Dissemination In Mosquitoes Research Articles

Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.

A microsporidian impairs Plasmodium falciparum transmission in Anopheles arabiensis mosquitoes

A possible malaria control approach involves the dissemination in mosquitoes of inherited symbiotic microbes to block Plasmodium transmission. However, in the Anopheles gambiae complex, the primary African vectors of malaria, there are limited reports of inherited symbionts that impair transmission. We show that a vertically transmitted microsporidian symbiont (Microsporidia MB) in the An. gambiae complex can impair Plasmodium transmission. Microsporidia MB is present at moderate prevalence in geographically dispersed populations of An. arabiensis in Kenya, localized to the mosquito midgut and ovaries, and is not associated with significant reductions in adult host fecundity or survival. Field-collected Microsporidia MB infected An. arabiensis tested negative for P. falciparum gametocytes and, on experimental infection with P. falciparum, sporozoites aren’t detected in Microsporidia MB infected mosquitoes. As a microbe that impairs Plasmodium transmission that is non-virulent and vertically transmitted, Microsporidia MB could be investigated as a strategy to limit malaria transmission.

Increased Mosquito Midgut Infection by Dengue Virus Recruitment of Plasmin Is Blocked by an Endogenous Kazal-type Inhibitor.

SummaryDengue symptoms include alteration of blood coagulation and fibrinolysis, causing severe hemorrhage and death. Here, we demonstrate that higher concentration of plasmin, the human fibrinolytic factor, in blood meal enhances dengue virus (DENV) infection in mosquito midgut and dissemination in mosquitoes. We also show that mosquitoes express a plasmin-selective Kazal-type inhibitor (AaTI) in the midgut to inhibit plasmin proteolysis and revert the enhanced infection. Using bio-layer interferometry, we show that DENV, plasmin, and AaTI interact to form a tripartite complex. Eventually, plasmin increases midgut internalization of dextran molecules and this is reverted by AaTI. Our study demonstrates that (1) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glycocalyx and enhancing DENV internalization and (2) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes.

Dengue Virus Recruitment of Plasmin Proteolysis Increases Mosquito Midgut Internalization, Enhancing Infection Onset, and this Can Be Blocked by an Endogenous Kazal-Type Inhibitor

Deciphering the impact of dengue pathogenesis on vector transmission is critical for understanding virulence evolution, epidemiology and developing transmission-blocking tools. Dengue infection can alter blood coagulation and fibrinolysis pathways, resulting in severe hemorrhage and death. Here, we demonstrate that when plasmin, the human fibrinolytic factor, is added to blood meal, it enhances DENV infection in mosquito midgut, resulting in higher dissemination in mosquitoes. In addition, the mosquito midgut-expressed AaTI, a plasmin-selective mosquito Kazal-type inhibitor, reverted this enhanced infection by inhibiting plasmin proteolysis in the midgut lumen. Using bio-layer interferometry, we showed that DENV, plasmin and AaTI interact to form a tripartite complex. Eventually, plasmin supplementation in infectious blood meal increased midgut internalization of dextran molecules and this was reverted by AaTI. Our study demonstrates that: (a) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glyocalyx and enhancing DENV infection onset, and (b) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes.

First evidence of the presence of genotype-1 of Japanese encephalitis virus in Culex gelidus in Indonesia

BackgroundJapanese encephalitis has become a public health threat in Indonesia. Three genotypes have been recorded in Indonesia, i.e. genotype II (GII), genotype III (GIII) and genotype IV (GIV). Genotype I (GI) and genotype V (GV) have never been reported in Indonesia.ResultsA Japanese encephalitis virus (JEV) belonging to the genotype I-a (GI-a) has been isolated for the first time from a Culex gelidus mosquito in the Province of Jambi, Indonesia. This virus is related to a 1983 isolate from Thailand whereas the infected Cx. gelidus mosquito belonged to a Chinese haplotype.ConclusionsSurveillance of JEV and mosquito dissemination is recommended.

Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes.

BackgroundChikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear.Methodology / Principal findingsHere, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti.Conclusions / SignificanceThe P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies.

Mutagenesis analysis of T380R mutation in the envelope protein of yellow fever virus

BackgroundThe RGD motif in the mosquito-borne flaviviruses envelope protein domain III (EDIII) FG loop was shown to bind negatively charged cellular molecules and mediate virus entry in mammals. However, its importance in virus entry in the mosquito has not yet been defined. The sequences of RGD motifs are conserved in JEV-serocomplex members primarily transmitted by Culex mosquitoes but absent from members of the DENV serocomplex, which utilize Aedes mosquitoes as vectors. Interestingly, the RGD sequence is present in the attenuated 17D strain of yellow fever virus as a result of the T380R mutation in the EDIII of Asibi strain following extensive in vitro passage in mice and chicken embryos and was found to contribute to the more rapid clearance in mice challenged with 17D. However, viral infectivity and dissemination in mosquitoes had not been evaluated for this mutant.FindingsThe study utilized the reverse genetics system of YFV and Ae. aegypti RexD WE mosquitoes to assess the impact of a T380R mutation in YFV Asibi and 17D/Asibi M-E chimera. The T380R mutation led to higher infection rates but similar dissemination rates when introduced into the YFV Asibi strain and 17D/Asibi M-E chimera.ConclusionsWhile the increase of the positive charge in EDIII may reduce the virulence of YFV in mice, this mutation favored the establishment of the viral infection in Ae. aegypti. However, such gain in viral infectivity did not increase dissemination in infected mosquitoes.

The Rift Valley fever accessory proteins NSm and P78/NSm-GN are distinct determinants of virus propagation in vertebrate and invertebrate hosts

Rift Valley fever virus (RVFV) is an enzootic virus circulating in Africa that is transmitted to its vertebrate host by a mosquito vector and causes severe clinical manifestations in humans and ruminants. RVFV has a tripartite genome of negative or ambisense polarity. The M segment contains five in-frame AUG codons that are alternatively used for the synthesis of two major structural glycoproteins, GN and GC, and at least two accessory proteins, NSm, a 14-kDa cytosolic protein, and P78/NSm-GN, a 78-kDa glycoprotein. To determine the relative contribution of P78 and NSm to RVFV infectivity, AUG codons were knocked out to generate mutant viruses expressing various sets of the M-encoded proteins. We found that, in the absence of the second AUG codon used to express NSm, a 13-kDa protein corresponding to an N-terminally truncated form of NSm, named NSm′, was synthesized from AUG 3. None of the individual accessory proteins had any significant impact on RVFV virulence in mice. However, a mutant virus lacking both NSm and NSm′ was strongly attenuated in mice and grew to reduced titers in murine macrophages, a major target cell type of RVFV. In contrast, P78 was not associated with reduced viral virulence in mice, yet it appeared as a major determinant of virus dissemination in mosquitoes. This study demonstrates how related accessory proteins differentially contribute to RVFV propagation in mammalian and arthropod hosts.

Effects of Manipulating Apoptosis on Sindbis Virus Infection of Aedes aegypti Mosquitoes

Improved control of vector-borne diseases requires an understanding of the molecular factors that determine vector competence. Apoptosis has been shown to play a role in defense against viruses in insects and mammals. Although some observations suggest a correlation between apoptosis and resistance to arboviruses in mosquitoes, there is no direct evidence tying apoptosis to arbovirus vector competence. To determine whether apoptosis can influence arbovirus replication in mosquitoes, we manipulated apoptosis in Aedes aegypti mosquitoes by silencing the expression of genes that either positively or negatively regulate apoptosis. Silencing of the A. aegypti anti-apoptotic gene iap1 (Aeiap1) caused apoptosis in midgut epithelium, alterations in midgut morphology, and 60 to 70% mosquito mortality. Mortality induced by Aeiap1 silencing was rescued by cosilencing the initiator caspase gene Aedronc, indicating that the mortality was due to apoptosis. When mosquitoes which had been injected with Aeiap1 double-stranded RNA (dsRNA) were orally infected with Sindbis virus (SINV), increased midgut infection and virus dissemination to other organs were observed. This increase in virus infection may have been due to the effects of widespread apoptosis on infection barriers or innate immunity. In contrast, silencing the expression of Aedronc, which would be expected to inhibit apoptosis, reduced SINV midgut infection and virus dissemination. Thus, our data suggest that some level of caspase activity and/or apoptosis may be necessary for efficient virus replication and dissemination in mosquitoes. This is the first study to directly test the roles of apoptosis and caspases in determining mosquito vector competence for arboviruses.

A role for endosomal proteins in alphavirus dissemination in mosquitoes

Little is known about endosomal pathway proteins involved in arthropod-borne virus (arbovirus) assembly and cell-to-cell spread in vector mosquitoes. UNC93A and synaptic vesicle-2 (SV2) proteins are involved in intracellular transport in mammals. They show amino acid sequence conservation from mosquitoes to humans, and their transcripts are highly enriched in Aedes aegypti during arbovirus infection. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant alphavirus Sindbis MRE16-enhanced green fluorescent protein (SINV; family Togaviridae) resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Further, UNC93A silencing, but not SV2 silencing, resulted in a 10-fold reduction in viral titres at 4 days post-infection. Together, these data support a role for UNC93A and SV2 in virus assembly or budding. Cis-regulatory elements (CREs) were identified at the 5'-ends of genes from the original data set in which SV2 and UNC93A were identified. Common CREs at the 5'-end genomic regions of a subset of enriched transcripts support the hypothesis that UNC93A transcription may be co-regulated with that of other ion transport and endosomal trafficking proteins.

Glycosylation of the West Nile Virus Envelope Protein Increases In Vivo and In Vitro Viral Multiplication in Birds

Many West Nile (WN) virus isolates associated with significant outbreaks possess a glycosylation site on the envelope (E) protein. E-protein glycosylated variants of New York (NY) strains of WN virus are more neuroinvasive in mice than the non-glycosylated variants. To determine how E protein glycosylation affects the interactions between WN virus and avian hosts, we inoculated young chicks with NY strains of WN virus containing either glycosylated or non-glycosylated variants of the E protein. The glycosylated variants were more virulent and had higher viremic levels than the non-glycosylated variants. The glycosylation status of the variant did not affect viral multiplication and dissemination in mosquitoes in vivo. Glycosylated variants showed more heat-stable propagation than non-glycosylated variants in mammalian (BHK) and avian (QT6) cells but not in mosquito (C6/36) cells. Thus, E-protein glycosylation may be a requirement for efficient transmission of WN virus from avian hosts to mosquito vectors.

Role of the yellow fever virus structural protein genes in viral dissemination from the Aedes aegypti mosquito midgut

Live-attenuated virus vaccines are key components in controlling arboviral diseases, but they must not disseminate in or be transmitted by mosquito vectors. Although the cycles in which many mosquito-borne viruses are transmitted are well understood, the role of viral genetics in these processes has not been fully elucidated. Yellow fever virus (YFV) is an important arbovirus and the prototype member of the family Flaviviridae. Here, YFV was used in Aedes aegypti mosquitoes as a model to investigate the genetic basis of infection and dissemination in mosquitoes. Viruses derived from infectious clones and chimeric viruses with defined sequential manipulations were used to investigate the influence of specific sequences within the membrane and envelope structural protein genes on dissemination of virus from the mosquito midgut. Substitution of domain III of the envelope protein from a midgut-restricted YFV into a wild-type YFV resulted in a marked decrease in virus dissemination, suggesting an important role for domain III in this process. However, synergism between elements within the flavivirus structural and non-structural protein genes may be necessary for efficient virus escape from the mosquito midgut.

A live attenuated recombinant dengue-4 virus vaccine candidate with restricted capacity for dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes.

2Adelta30 is a live dengue-4 virus vaccine candidate with a 30-nucleotide deletion in its 3'-untranslated region. To assess the transmissibility of 2Adelta30 by mosquitoes, we compared its in vivo replication in mosquitoes with that of its wild type DEN-4 parent. Both the vaccine candidate and wild type virus were equally able to infect the mosquito Toxorhynchites splendens after intrathoracic inoculation. Relative to its wild type parent, 2Adelta30 was slightly restricted in its ability to infect the midgut of Aedes aegypti mosquitoes fed on an artificial blood meal and was even more restricted in its ability to disseminate from the midgut to the salivary glands. Thus, the 30-nucleotide deletion rendered the vaccine candidate more sensitive than its wild type parent to the mosquito midgut escape barrier. Most significantly, 2Adelta30 was not transmitted to 352 Ae. albopictus mosquitoes fed on 10 vaccinees, all of whom were infected with the vaccine candidate.