Vertical transmission of naturally occurring Bunyamwera and insect-specific flavivirus infections in mosquitoes from islands and mainland shores of Lakes Victoria and Baringo in Kenya.

BackgroundMany arboviruses transmitted by mosquitoes have been implicated as causative agents of both human and animal illnesses in East Africa. Although epidemics of arboviral emerging infectious diseases have risen in frequency in recent years, the extent to which mosquitoes maintain pathogens in circulation during inter-epidemic periods is still poorly understood. This study aimed to investigate whether arboviruses may be maintained by vertical transmission via immature life stages of different mosquito vector species.MethodologyWe collected immature mosquitoes (egg, larva, pupa) on the shores and islands of Lake Baringo and Lake Victoria in western Kenya and reared them to adults. Mosquito pools (≤25 specimens/pool) of each species were screened for mosquito-borne viruses by high-resolution melting analysis and sequencing of multiplex PCR products of genus-specific primers (alphaviruses, flaviviruses, phleboviruses and Bunyamwera-group orthobunyaviruses). We further confirmed positive samples by culturing in baby hamster kidney and Aedes mosquito cell lines and re-sequencing.Principal findingsCulex univittatus (2/31pools) and Anopheles gambiae (1/77 pools) from the Lake Victoria region were positive for Bunyamwera virus, a pathogenic virus that is of public health concern. In addition, Aedes aegypti (3/50), Aedes luteocephalus (3/13), Aedes spp. (2/15), and Culex pipiens (1/140) pools were positive for Aedes flaviviruses at Lake Victoria, whereas at Lake Baringo, three pools of An. gambiae mosquitoes were positive for Anopheles flavivirus. These insect-specific flaviviruses (ISFVs), which are presumably non-pathogenic to vertebrates, were found in known medically important arbovirus and malaria vectors.ConclusionsOur results suggest that not only ISFVs, but also a pathogenic arbovirus, are naturally maintained within mosquito populations by vertical transmission, even in the absence of vertebrate hosts. Therefore, virus and vector surveillance, even during inter-epidemics, and the study of vector-arbovirus-ISFV interactions, may aid in identifying arbovirus transmission risks, with the potential to inform control strategies that lead to disease prevention

Number of bloodmeal sources of mosquito species sampled in Baringo County.

<p>N = Number of mosquitoes analyzed, ND = Not determined.</p><p>^#mixed blood meal cases.</p><p>*virus positive cases.</p><p>Number of bloodmeal sources of mosquito species sampled in Baringo County.</p

Melt rate profiles of Bunyamwera S segment amplicons detected in blood-fed mosquitoes.

<p>Melt rate profiles of Bunyamwera S segment amplicons detected in blood-fed mosquitoes.</p

Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface

<div><p>The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (<i>cyt b</i>) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of <i>cyt b</i> PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both <i>cyt b</i> and 16S ribosomal RNA genes. Among 445 blood-fed <i>Aedeomyia</i>, <i>Aedes</i>, <i>Anopheles</i>, <i>Culex</i>, <i>Mansonia</i>, and <i>Mimomyia</i> mosquitoes from Kenya’s Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both <i>cyt b</i> and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a <i>Culex pipiens</i> from Mbita that had fed on a goat and a human and in two <i>Mansonia africana</i> mosquitoes from Baringo that each had fed on a rodent (<i>Arvicanthis niloticus</i>) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in <i>Culex</i>, <i>Aedeomyia</i>, <i>Anopheles</i> and <i>Mansonia</i> mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies.</p></div

Number of bloodmeal sources of mosquito species sampled in Homa Bay County.

<p>N = Number of mosquitoes analyzed, ND = Not determined.</p><p>^#mixed blood meal cases.</p><p>Number of bloodmeal sources of mosquito species sampled in Homa Bay County.</p

Numbers of blood-fed mosquito species captured on sampling sites of Homa Bay and Baringo Counties of Kenya.

<p>Numbers of blood-fed mosquito species captured on sampling sites of Homa Bay and Baringo Counties of Kenya.</p

Vertebrate mitochondrial genomes aligned for primer design.

<p>Vertebrate mitochondrial genomes aligned for primer design.</p

Melt rates of mosquito <i>cyt b</i> amplicons alongside selected vertebrate bloodmeal amplicons.

<p>Species in the legend are ordered from their lowest to highest melting temperatures.</p

Number of bloodmeal sources of mosquito species sampled in Baringo County.

<p>N = Number of mosquitoes analyzed, ND = Not determined.</p><p>^#mixed blood meal cases.</p><p>*virus positive cases.</p><p>Number of bloodmeal sources of mosquito species sampled in Baringo County.</p