Identification and RNAi Profile of a Novel Iflavirus Infecting Senegalese Aedes vexans arabiensis Mosquitoes.

The inland floodwater mosquito Aedes vexans (Meigen, 1830) is a competent vector of numerous arthropod-borne viruses such as Rift Valley fever virus (Phenuiviridae) and Zika virus (Flaviviridae). Aedes vexans spp. have widespread Afrotropical distribution and are common European cosmopolitan mosquitoes. We examined the virome of Ae. vexans arabiensis samples from Barkédji village, Senegal, with small RNA sequencing, bioinformatic analysis, and RT-PCR screening. We identified a novel 9494 nt iflavirus (Picornaviridae) designated here as Aedes vexans iflavirus (AvIFV). Annotation of the AvIFV genome reveals a 2782 amino acid polyprotein with iflavirus protein domain architecture and typical iflavirus 5' internal ribosomal entry site and 3' poly-A tail. Aedes vexans iflavirus is most closely related to a partial virus sequence from Venturia canescens (a parasitoid wasp) with 56.77% pairwise amino acid identity. Analysis of AvIFV-derived small RNAs suggests that AvIFV is targeted by the exogenous RNA interference pathway but not the PIWI-interacting RNA response, as ~60% of AvIFV reads corresponded to 21 nt Dicer-2 virus-derived small RNAs and the 24-29 nt AvIFV read population did not exhibit a "ping-pong" signature. The RT-PCR screens of archival and current (circa 2011-2020) Ae. vexans arabiensis laboratory samples and wild-caught mosquitoes from Barkédji suggest that AvIFV is ubiquitous in these mosquitoes. Further, we screened wild-caught European Ae. vexans samples from Germany, the United Kingdom, Italy, and Sweden, all of which tested negative for AvIFV RNA. This report provides insight into the diversity of commensal Aedes viruses and the host RNAi response towards iflaviruses.

Biological and phylogenetic characteristics of West African lineages of West Nile virus.

The West Nile virus (WNV), isolated in 1937, is an arbovirus (arthropod-borne virus) that infects thousands of people each year. Despite its burden on global health, little is known about the virus' biological and evolutionary dynamics. As several lineages are endemic in West Africa, we obtained the complete polyprotein sequence from three isolates from the early 1990s, each representing a different lineage. We then investigated differences in growth behavior and pathogenicity for four distinct West African lineages in arthropod (Ap61) and primate (Vero) cell lines, and in mice. We found that genetic differences, as well as viral-host interactions, could play a role in the biological properties in different WNV isolates in vitro, such as: (i) genome replication, (ii) protein translation, (iii) particle release, and (iv) virulence. Our findings demonstrate the endemic diversity of West African WNV strains and support future investigations into (i) the nature of WNV emergence, (ii) neurological tropism, and (iii) host adaptation.

Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

BACKGROUND: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. CONCLUSIONS/SIGNIFICANCE: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.

Distinct lineages of Ebola virus in Guinea during the 2014 West African epidemic.

An epidemic of Ebola virus disease of unprecedented scale has been ongoing for more than a year in West Africa. As of 29 April 2015, there have been 26,277 reported total cases (of which 14,895 have been laboratory confirmed) resulting in 10,899 deaths. The source of the outbreak was traced to the prefecture of Guéckédou in the forested region of southeastern Guinea. The virus later spread to the capital, Conakry, and to the neighbouring countries of Sierra Leone, Liberia, Nigeria, Senegal and Mali. In March 2014, when the first cases were detected in Conakry, the Institut Pasteur of Dakar, Senegal, deployed a mobile laboratory in Donka hospital to provide diagnostic services to the greater Conakry urban area and other regions of Guinea. Through this process we sampled 85 Ebola viruses (EBOV) from patients infected from July to November 2014, and report their full genome sequences here. Phylogenetic analysis reveals the sustained transmission of three distinct viral lineages co-circulating in Guinea, including the urban setting of Conakry and its surroundings. One lineage is unique to Guinea and closely related to the earliest sampled viruses of the epidemic. A second lineage contains viruses probably reintroduced from neighbouring Sierra Leone on multiple occasions, while a third lineage later spread from Guinea to Mali. Each lineage is defined by multiple mutations, including non-synonymous changes in the virion protein 35 (VP35), glycoprotein (GP) and RNA-dependent RNA polymerase (L) proteins. The viral GP is characterized by a glycosylation site modification and mutations in the mucin-like domain that could modify the outer shape of the virion. These data illustrate the ongoing ability of EBOV to develop lineage-specific and potentially phenotypically important variation.

Validation of a structural comparison of the antigenic characteristics of Usutu virus and West Nile virus envelope proteins.

Cross-reactions observed in serological assays between Usutu virus (USUV), the USUV outlier subtype strain CAR_1969 and West Nile virus (WNV) suggest that they share antigenic features amongst their structural outer proteins especially envelope (E) proteins. To investigate the molecular background of this observation, we compared the E protein sequences of seven USUV strains, USUV subtype strain CAR_1969 and WNV strain 2471, focusing on the binding site defined by the WNV neutralizing antibody E16. USUV SouthAfrica_1959 differs from WNV 2741 in three of four residues critical for E16 antibody binding and five of the 12 additionally involved residues. In contrast, USUV subtype CAR_1969 differs from WNV 2741 in two critical residues and five additional residues. Furthermore, USUV subtype CAR_1969 differs from other USUV strains in two critical residues. E16 antibody binding has previously been shown to be highly specific for WNV; thus, the observed variation in amino acid residues suggests that the region corresponding to the WNV E16 epitope is probably not responsible for the observed cross-reactions between WNV and USUV. Seroneutralisation assays confirmed these findings for WNV and USUV, however, showed occurring cross-reactivity between WNV and USUV subtype CAR_1969 at high antibody titers. The sequence diversity in this region might also explain some of the observed different antigenic characteristics of USUV strains and USUV subtype CAR_1969. A therapeutic effect of E16 antibody has been described in WNV infected mice; therefore, a USUV specific antibody generated against the region corresponding to the WNV E16 binding site might represent an approach for treating USUV infections.

Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite Plasmodium falciparum.

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.