Rebecca C. Christofferson

Estimating the Magnitude and Direction of Altered Arbovirus Transmission Due to Viral Phenotype

Vectorial capacity is a measure of the transmission potential of a vector borne pathogen within a susceptible population. Vector competence, a component of the vectorial capacity equation, is the ability of an arthropod to transmit an infectious agent following exposure to that agent. Comparisons of arbovirus strain-specific vector competence estimates have been used to support observed or hypothesized differences in transmission capability. Typically, such comparisons are made at a single time point during the extrinsic incubation period, the time in days it takes for the virus to replicate and disseminate to the salivary glands. However, vectorial capacity includes crucial parameters needed to effectively evaluate transmission capability, though often this is based on the discrete vector competence values. Utilization of the rate of change of vector competence over a range of days gives a more accurate measurement of the transmission potential. Accordingly, we investigated the rate of change in vector competence of dengue virus in Aedes aegypti mosquitoes and the resulting vectorial capacity curves. The areas under the curves represent the effective vector competence and the cumulative transmission potentials of arboviruses within a population of mosquitoes. We used the calculated area under the curve for each virus strain and the corresponding variance estimates to test for differences in cumulative transmission potentials between strains of dengue virus based on our dynamic model. To further characterize differences between dengue strains, we devised a displacement index interpreted as the capability of a newly introduced strain to displace the established, dominant circulating strain. The displacement index can be used to better understand the transmission dynamics in systems where multiple strains/serotypes circulate or even multiple arbovirus species. The use of a rate of a rate of change based model of vectorial capacity and the informative calculations of the displacement index will lead to better measurements of the differences in transmission potential of arboviruses.

An explosive epidemic of DENV-3 in Cairns, Australia.

From November 2008-May 2009 Cairns Queensland Australia was struck by an explosive epidemic of DENV-3 that exceeded the capacity of highly skilled dengue control team to control it. We describe the environmental, virological and entomological factors associated with this outbreak to better understand the circumstances leading to its occurrence. Patient interviews, serological results and viral sequencing strongly suggest that the imported index case was infected in Kalimantan, Indonesia. A delay in notification of 27 days from importation of the index case until Queensland Health was notified of dengue transmission allowed the virus to amplify and spread unchecked through November 2008. Unseasonably warm weather, with daily mean temperatures exceeding 30°C, occurred in late November and would have shortened the extrinsic incubation period of the virus and enhanced transmission. Analysis of case movements early in the outbreak indicated that the total incubation period was as low as 9–11 days. This was supported by laboratory vector competence studies that found transmission by Aedes aegypti occurred within 5 days post exposure at 28°C. Effective vector competence rates calculated from these transmission studies indicate that early transmission contributed to the explosive dengue transmission observed in this outbreak. Collections from BG sentinel traps and double sticky ovitraps showed that large populations of the vector Ae. aegypti occurred in the transmission areas from November – December 2008. Finally, the seasonal movement of people around the Christmas holiday season enhanced the spread of DENV-3. These results suggest that a strain of DENV-3 with an unusually rapid transmission cycle was able to outpace vector control efforts, especially those reliant upon delayed action control such as lethal ovitraps.

Zika Virus Emergence and Expansion: Lessons Learned from Dengue and Chikungunya May Not Provide All the Answers.

Following the emergence of Zika in the past decade, there are lessons to be learned from similar emergence events of dengue (DENV) and chikungunya (CHIKV). Specifically, as Zika emerges in the Americas there is a natural tendency to apply the knowledge base of DENV and CHIKV to mitigation and control of a virus with such a similar transmission system. However, there are marked differences that may preclude such broad stroke application of this knowledge base without making potentially faulty assumptions. Herein, Zika virus (ZIKV) transmission is reviewed, and the commonalities among these three arboviruses are discussed. Importantly, the divergence of this particular arbovirus is discussed, as is the need to develop ZIKV-specific knowledge base for mitigation of this disease. Specifically reviewed are 1) emergence and persistence patterns, 2) genetic and phenotypic diversity, 3) vector host range, and finally, 4) alternate transmission routes and added complexity of ZIKV transmission and presentation.

Analysis of Early Dengue Virus Infection in Mice as Modulated by Aedes aegypti Probing

ABSTRACT Dengue virus (DENV), the etiologic agent of dengue fever, is transmitted during probing of human skin by infected-mosquito bite. The expectorated viral inoculum also contains an assortment of mosquito salivary proteins that have been shown to modulate host hemostasis and innate immune responses. To examine the potential role of mosquito probing in DENV establishment within the vertebrate host, we inoculated mice intradermally with DENV serotype 2 strain 1232 at sites where Aedes aegypti had or had not probed immediately prior. We assayed these sites 3 h postinoculation with transcript arrays for the Toll-like receptor (TLR), RIG-I-like receptor, and NOD-like receptor signaling pathways of the innate immune system. We then chose TLR7, transcription factor p65 (RelA), gamma interferon (IFN-γ), and IFN-γ-inducible protein 10 (IP-10) from the arrays for further investigation and assayed these transcripts at 10 min, 3 h, and 6 h postinoculation. The transcripts for TLR7, RelA, IFN-γ, and IP-10 were significantly downregulated between 2- and 3-fold in the group subjected to mosquito probing relative to the virus-only inoculation group at 3 h postinoculation. A reduction in these transcripts could indicate reduced DENV recognition and antigen presentation and diminished inhibition of viral replication and spread. Further, mosquito probing resulted in viremia titers significantly higher than those in mice that did not receive probing. A. aegypti probing has a significant effect on the innate immune response to DENV infection and generates an early immune environment more permissive to the establishment of infection.

Effect of Dengue-2 Virus Infection on Protein Expression in the Salivary Glands of Aedes aegypti Mosquitoes

Dengue virus (DENV) is the most important mosquito-transmitted flavivirus that is transmitted throughout the tropical and subtropical regions of the world. The primary mosquito vector of DENV in urban locations is Aedes aegypti. Key to understanding the transmission of DENV is the relationship between pathogen and vector. Accordingly, we report our preliminary characterization of the differentially expressed proteins from Ae. aegypti mosquitoes after DENV infection. We investigated the virus-vector interaction through changes in the proteome of the salivary glands of mosquitoes with disseminated DENV serotype 2 (DENV-2) infections using two-dimensional gel electrophoresis and identification by mass spectrometry. Our findings indicate that DENV-2 infection in the Ae. aegypti salivary gland alters the expression of structural, secreted, and metabolic proteins. These changes in the salivary gland proteome highlight the virally influenced environment caused by a DENV-2 infection and warrant additional investigation to determine if these differences extend to the expectorated saliva.

Utility of a Dengue-Derived Monoclonal Antibody to Enhance Zika Infection In Vitro

Introduction: Zika virus (ZIKV) has emerged in dengue (DENV) endemic areas, where these two related flaviviruses continue to co-circulate. DENV is a complex of four serotypes and infections can progress to severe disease. It is thought that this is mediated by antibody dependent enhancement (ADE) whereby antibodies from a primary DENV infection are incapable of neutralizing heterologous DENV infections with another serotype. ADE has been demonstrated among other members of the Flavivirus group. Methods: We utilize an in vitro ADE assay developed for DENV to determine whether ZIKV is enhanced by a commonly available DENV serotype 2-derived monoclonal antibody (4G2). Results: We show that ZIKV infection in vitro is enhanced in the presence of the 4G2 mAb. Discussion: Our results demonstrate that ADE between ZIKV and DENV is possible and that the 4G2 antibody is a useful tool for the effects of pre-existing anti-DENV antibodies during ZIKV infections.

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.

Development of a transmission model for dengue virus

BackgroundDengue virus (DENV) research has historically been hampered by the lack of a susceptible vertebrate transmission model. Recently, there has been progress towards such models using several varieties of knockout mice, particularly those deficient in type I and II interferon receptors. Based on the critical nature of the type I interferon response in limiting DENV infection establishment, we assessed the permissiveness of a mouse strain with a blunted type I interferon response via gene deficiencies in interferon regulatory factors 3 and 7 (IRF3/7 −/− −/−) with regards to DENV transmission success. We investigated the possibility of transmission to the mouse by needle and infectious mosquito, and subsequent transmission back to mosquito from an infected animal during its viremic period.MethodsMice were inoculated subcutaneously with non-mouse adapted DENV-2 strain 1232 and serum was tested for viral load and cytokine production each day. Additionally, mosquitoes were orally challenged with the same DENV-2 strain via artificial membrane feeder, and then allowed to forage or naïve mice. Subsequently, we determined acquisition potential by allowing naïve mosquitoes on forage on exposed mice during their viremic period.ResultsBoth needle inoculation and infectious mosquito bite(s) resulted in 100% infection. Significant differences between these groups in viremia on the two days leading to peak viremia were observed, though no significant difference in cytokine production was seen. Through our determination of transmission and acquisition potentials, the transmission cycle (mouse-to mosquito-to mouse) was completed. We confirmed that the IRF3/7 −/− −/− mouse supports DENV replication and is competent for transmission experiments, with the ability to use a non-mouse adapted DENV-2 strain. A significant finding of this study was that this IRF3/7 −/− −/− mouse strain was able to be infected by and transmit virus to mosquitoes, thus providing means to replicate the natural transmission cycle of DENV.ConclusionAs there is currently no approved vaccine for DENV, public health monitoring and a greater understanding of transmission dynamics leading to outbreak events are critical. The further characterization of DENV using this model will expand knowledge of key entomological, virological and immunological components of infection establishment and transmission events.

Chikungunya viral fitness measures within the vector and subsequent transmission potential.

Given the recent emergence of chikungunya in the Americas, the accuracy of forecasting and prediction of chikungunya transmission potential in the U.S. requires urgent assessment. The La Reunion-associated sub-lineage of chikungunya (with a valine substitution in the envelope protein) was shown to increase viral fitness in the secondary vector, Ae. albopictus. Subsequently, a majority of experimental and modeling efforts focused on this combination of a sub-lineage of the East-Central-South African genotype (ECSA-V) – Ae. albopictus, despite the Asian genotype being the etiologic agent of recent chikungunya outbreaks world-wide. We explore a collection of data to investigate relative transmission efficiencies of the three major genotypes/sub-lineages of chikungunya and found difference in the extrinsic incubation periods to be largely overstated. However, there is strong evidence supporting the role of Ae. albopictus in the expansion of chikungunya that our R0 calculations cannot attribute to fitness increases in one vector over another. This suggests other ecological factors associated with the Ae. albopictus-ECSA-V cycle may drive transmission intensity differences. With the apparent bias in literature, however, we are less prepared to evaluate transmission where Ae. aegypti plays a significant role. Holistic investigations of CHIKV transmission cycle(s) will allow for more complete assessment of transmission risk in areas affected by either or both competent vectors.

Towards an early warning system for forecasting human west nile virus incidence.

We have identified environmental and demographic variables, available in January, that predict the relative magnitude and spatial distribution of West Nile virus (WNV) for the following summer. The yearly magnitude and spatial distribution for WNV incidence in humans in the United States (US) have varied wildly in the past decade. Mosquito control measures are expensive and having better estimates of the expected relative size of a future WNV outbreak can help in planning for the mitigation efforts and costs. West Nile virus is spread primarily between mosquitoes and birds; humans are an incidental host. Previous efforts have demonstrated a strong correlation between environmental factors and the incidence of WNV. A predictive model for human cases must include both the environmental factors for the mosquito-bird epidemic and an anthropological model for the risk of humans being bitten by a mosquito. Using weather data and demographic data available in January for every county in the US, we use logistic regression analysis to predict the probability that the county will have at least one WNV case the following summer. We validate our approach and the spatial and temporal WNV incidence in the US from 2005 to 2013. The methodology was applied to forecast the 2014 WNV incidence in late January 2014. We find the most significant predictors for a county to have a case of WNV to be the mean minimum temperature in January, the deviation of this minimum temperature from the expected minimum temperature, the total population of the county, publicly available samples of local bird populations, and if the county had a case of WNV the previous year.