The interferon-stimulated gene IFITM3 restricts West Nile virus infection and pathogenesis

The interferon-induced transmembrane protein (IFITM) family of proteins inhibit infection of several different enveloped viruses in cell culture by virtue of their ability to restrict entry and fusion from late endosomes. As few studies have evaluated the importance of Ifitm3 in vivo in restricting viral pathogenesis, we investigated its significance as an antiviral gene against West Nile virus (WNV), an encephalitic flavivirus, in cells and mice. Ifitm3(−/−) mice were more vulnerable to lethal WNV infection, and this was associated with greater virus accumulation in peripheral organs and central nervous system tissues. As no difference in viral burden in the brain or spinal cord was observed after direct intracranial inoculation, Ifitm3 likely functions as an antiviral protein in nonneuronal cells. Consistent with this, Ifitm3(−/−) fibroblasts but not dendritic cells resulted in higher yields of WNV in multistep growth analyses. Moreover, transcomplementation experiments showed that Ifitm3 inhibited WNV infection independently of Ifitm1, Ifitm2, Ifitm5, and Ifitm6. Beyond a direct effect on viral infection in cells, analysis of the immune response in WNV-infected Ifitm3(−/−) mice showed decreases in the total number of B cells, CD4(+) T cells, and antigen-specific CD8(+) T cells. Finally, bone marrow chimera experiments demonstrated that Ifitm3 functioned in both radioresistant and radiosensitive cells, as higher levels of WNV were observed in the brain only when Ifitm3 was absent from both compartments. Our analyses suggest that Ifitm3 restricts WNV pathogenesis likely through multiple mechanisms, including the direct control of infection in subsets of cells. IMPORTANCE As part of the mammalian host response to viral infections, hundreds of interferon-stimulated genes (ISGs) are induced. The inhibitory activity of individual ISGs varies depending on the specific cell type and viral pathogen. Among ISGs, the genes encoding interferon-induced transmembrane protein (IFITM) have been reported to inhibit multiple families of viruses in cell culture. However, few reports have evaluated the impact of IFITM genes on viral pathogenesis in vivo. In this study, we characterized the antiviral activity of Ifitm3 against West Nile virus (WNV), an encephalitic flavivirus, using mice with a targeted gene deletion of Ifitm3. Based on extensive virological and immunological analyses, we determined that Ifitm3 protects mice from WNV-induced mortality by restricting virus accumulation in peripheral organs and, subsequently, in central nervous system tissues. Our data suggest that Ifitm3 restricts WNV pathogenesis by multiple mechanisms and functions in part by controlling infection in different cell types

Persistence of Pathogens with Short Infectious Periods in Seasonal Tick Populations: The Relative Importance of Three Transmission Routes

BACKGROUND: The flaviviruses causing tick-borne encephalitis (TBE) persist at low but consistent levels in tick populations, despite short infectious periods in their mammalian hosts and transmission periods constrained by distinctly seasonal tick life cycles. In addition to systemic and vertical transmission, cofeeding transmission has been proposed as an important route for the persistence of TBE-causing viruses. Because cofeeding transmission requires ticks to feed simultaneously, the timing of tick activity may be critical to pathogen persistence. Existing models of tick-borne diseases do not incorporate all transmission routes and tick seasonality. Our aim is to evaluate the influence of seasonality on the relative importance of different transmission routes by using a comprehensive mathematical model. METHODOLOGY/PRINCIPAL FINDINGS: We developed a stage-structured population model that includes tick seasonality and evaluated the relative importance of the transmission routes for pathogens with short infectious periods, in particular Powassan virus (POWV) and the related "deer tick virus," emergent encephalitis-causing flaviviruses in North America. We used the next generation matrix method to calculate the basic reproductive ratio and performed elasticity analyses. We confirmed that cofeeding transmission is critically important for such pathogens to persist in seasonal tick populations over the reasonable range of parameter values. At higher but still plausible rates of vertical transmission, our model suggests that vertical transmission can strongly enhance pathogen prevalence when it operates in combination with cofeeding transmission. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that the consistent prevalence of POWV observed in tick populations could be maintained by a combination of low vertical, intermediate cofeeding and high systemic transmission rates. When vertical transmission is weak, nymphal ticks support integral parts of the transmission cycle that are critical for maintaining the pathogen. We also extended the model to pathogens that cause chronic infections in hosts and found that cofeeding transmission could contribute to elevating prevalence even in these systems. Therefore, the common assumption that cofeeding transmission is not relevant in models of chronic host infection, such as Lyme disease, could lead to underestimating pathogen prevalence

West Nile Virus Genetic Diversity is Maintained during Transmission by Culex pipiens quinquefasciatus Mosquitoes

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes

RNAi Targeting of West Nile Virus in Mosquito Midguts Promotes Virus Diversification

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification

West Nile Virus Experimental Evolution in vivo and the Trade-off Hypothesis

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong

Antecedent Avian Immunity Limits Tangential Transmission of West Nile Virus to Humans

Background: West Nile virus (WNV) is a mosquito-borne flavivirus maintained and amplified among birds and tangentially transmitted to humans and horses which may develop terminal neuroinvasive disease. Outbreaks typically have a three-year pattern of silent introduction, rapid amplification and subsidence, followed by intermittent recrudescence. Our hypothesis that amplification to outbreak levels is contingent upon antecedent seroprevalence within maintenance host populations was tested by tracking WNV transmission in Los Angeles, California from 2003 through 2011. Methods: Prevalence of antibodies against WNV was monitored weekly in House Finches and House Sparrows. Tangential or spillover transmission was measured by seroconversions in sentinel chickens and by the number of West Nile neuroinvasive disease (WNND) cases reported to the Los Angeles County Department of Public Health. Results: Elevated seroprevalence in these avian populations was associated with the subsidence of outbreaks and in the antecedent dampening of amplification during succeeding years. Dilution of seroprevalence by recruitment resulted in the progressive loss of herd immunity following the 2004 outbreak, leading to recrudescence during 2008 and 2011. WNV appeared to be a significant cause of death in these avian species, because the survivorship of antibody positive birds significantly exceeded that of antibody negative birds. Cross-correlation analysis showed that seroprevalence was negatively correlated prior to the onset of human cases and then positively correlated, peaking at 4–6 weeks after the onse

Does reservoir host mortality enhance transmission of West Nile virus?

<p>Abstract</p> <p>Background</p> <p>Since its 1999 emergence in New York City, West Nile virus (WNV) has become the most important and widespread cause of mosquito-transmitted disease in North America. Its sweeping spread from the Atlantic to the Pacific coast was accompanied by widespread mortality among wild birds, especially corvids. Only sporadic avian mortality had previously been associated with this infection in the Old World. Here, we examine the possibility that reservoir host mortality may intensify transmission, both by concentrating vector mosquitoes on remaining hosts and by preventing the accumulation of "herd immunity".</p> <p>Results</p> <p>Inspection of the Ross-Macdonald expression of the basic reproductive number (<it>R</it>₀) suggests that this quantity may increase with reservoir host mortality. Computer simulation confirms this finding and indicates that the level of virulence is positively associated with the numbers of infectious mosquitoes by the end of the epizootic. The presence of reservoir incompetent hosts in even moderate numbers largely eliminated the transmission-enhancing effect of host mortality. Local host die-off may prevent mosquitoes to "waste" infectious blood meals on immune host and may thus facilitate perpetuation and spread of transmission.</p> <p>Conclusion</p> <p>Under certain conditions, host mortality may enhance transmission of WNV and similarly maintained arboviruses and thus facilitate their emergence and spread. The validity of the assumptions upon which this argument is built need to be empirically examined.</p

Temperature, Viral Genetics, and the Transmission of West Nile Virus by Culex pipiens Mosquitoes

The distribution and intensity of transmission of vector-borne pathogens can be strongly influenced by the competence of vectors. Vector competence, in turn, can be influenced by temperature and viral genetics. West Nile virus (WNV) was introduced into the United States of America in 1999 and subsequently spread throughout much of the Americas. Previously, we have shown that a novel genotype of WNV, WN02, first detected in 2001, spread across the US and was more efficient than the introduced genotype, NY99, at infecting, disseminating, and being transmitted by Culex mosquitoes. In the current study, we determined the relationship between temperature and time since feeding on the probability of transmitting each genotype of WNV. We found that the advantage of the WN02 genotype increases with the product of time and temperature. Thus, warmer temperatures would have facilitated the invasion of the WN02 genotype. In addition, we found that transmission of WNV accelerated sharply with increasing temperature, T, (best fit by a function of T4) showing that traditional degree-day models underestimate the impact of temperature on WNV transmission. This laboratory study suggests that both viral evolution and temperature help shape the distribution and intensity of transmission of WNV, and provides a model for predicting the impact of temperature and global warming on WNV transmission

African tropical rainforest net carbon dioxide fluxes in the twentieth century

The African humid tropical biome constitutes the second largest rainforest region, significantly impacts global carbon cycling and climate, and has undergone major changes in functioning owing to climate and land-use change over the past century. We assess changes and trends in CO2 fluxes from 1901 to 2010 using nine land surface models forced with common driving data, and depict the inter-model variability as the uncertainty in fluxes. The biome is estimated to be a natural (no disturbance) net carbon sink (−0.02 kg C m−2 yr−1 or −0.04 Pg C yr−1, p < 0.05) with increasing strength fourfold in the second half of the century. The models were in close agreement on net CO2 flux at the beginning of the century (σ1901 = 0.02 kg C m−2 yr−1), but diverged exponentially throughout the century (σ2010 = 0.03 kg C m−2 yr−1). The increasing uncertainty is due to differences in sensitivity to increasing atmospheric CO2, but not increasing water stress, despite a decrease in precipitation and increase in air temperature. However, the largest uncertainties were associated with the most extreme drought events of the century. These results highlight the need to constrain modelled CO2 fluxes with increasing atmospheric CO2 concentrations and extreme climatic events, as the uncertainties will only amplify in the next century

A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16–WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti–WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction