Changing patterns of West Nile virus transmission: altered vector competence and host susceptibility

West Nile virus (WNV) is a flavivirus (Flaviviridae) transmitted between Culex spp. mosquitoes and avian hosts. The virus has dramatically expanded its geographic range in the past ten years. Increases in global commerce, climate change, ecological factors and the emergence of novel viral genotypes likely play significant roles in the emergence of this virus; however, the exact mechanism and relative importance of each is uncertain. Previously WNV was primarily associated with febrile illness of children in endemic areas, but it was identified as a cause of neurological disease in humans in 1994. This modulation in disease presentation could be the result of the emergence of a more virulent genotype as well as the progression of the virus into areas in which the age structure of immunologically naïve individuals makes them more susceptible to severe neurological disease. Since its introduction to North America in 1999, a novel WNV genotype has been identified that has been demonstrated to disseminate more rapidly and with greater efficiency at elevated temperatures than the originally introduced strain, indicating the potential importance of temperature as a selective criteria for the emergence of WNV genotypes with increased vectorial capacity. Even prior to the North American introduction, a mutation associated with increased replication in avian hosts, identified to be under adaptive evolutionary pressure, has been identified, indicating that adaptation for increased replication within vertebrate hosts could play a role in increased transmission efficiency. Although stable in its evolutionary structure, WNV has demonstrated the capacity for rapidly adapting to both vertebrate hosts and invertebrate vectors and will likely continue to exploit novel ecological niches as it adapts to novel transmission foci

Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae.

BackgroundSusceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.MethodsWe introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.ResultsThe MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.ConclusionMEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway

Animal Models of Zika Virus Sexual Transmission

ZIKV was first identified in the 1940s as a mosquito-borne virus; however, sexual transmission, which is uncommon for arboviruses, was demonstrated more than 60 years later. Tissue culture and animal models have allowed scientists to study how this transmission is possible. Immunocompromised mice infected with ZIKV had high viral loads in their testes, and infection of immunocompetent female mice was achieved following intravaginal inoculation or inoculation via mating with an infected male. These mouse studies lead researchers to investigate the individual components of the male reproductive system. In cell culture and mouse models, ZIKV can persist in Sertoli and germ cells of the testes and epithelial cells in the epididymis, which may lead to sexual transmission even after ZIKV has been cleared from other tissues. ZIKV has also been studied in nonhuman primates (NHPs), which appears to mimic the limited human epidemiological data, with low rates of symptomatic individuals and similar clinical signs. Although refinement is needed, these animal models have proven to be key in ZIKV research and continue to help uncovering the mechanisms of sexual transmission. This review will focus on the animal models used to elucidate the mechanisms of sexual transmission and persistence of flaviviruses

Evidence of Efficient Transovarial Transmission of Culex Flavivirus by Culex pipiens (Diptera: Culicidae)

This study determined the transovarial transmission (TOT) potential and tissue tropisms of Culex flavivirus (CxFV), an insect-specific flavivirus, in Culex pipiens (L.). Several hundred mosquito egg rafts were collected in the field, transferred to the insectaries, reared to the fourth larval instar, and identified using morphological characteristics. Cx. pipiens were reared to adults, allowed to oviposit in individual containers, and tested for CxFV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. Eighteen CxFV RNA-positive females were identified from 26 females that oviposited viable egg rafts. Thirty F1 adults from each positive female were individually tested by RT-PCR for CxFV RNA. Viral RNA was detected in 526 of 540 progeny, and thus, the filial infection rate was 97.4%. Because all 18 positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that efficient TOT of CxFV occurs in nature. To define the tissue tropisms of CxFV, different tissues (salivary glands, ovaries, testes, head, fat bodies, and midguts) were removed from the remainder of the F1 and tested by RT-PCR for CxFV RNA. Viral RNA was detected in all tissues. Additionally, uninfected laboratory-colonized Cx. pipiens were infected with CxFV by needle inoculation, and ovaries were collected at 4, 6, 8, and 12 d postinoculation and tested for CxFV RNA by RT-PCR. Viral RNA was detected at all time points, demonstrating that CxFV infects the ovaries as early as 4 d postinoculation. Surprisingly, however, we were unable to demonstrate transovarial transmission despite the presence of viral RNA in the ovaries. Nevertheless, the experiments performed with field-infected Cx. pipiens demonstrate that TOT is an efficient mechanism by which CxFV is maintained in mosquitoes in nature

Increases in the competitive fitness of West Nile virus isolates after introduction into California

To investigate the phenotypic evolution of West Nile virus (WNV) in California, we competed sixteen isolates made during 2007-08 against COAV997-5nt, a genetically marked clone from the founding 2003 California isolate COAV997-2003. Using in vivo fitness competitions in House Finches (HOFI) and Culex tarsalis mosquitoes, we found that the majority of WNV WN02 and SW03 genotype isolates exhibited elevated replicative fitness in both hosts compared to COAV997-5nt. Increased replicative capacity in HOFIs was not associated with increased mortality, indicating that these isolates had not gained avian virulence. One WN02 isolate from Coachella Valley, a region geographically close to the isolation of COAV997, showed neutral fitness in HOFIs and reduced fitness in Cx. tarsalis. Two isolates from Kern County and Sacramento/Yolo County out-competed COAV997-nt in HOFIs, but were transmitted less efficiently by Cx. tarsalis. Competition demonstrated neutral or increased fitness that appeared independent of both WN02 and SW03 genotypes

West Nile virus growth is independent of autophagy activation

AbstractWest Nile virus (WNV) is an arthropod-borne virus with a worldwide distribution that causes neurologic disease and death. Autophagy is a cellular homeostatic mechanism involved in antiviral responses but can be subverted to support viral growth as well. We show that autophagy is induced by WNV infection in cell culture and in primary neuron cultures. Following WNV infection, lysosomes co-localize with autophagosomes resulting in LC3B-II turnover and autolysosomal acidification. However, activation or inhibition of autophagy has no significant effect on WNV growth but pharmacologic inhibition of PI3 kinases associated with autophagy reduce WNV growth. Basal levels of p62/sequestosome1(SQSTM1) do not significantly change following WNV-induced autophagy activation, but p62 is turned over or degraded by autophagy activation implying that p62 expression is increased following WNV-infection. These data show that WNV-induces autophagy but viral growth is independent of autophagy activation suggesting that WNV-specific interactions with autophagy have diverged from other flaviviruses

Avian Hosts for West Nile Virus in St. Tammany Parish, Louisiana, 2002

West Nile virus (WNV) infections in free-ranging birds were studied in Slidell, St. Tammany Parish, Louisiana, after a human encephalitis outbreak peaked there in July 2002. Seroprevalence in resident, free-ranging wild birds in one suburban site was 25% and 24% in August and October, respectively, indicating that most transmission had ceased by early August. Mortality rates, seroprevalence rates, host competence, and crude population estimates were used in mathematical models to predict actual infection rates, population impacts, and importance as amplifying hosts for several common passerine birds. Northern cardinal (Cardinalis cardinalis) and house sparrow (Passer domesticus) were the principal amplifying hosts, but blue jay (Cyanocitta cristata) and northern mockingbird (Mimus polyglottos) also contributed. The blue jay population was reduced by an estimated 47%. A variety of passerine bird species combined to play an important role as amplifying hosts in the WNV transmission cycle