DPI), with necropsy and histopathology specimens collected. Sera and organ pools, blood and oronasorectal swabs were tested for RVFV antibodies and RNA. Results confirmed that pigs can be experimentally infected with RVFV, although subclinically, and that pregnant sows can abort following infection. Presence of viral RNA in oronasorectal swab pools on 28 DPI suggest that pigs may shed RVFV for at least one month. It is concluded that precautions should be applied when handling pig body fluids and carcasses during RVF outbreaks.

Phlebovirus diversity in ticks from livestock in arid ecologies in Kenya.

Phleboviruses are emerging pathogens of public health importance. However, their association with ticks is poorly described, particularly in Africa. Here, adult ticks infesting cattle, goats and sheep were collected in two dryland pastoralist ecosystems of Kenya (Baringo and Kajiado counties) and were screened for infection with phleboviruses. Ticks mainly belonged to the species Rhipicephalus appendiculatus, Hyalomma impeltatum, and Hyalomma rufipes. A fragment of the RNA-dependent RNA polymerase (RdRp) gene was identified in thirty of 671 tick pools, of which twenty-nine were from livestock sampled in Baringo county. Phylogenetic analyses revealed that twenty-five sequences were falling in three clades within the group of tick-associated phleboviruses. The sequences of the three clades showed nucleotide distances 8%, 19% and 22%, respectively, to previously known viruses suggesting that these sequence fragments may belong to three distinct viruses. Viruses of the group of tick-associated phleboviruses have been found in several countries and continents but so far have not been associated with disease in humans or animals. In addition, five sequences were found to group with the sandfly-associated phleboviruses Bogoria virus, Perkerra virus and Ntepes virus recently detected in the same region. Further studies are needed to investigate the transmission and maintenance cycles of these viruses, as well as to assess their potential to infect vertebrates.

Analyses of Mosquito Species Composition, Blood-Feeding Habits and Infection with Insect-Specific Flaviviruses in Two Arid, Pastoralist-Dominated Counties in Kenya.

Insect-specific flaviviruses (ISFs), although not known to be pathogenic to humans and animals, can modulate the transmission of arboviruses by mosquitoes. In this study, we screened 6665 host-seeking, gravid and blood-fed mosquitoes for infection with flaviviruses and assessed the vertebrate hosts of the blood-fed mosquitoes sampled in Baringo and Kajiado counties; both dryland ecosystem counties in the Kenyan Rift Valley. Sequence fragments of two ISFs were detected. Cuacua virus (CuCuV) was found in three blood-fed Mansonia (Ma.) africana. The genome was sequenced by next-generation sequencing (NGS), confirming 95.8% nucleotide sequence identity to CuCuV detected in Mansonia sp. in Mozambique. Sequence fragments of a potential novel ISF showing nucleotide identity of 72% to Aedes flavivirus virus were detected in individual blood-fed Aedes aegypti, Anopheles gambiae s.l., Ma. africana and Culex (Cx.) univittatus, all having fed on human blood. Blood-meal analysis revealed that the collected mosquitoes fed on diverse hosts, primarily humans and livestock, with a minor representation of wild mammals, amphibians and birds. The potential impact of the detected ISFs on arbovirus transmission requires further research.

An Updated Review of Ornithodoros Ticks as Reservoirs of African Swine Fever in Sub-Saharan Africa and Madagascar.

This updated review provides an overview of the available information on Ornithodoros ticks as reservoirs and biological vectors of the ASF virus in Africa and Indian Ocean islands in order to update the current knowledge in this field, inclusive of an overview of available methods to investigate the presence of ticks in the natural environment and in domestic pig premises. In addition, it highlights the major areas of research that require attention in order to guide future investigations and fill knowledge gaps. The available information suggests that current knowledge is clearly insufficient to develop risk-based control and prevention strategies, which should be based on a sound understanding of genotype distribution and the potential for spillover from the source population. Studies on tick biology in the natural and domestic cycle, including genetics and systematics, represent another important knowledge gap. Considering the rapidly changing dynamics affecting the African continent (demographic growth, agricultural expansion, habitat transformation), anthropogenic factors influencing tick population distribution and ASF virus (ASFV) evolution in Africa are anticipated and have been recorded in southern Africa. This dynamic context, together with the current global trends of ASFV dissemination, highlights the need to prioritize further investigation on the acarological aspects linked with ASF ecology and evolution.

Viral diversity and blood-feeding patterns of Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae).

Introduction: Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of arboviral pathogens that primarily affect livestock represented by Schmallenberg virus (SBV), epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). In Kenya, studies examining the bionomic features of Culicoides including species diversity, blood-feeding habits, and association with viruses are limited. Methods: Adult Culicoides were surveyed using CDC light traps in two semi-arid ecologies, Baringo and Kajiado counties, in Kenya. Blood-fed specimens were analysed through polymerase chain reaction (PCR) and sequencing of cytochrome oxidase subunit 1 (cox1) barcoding region. Culicoides pools were screened for virus infection by generic RT-PCR and next-generation sequencing (NGS). Results: Analysis of blood-fed specimens confirmed that midges had fed on cattle, goats, sheep, zebra, and birds. Cox1 barcoding of the sampled specimens revealed the presence of known vectors of BTV and epizootic hemorrhagic disease virus (EHDV) including species in the Imicola group (Culicoides imicola) and Schultzei group (C. enderleni, C. kingi, and C. chultzei). Culicoides leucostictus and a cryptic species distantly related to the Imicola group were also identified. Screening of generated pools (11,006 individuals assigned to 333 pools) by generic RT-PCR revealed presence of seven phylogenetically distinct viruses grouping in the genera Goukovirus, Pacuvirus and Orthobunyavirus. The viruses showed an overall minimum infection rate (MIR) of 7.0% (66/333, 95% confidence interval (CI) 5.5-8.9). In addition, full coding sequences of two new iflaviruses, tentatively named Oloisinyai_1 and Oloisinyai_2, were generated by next-generation sequencing (NGS) from individual homogenate of Culicoides pool. Conclusion: The results indicate a high genetic diversity of viruses in Kenyan biting midges. Further insights into host-vector-virus interactions as well as investigations on the potential clinical significance of the detected viruses are warranted.

Risk assessment of urban yellow fever virus transmission in Kenya: is Aedes aegypti an efficient vector?

The absence of urban yellow fever epidemics in East Africa remains a mystery amidst the proliferation of Aedes aegypti in this region. To understand the transmission dynamics of the disease, we tested urban (Mombasa, Kisumu, and Nairobi) Aedes mosquito populations in Kenya for their susceptibility to an East African yellow fever virus (YFV) genotype. Overall, 22% (n = 805) of the Ae. aegypti that were orally challenged with an infectious dose of YFV had a midgut infection, with comparable rates for Mombasa and Kisumu (χ2 = 0.35, df = 1, P = 0.55), but significantly lower rates for Nairobi (χ2 ≥ 11.08, df = 1, P ≤ 0.0009). Variations in YFV susceptibility (midgut infection) among Ae. aegypti subspecies were not associated with discernable cytochrome c oxidase subunit 1 gene haplotypes. Remarkably, no YFV dissemination or transmission was observed among the orally challenged Ae. aegypti populations. Moreover, Ae. aegypti mosquitoes that were intrathoracically inoculated with YFV failed to transmit the virus via capillary feeding. In contrast, dissemination (oral exposure) and transmission (intrathoracic inoculation) of YFV was observed among a few peri-domestic Ae. bromeliae mosquitoes (n = 129) that were assessed from these urban areas. Our study highlights an inefficient urban Ae. aegypti population, and the potential for Ae. bromeliae in sustaining an urban YFV transmission in Kenya. An assessment of urban Ae. aegypti susceptibility to other YFV genotypes, and vector potential of urban Ae. bromeliae populations in Kenya is recommended to guide cost-effective vaccination.

Haemoplasma Prevalence and Diversity in Three Invasive Rattus Species from Gauteng Province, South Africa.

Invasive Rattus species are carriers of haemotropic Mycoplasmas (haemoplasmas) globally, but data from Africa are lacking. Using a PCR-sequencing approach, we assessed haemoplasma prevalence and diversity in kidney and buccal swabs collected from three invasive Rattus species (Rattus rattus, R. norvegicus and R. tanezumi) in Gauteng Province, South Africa. Whilst the overall sequence-confirmed haemoplasma prevalence was 38.4%, infection rates in R. rattus (58.3%) were significantly higher (χ2 = 12.96; df = 2; n = 99 p < 0.05) than for R. tanezumi (14.3%). Differences between host sex (χ2 = 3.59 × 10−31; df = 1; n = 99; p = 1.00) and age (χ2 = 4.28; df = 2; n = 99; p = 0.12) were not significant. Whilst buccal (1.01%) and ectoparasite positivity (2.13%) were low, these results suggest that multiple transmission routes are possible. Three phylogenetically distinct lineages, consistent with global rat-associated strains described to date, were detected, namely, 'Candidatus Mycoplasma haemomuris subsp. Ratti', and two Rattus-specific haemoplasmas that are yet to be formally described. These results expand the known distribution of invasive rat-associated haemoplasmas and highlight the potential for pathogen co-invasion of new territories together with invading rodent hosts.