Mosquito RNA Research Articles

Predicting novel mosquito-associated viruses from metatranscriptomic dark matter.

The exponential growth of metatranscriptomic studies dedicated to arboviral surveillance in mosquitoes has yielded an unprecedented volume of unclassified sequences referred to as the virome dark matter. Mosquito-associated viruses are classified based on their host range into Mosquito-specific viruses (MSV) or Arboviruses. While MSV replication is restricted to mosquito cells, Arboviruses infect both mosquito vectors and vertebrate hosts. We developed the MosViR pipeline designed to identify complex genomic discriminatory patterns for predicting novel MSV or Arboviruses from viral contigs as short as 500 bp. The pipeline combines the predicted probability score from multiple predictive models, ensuring a robust classification with Area Under ROC (AUC) values exceeding 0.99 for test datasets. To assess the practical utility of MosViR in actual cases, we conducted a comprehensive analysis of 24 published mosquito metatranscriptomic datasets. By mining this metatranscriptomic dark matter, we identified 605 novel mosquito-associated viruses, with eight putative novel Arboviruses exhibiting high probability scores. Our findings highlight the limitations of current homology-based identification methods and emphasize the potentially transformative impact of the MosViR pipeline in advancing the classification of mosquito-associated viruses. MosViR offers a powerful and highly accurate tool for arboviral surveillance and for elucidating the complexities of the mosquito RNA virome.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Molecular Detection and Genotyping of Dengue Viruses: Current Techniques and Future Prospect

The Dengue virus is a prevalent mosquito-borne disease, and its global incidence has been steadily increasing due to the favorable environmental conditions that promote mosquito breeding, primarily influenced by rising temperatures. Bangladesh has been particularly hard-hit by an intense and ongoing outbreak, resulting in a surge of cases and fatalities. Effective management of this disease necessitates the implementation of robust public health measures, including rigorous surveillance and early diagnosis. While serological tests are commonly employed in clinical diagnosis, molecular methods hold a critical role in identifying the specific Dengue virus strain, thereby contributing to a more comprehensive understanding of disease severity. This article serves as an extensive review, delving into various molecular testing techniques employed for both surveillance and clinical diagnosis. It offers valuable insights for research and clinical laboratories engaged in the detection of Dengue virus RNA in mosquitoes, environmental samples, and clinical specimens. The methods covered encompass a spectrum of approaches, including conventional PCR, isothermal amplification, real-time RT-PCR, Sanger sequencing, and whole-genome sequencing, providing a holistic overview of the available techniques. These methods play pivotal roles in clinical diagnosis, outbreak analysis, vector surveillance, and vaccine development. Furthermore, the article underscores the importance of integrating these techniques into existing healthcare systems, emphasizing their significance in ensuring precise dengue diagnostics to enhance the efficiency of disease management. These molecular methods are indispensable tools that contribute to accurate diagnosis, enable effective outbreak investigation, facilitate vector surveillance, and support vaccine preparation, thereby enhancing the overall management and control of dengue, ultimately working toward mitigating its impact on public health.
 Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 41-49

Case report: Zika surveillance complemented with wastewater and mosquito testing

In June 2023, a local cluster of 15 Zika cases was reported in a neighbourhood in Northeastern Singapore. The last significant local transmission of Zika virus (ZIKV) with more than 450 cases was in 2016-2017. To monitor the situation and mitigate further transmission, case, entomological and wastewater-based surveillance were carried out. Primary healthcare practitioners and the community were alerted to encourage timely case identification. Surveillance was enhanced through testing of Aedes mosquitoes collected from the National Gravitrap surveillance system, and wastewater samples were collected from a network of autosamplers deployed at manholes across the country. ZIKV RNA was detected in mosquito pools (3/43; 7%) and individual mosquitoes (3/82; 3.7%) captured, and in wastewater samples (13/503) collected from the vicinity of the cluster of cases. Respective samples collected from other sites across the country were negative. The peak detection of ZIKV RNA in mosquitoes and wastewater coincided temporally with the peak in the number of cases in the area (15-25 May 2023). The restriction of ZIKV signals from wastewater and mosquitoes within the neighbourhood suggested limited ZIKV transmission. The subsequent waning of signals suggested effectiveness of control measures. We demonstrate the utility of wastewater-based surveillance of ZIKV, which complements existing case- and entomological-based surveillance. The non-intrusive approach is particularly useful to monitor diseases such as Zika, which generally causes silent or mild infections, but may cause severe outcomes such as congenital Zika syndrome. This study was funded by Singapore's Ministry of Finance and the National Environment Agency, Singapore.

Evaluation of long-term preservation methods for viral RNA in mosquitoes at room temperature

Mosquitoes are important vectors of various pathogenic viruses. Almost all viruses transmitted by mosquitoes are RNA viruses. Therefore, to detect viral genes, mosquito samples must be kept at low temperatures to prevent RNA degradation. However, prolonged transport from the field to laboratory can pose challenges for temperature control. The aim of this study was to evaluate methods for preserving viral RNA in mosquito bodies at room temperature. Virus-infected mosquito samples were immersed in ethanol, propylene glycol, and a commercially available nucleic acid preservation reagent at room temperature, and viral RNA stability was compared. As a result, for the two RNA viruses (San Gabriel mononegavirus and dengue virus 1) subjected to this experiment, no significant decrease in the viral RNA was observed for at least eight weeks after immersion in the reagents, and the amount of RNA remaining was equivalent to that of samples stored at − 80 °C. These results indicate that immersion storage in these reagents used in this study is effective in preserving viral RNA in mosquitoes under room temperature conditions and is expected to be implemented in epidemiologic surveillance that is not limited by the cold chain from the field to the laboratory.

Viral RNA in Mosquitoes (Diptera: Culicidae) Collected between 2019 and 2021 in Germany.

Due to globalisation and climate change, mosquito-borne pathogens are emerging in new areas on all continents, including Europe, which has recently faced outbreaks of dengue, chikungunya and West Nile fever. The present study complements previous investigations to evaluate the circulation of mosquito-borne viruses in Germany, with the aim of identifying potential vector species and risk areas. Mosquitoes collected from 2019 to 2021 and identified to species or species group level were screened for viruses of the families Flaviviridae, Peribunyaviridae and the genus Alphavirus of the family Togaviridae. In total, 22,528 mosquitoes were examined, thus providing the most comprehensive study on West Nile virus (WNV) circulation so far in the German mosquito population. Usutu virus (USUV) RNA was detected in six samples, Sindbis virus (SINV) RNA in 21 samples and WNV RNA in 11 samples. Samples containing RNA of USUV and WNV consisted of mosquitoes collected in the East German federal states of Brandenburg, Saxony and Saxony-Anhalt, while samples with RNA of SINV originated from more widespread locations. Although minimum infection rates have remained relatively low, the intensity of virus circulation appears to be increasing compared to previous studies. Continuous mosquito screening contributes to the early detection of the introduction and spread of mosquito-borne pathogens.

Metagenomic analysis of coastal Kenya female Aedes aegypti mosquito RNA metaviromes reveal presence of diverse insect specific viruses

Background: Coastal Kenya has experienced repeated outbreaks of Chikungunya (CHIKV) and Dengue (DENV) viruses mediated by competent Aedes aegypti mosquito populations. These mosquitoes harbor insect specific viruses (ISVs), some of which can prevent arboviral transmission. However, there has been no systematic molecular entomological surveillance in coastal Kenya and the diversity of viruses in local Aedes aegypti populations remains largely unknown. Methods: To obtain a snapshot of the Aedes aegypti viromes from coastal Kenya, we took advantage of a cross-sectional survey of mosquitoes to determine the prevalence of Zika virus. We collected adult mosquitoes using lured Biogent’s sentinel traps at 16 different localities along the Kenyan coast between May to September 2017. Pools of 20 female Aedes aegypti mosquitoes were generated following grouping by morphological characteristics. Presence of arboviruses in the mosquito pools was determined using virus-specific and genera-specific primers with real-time PCR. Metagenomic next generation sequencing (mNGS) on Illumina Miseq and analysis was used to characterize the virome. Results: A total of 16,520 female Aedes aegypti grouped into 826 pools were analysed. Flaviviruses were detected in 69/826 (8.4%) pools by real time PCR. Sequencing generated 8,459/971,754 (0.87%) clean reads that were taxonomically assigned to 16 and 28 viral families and species, respectively. The family Phenuiviridae represented by Phasi Charoen-like phasivirus (PCLV) species was the most abundant, detected in 64/73 (87%) mosquito pools. No pathogenic viruses were identified by mNGS. Phylogenetic analysis revealed local PCLV and Cell fusing agent virus (CFAV) were distinct from global sequences. Conclusions: Our data provides information about virus diversity and composition of the Aedes aegypti mosquitoes from coastal Kenya and contributes to the body of knowledge of the Aedes aegypti virome. To the best of our knowledge, this is the first study to provide this information from this region.

Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Optimization of RNA Extraction from Aedes aegypti and Aedes albopictus

RNA extraction is the critical initial stage in analyzing certain gene expressions, further analysis using Real Time PCR technology, and performing virus detection. However, the process of extracting RNA is often hampered by the risk of contamination, resulting in low concentrations of RNA and low purity of RNA. This is often an obstacle in extracting mosquito RNA especially detecting Dengue Virus (Den-V). Dengue virus (Den-V) can cause dangerous diseases in humans such as Dengue Fever (DHF) which is transmitted through the bites of Aedes aegypti and Aedes albopictus mosquitoes. This study aims to find out the effective steps for extracting RNA from Aedes aegypti and Aedes albopictus mosquitoes. The method being compared is a commercial RNA extraction kit with modification (addition of β-mercaptoethanol) and without modification. The results showed that the best DNA concentration and purity were obtained in mosquito samples from modified process. The purity ratio of RNA extracted without modification was 1.971 (0.021 ± 0.800) while with modification it was 2.003 (0.011 ± 0.112). Aedes aegypti had a better average concentration of 7.146 µg/ml for unmodified RNA and 7.613 µg/ml for modified RNA. This research is expected to be a reference for further studies on viruses in Aedes aegypti and Aedes albopictus.

Metagenomic analysis of coastal Kenya female Aedes aegypti mosquito RNA metaviromes reveal presence of diverse insect specific viruses

Background: Coastal Kenya has experienced repeated outbreaks of Chikungunya (CHIKV) and Dengue (DENV) viruses mediated by competent Aedes aegypti mosquito populations. These mosquitoes harbor insect specific viruses (ISVs), some of which can prevent arboviral transmission. However, there has been no systematic molecular entomological surveillance in coastal Kenya and the diversity of viruses in local Aedes aegypti populations remains largely unknown. Methods: To obtain a snapshot of the Aedes aegypti viromes from coastal Kenya, we took advantage of a cross-sectional survey of mosquitoes to determine the prevalence of Zika virus. We collected adult mosquitoes using lured Biogent’s sentinel traps at 16 different localities along the Kenyan coast between May to September 2017. Pools of 20 female Aedes aegypti mosquitoes were generated following grouping by morphological characteristics. Presence of arboviruses in the mosquito pools was determined using virus-specific and genera-specific primers with real-time PCR. Metagenomic next generation sequencing (mNGS) on Illumina Miseq and analysis was used to characterize the virome. Results: A total of 16,520 female Aedes aegypti grouped into 826 pools were analysed. Flaviviruses were detected in 69/826 (8.4%) pools by real time PCR. Sequencing generated 8,459/971,754 (0.87%) clean reads that were taxonomically assigned to 16 and 28 viral families and species, respectively. The family Phenuiviridae represented by Phasi Charoen-like phasivirus (PCLV) species was the most prevalent, detected in 64/73 (87%) mosquito pools. No pathogenic viruses were identified by mNGS. Phylogenetic analysis revealed local PCLV and Cell fusing agent virus (CFAV) were distinct from global sequences. Conclusions: Our data provides information about virus diversity and composition of the Aedes aegypti mosquitoes from coastal Kenya and contributes to the body of knowledge of the Aedes aegypti virome. To the best of our knowledge, this is the first study to provide this information from this region.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

New records of California serogroup viruses in Aedes mosquitoes and first detection in simulioidae flies from Northern Canada and Alaska

An expected consequence of climate warming is an expansion of the geographical distribution of biting insects and associated arthropod-borne viruses (arboviruses). Emerging and re-emerging arboviruses that can affect human and animal health are likely to pose significant consequences for Northern communities where access to health resources is limited. In the North American Arctic, little is known about arboviruses. Thus, in 2019, we sampled biting insects in Nunavik, Northern Québec (Kuujjuaq), Nunavut (Igloolik, Karrak Lake and Cambridge Bay), Northwest Territories (Igloolik and Yellowknife) and Alaska (Fairbanks). The main objective was to detect the presence of California serogroup (CSG) viruses– a widespread group of arboviruses across North America and that are known to cause a wide range of symptoms, ranging from mild febrile illness to fatal encephalitis. Biting insects were captured twice daily for a 7-day period in mid-summer, using a standardised protocol consisting of 100 figure-eight movements of a sweep net. Captured specimens were separated by genus (mosquitoes) or by superfamily (other insects) and then grouped into pools of 75 by geographical locations. In total, 5079 Aedes mosquitoes and 1014 Simulioidae flies were caught. We report the detection of CSG viruses RNA in mosquitoes captured in Nunavut (Karrak Lake) and Nunavik (Kuujjuaq). We also report, for the first time in North America, the presence of CSG viruses RNA in Simulioidae flies. These results highlight the use of biting insects for tracking any future emergence of arboviruses in the North, thereby providing key information for public health in Northern communities.

A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Design and Use of Chikungunya Virus Replication Templates Utilizing Mammalian and Mosquito RNA Polymerase I-Mediated Transcription.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive-sense RNA genome that also serves as the mRNA for four nonstructural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication-competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce noncapped RNA templates, which are poorly translated relative to CHIKV replicase-generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the presence of replicase relative to background. Using this highly sensitive assay to analyze CHIKV nsP1 functionality, several mutations that severely reduced, but did not completely block, CHIKV replicase activity were identified: (i) nsP1 tagged at its N terminus with enhanced green fluorescent protein; (ii) mutations D63A and Y248A, blocking the RNA capping; and (iii) mutation R252E, affecting nsP1 membrane anchoring. In contrast, a mutation in the nsP1 palmitoylation site completely inactivated CHIKV replicase in both human and mosquito cells and was lethal for the virus. Our data confirm that this novel system provides a valuable tool to study CHIKV replicase, RNA replication, and virus-host interactions.IMPORTANCE Chikungunya virus (CHIKV) is a medically important pathogen responsible for recent large-scale epidemics. The development of efficient therapies against CHIKV has been hampered by gaps in our understanding of how nonstructural proteins (nsPs) function to form the viral replicase and replicate virus RNA. Here we describe an extremely sensitive assay to analyze the effects of mutations on the virus RNA synthesis machinery in cells of both mammalian (host) and mosquito (vector) origin. Using this system, several lethal mutations in CHIKV nsP1 were shown to reduce but not completely block the ability of its replicase to synthesize viral RNAs. However, in contrast to related alphaviruses, CHIKV replicase was completely inactivated by mutations preventing palmitoylation of nsP1. These data can be used to develop novel, virus-specific antiviral treatments.

Usutu Virus RNA in Mosquitoes, Israel, 2014-2015.

We identified Usutu virus (USUV) RNA in 6 pools of mosquitoes trapped in northern Israel during 2014–2015. These Israeli strains were most similar to strains identified in Senegal and Germany, which further elucidates common ancestry and evolutionary dynamics of USUV. Our findings suggest that human infection with USUV might occur in Israel.

High-Resolution Metatranscriptomics Reveals the Ecological Dynamics of Mosquito-Associated RNA Viruses in Western Australia.

Mosquitoes harbor a high diversity of RNA viruses, including many that impact human health. Despite a growing effort to describe the extent and nature of the mosquito virome, little is known about how these viruses persist, spread, and interact with both their hosts and other microbes. To address this issue we performed a metatranscriptomics analysis of 12 Western Australian mosquito populations structured by species and geographic location. Our results identified the complete genomes of 24 species of RNA viruses from a diverse range of viral families and orders, among which 19 are newly described. Comparisons of viromes revealed a striking difference between the two mosquito genera, with viromes of mosquitoes of the Aedes genus exhibiting substantially less diversity and lower abundances than those of mosquitoes of the Culex genus, within which the viral abundance reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity between the two genera, although the viromes were very similar among the three Culex species studied, suggesting that the host taxon plays a major role in structuring virus diversity. In contrast, we found no evidence that geographic location played a major role in shaping RNA virus diversity, and several viruses discovered here exhibited high similarity (95 to 98% nucleotide identity) to those from Indonesia and China. Finally, using abundance-level and phylogenetic relationships, we were able to distinguish potential mosquito viruses from those present in coinfecting bacteria, fungi, and protists. In sum, our metatranscriptomics approach provides important insights into the ecology of mosquito RNA viruses.IMPORTANCE Studies of virus ecology have generally focused on individual viral species. However, recent advances in bulk RNA sequencing make it possible to utilize metatranscriptomic approaches to reveal both complete virus diversity and the relative abundance of these viruses. We used such a metatranscriptomic approach to determine key aspects of the ecology of mosquito viruses in Western Australia. Our results show that RNA viruses are some of the most important components of the mosquito transcriptome, and we identified 19 new virus species from a diverse set of virus families. A key result was that host genetic background plays a more important role in shaping virus diversity than sampling location, with Culex species harboring more viruses at higher abundance than those from Aedes mosquitoes.

Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes.

Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

Comparison of the Efficiency and Cost of West Nile Virus Surveillance Methods in California

Surveillance systems for West Nile virus (WNV) combine several methods to determine the location and timing of viral amplification. The value of each surveillance method must be measured against its efficiency and costs to optimize integrated vector management and suppress WNV transmission to the human population. Here we extend previous comparisons of WNV surveillance methods by equitably comparing the most common methods after standardization on the basis of spatial sampling density and costs, and by estimating optimal levels of sampling effort for mosquito traps and sentinel chicken flocks. In general, testing for evidence of viral RNA in mosquitoes and public-reported dead birds resulted in detection of WNV approximately 2-5 weeks earlier than serological monitoring of sentinel chickens at equal spatial sampling density. For a fixed cost, testing of dead birds reported by the public was found to be the most cost effective of the methods, yielding the highest number of positive results per $1000. Increased spatial density of mosquito trapping was associated with more precise estimates of WNV infection prevalence in mosquitoes. Our findings also suggested that the most common chicken flock size of 10 birds could be reduced to six to seven without substantial reductions in timeliness or sensitivity. We conclude that a surveillance system that uses the testing of dead birds reported by the public complemented by strategically timed mosquito and chicken sampling as agency resources allow would detect viral activity efficiently in terms of effort and costs, so long as susceptible bird species that experience a high mortality rate from infection with WNV, such as corvids, are present in the area.

Detection and Early Warning of West Nile Virus Circulation in Central Macedonia, Greece, Using Sentinel Chickens and Mosquitoes

Following the West Nile Virus (WNV) epidemic in 2010 in Central Macedonia, Greece, which resulted in 197 human neuroinvasive disease cases, we determined the seasonal appearance and prevalence of the virus in 2011 by testing weekly for WNV genomic RNA in mosquitoes collected in carbon dioxide-baited traps, and for anti-WNV antibodies in sentinel chickens. Preliminary findings of the surveillance program regarding the circulation of "Nea Santa-Greece-2010" in sentinel chickens were rapidly communicated to public health authorities. In the present article, the full 2011 data produced by this surveillance program are presented. We detected enzootic circulation of WNV in chickens 1 month prior to the onset of the first human cases in 2011. Culex pipiens and Cx. modestus were abundant throughout the sampling period and at all sites of increased transmission. Molecular identification and phylogenetic analysis of WNV isolates from two chickens and one Cx. pipiens mosquito pool suggested that: (1) the virulent "Nea Santa-Greece-2010" WNV lineage 2 strain responsible for the 2010 epidemic was actively circulating in 2011, and (2) all Greek isolates belong to a distinct recent evolutionary clade. In Europe, where numerous strains of different virulence coexist, sequencing information for WNV is important for phylogeography and identification of virulent strains for human health risk assessment.

Evolution of mosquito-based arbovirus surveillance systems in Australia.

Control of arboviral disease is dependent on the sensitive and timely detection of elevated virus activity or the identification of emergent or exotic viruses. The emergence of Japanese encephalitis virus (JEV) in northern Australia revealed numerous problems with performing arbovirus surveillance in remote locations. A sentinel pig programme detected JEV activity, although there were a number of financial, logistical, diagnostic and ethical limitations. A system was developed which detected viral RNA in mosquitoes collected by solar or propane powered CO2-baited traps. However, this method was hampered by trap-component malfunction, microbial contamination and large mosquito numbers which overwhelmed diagnostic capabilities. A novel approach involves allowing mosquitoes within a box trap to probe a sugar-baited nucleic-acid preservation card that is processed for expectorated arboviruses. In a longitudinal field trial, both Ross River and Barmah Forest viruses were detected numerous times from multiple traps over different weeks. Further refinements, including the development of unpowered traps and use of yeast-generated CO2, could enhance the applicability of this system to remote locations. New diagnostic technology, such as next generation sequencing and biosensors, will increase the capacity for recognizing emergent or exotic viruses, while cloud computing platforms will facilitate rapid dissemination of data.