The Insect Microbiome Modulates Vector Competence for Arboviruses

Diseases caused by arthropod-borne viruses (arboviruses), such as Dengue, West Nile, and Chikungunya, constitute a major global health burden and are increasing in incidence and geographic range. The natural microbiota of insect vectors influences various aspects of host biology, such as nutrition, reproduction, metabolism, and immunity, and recent studies have highlighted the ability of insect-associated bacteria to reduce vector competence for arboviruses and other pathogens. This reduction can occur through mechanisms, such as immune response activation, resource competition, or the production of anti-viral molecules. Studying the interactions between insect vectors and their microbiota is an important step toward developing alternative strategies for arbovirus transmission control.     

NADP+-dependent farnesol dehydrogenase,a corpora allata enzyme involved in juvenile hormone synthesis

The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the “classical” NADP⁺-dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C₁₀ to C₁₅ isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes.     

Risk assessment of flavivirus transmission in Namibia

The role of arboviruses causing acute febrile illness in sub-Saharan Africa is receiving more attention. Reports of dengue in tourists were published nearly 10 years ago in Namibia, but the current epidemiology of arboviruses is unknown and surveys of mosquito vectors have not been carried out since the 1950s. To begin addressing this knowledge gap, a prospective cross-sectional study was conducted using samples from volunteer blood donors linked to questionnaire. Serum samples were tested using a Dengue IgG Indirect ELISA which measured exposure to dengue virus/flaviviruses. Entomological samples were collected from tires during the rainy season (February–March 2012) in six locations across Namibia's capital city, Windhoek. Among 312 blood donors tested, 25 (8.0%) were positive for dengue virus/flavivirus exposure. The only significant risk factor was age group with high exposure rates among those older than 50 (29%) compared with those below 40 years old (between 2.9% and 8.3%) (P < 0.002). Larvae and pupae of Aedes aegypti and Culex pipiens complex accounted for 100% of the 2751 samples collected, of which only 12.2% (n = 336) were Ae. aegypti. Each site demonstrated high variability of species composition between sampling times. While the significant dengue virus/flavivirus exposure rate among those above 50 years old is likely indicative of the West Nile epidemic in the 70s and 80s, the low exposure among those under 50 suggests that flaviviruses are still circulating in Namibia. While Ae. aegypti and C. pipiens sp. may play a role in future epidemics, the significance of presence may be reduced due to short rain periods, dry, arid, cold winters and policies and social understandings that limit non-structured storage and use of tires in low income areas. Future studies should further characterize the circulating arboviruses and investigate mosquito ecology nationally to map areas at higher risk for future arbovirus outbreaks.     

Vector-Virus Interactions and Transmission Dynamics of West Nile Virus

West Nile virus (WNV; Flavivirus; Flaviviridae) is the cause of the most widespread arthropod-borne viral disease in the world and the largest outbreak of neuroinvasive disease ever observed. Mosquito-borne outbreaks are influenced by intrinsic (e.g., vector and viral genetics, vector and host competence, vector life-history traits) and extrinsic (e.g., temperature, rainfall, human land use) factors that affect virus activity and mosquito biology in complex ways. The concept of vectorial capacity integrates these factors to address interactions of the virus with the arthropod host, leading to a clearer understanding of their complex interrelationships, how they affect transmission of vector-borne disease, and how they impact human health. Vertebrate factors including host competence, population dynamics, and immune status also affect transmission dynamics. The complexity of these interactions are further exacerbated by the fact that not only can divergent hosts differentially alter the virus, but the virus also can affect both vertebrate and invertebrate hosts in ways that significantly alter patterns of virus transmission. This chapter concentrates on selected components of the virus-vector-vertebrate interrelationship, focusing specifically on how interactions between vector, virus, and environment shape the patterns and intensity of WNV transmission.     

Natural colonization and adaptation of a mosquito species in Galápagos and its implications for disease threats to endemic wildlife

Emerging infectious diseases of wildlife have been recognized as a major threat to global biodiversity. Endemic species on isolated oceanic islands, such as the Galápagos, are particularly at risk in the face of introduced pathogens and disease vectors. The black salt-marsh mosquito (Aedes taeniorhynchus) is the only mosquito widely distributed across the Galápagos Archipelago. Here we show that this mosquito naturally colonized the Galápagos before the arrival of man, and since then it has evolved to represent a distinct evolutionary unit and has adapted to habitats unusual for its coastal progenitor. We also present evidence that A. taeniorhynchus feeds on reptiles in Galápagos in addition to previously reported mammal and bird hosts, highlighting the important role this mosquito might play as a bridge-vector in the transmission and spread of extant and newly introduced diseases in the Galápagos Islands. These findings are particularly pertinent for West Nile virus, which can cause significant morbidity and mortality in mammals (including humans), birds, and reptiles, and which recently has spread from an introductory focus in New York to much of the North and South American mainland and could soon reach the Galápagos Islands. Unlike Hawaii, there are likely to be no highland refugia free from invading mosquito-borne diseases in Galápagos, suggesting bleak outcomes to possible future pathogen introduction events.     

Utility of a Sequence-Independent,Single-Primer-Amplification (SISPA) and Nanopore Sequencing Approach for Detection and Characterization of Tick-Borne Viral Pathogens

Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.     

Identification of Essential Containers for Aedes Larval Breeding to Control Dengue in Dhaka,Bangladesh

Dengue fever (DF), one of the most important emerging arboviral diseases, is transmitted through the bite of container breeding mosquitoes Aedes aegypti and Aedes albopictus. A household entomological survey was conducted in Dhaka from August through October 2000 to inspect water-holding containers in indoor, outdoor, and rooftop locations for Aedes larvae. The objective of this study was to determine mosquito productivity of each container type and to identify some risk factors of households infested with Aedes larvae. Of 9,222 households inspected, 1,306 (14.2%) were positive for Aedes larvae. Of 38,777 wet containers examined, 2,272 (5.8%) were infested with Aedes larvae. Containers used to hold water, such as earthen jars, tanks, and drums were the most common containers for larval breeding. Tires in outdoor and rooftop locations of the households were also important for larval breeding. Although present in abundance, buckets were of less importance. Factors such as independent household, presence of a water storage system in the house, and fully/partly shaded outdoors were found to be significantly associated with household infestation of Aedes larvae. Identification and subsequent elimination of the most productive containers in a given area may potentially reduce mosquito density to below a level at which dengue transmission may be halted.     

An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle

Many pathogens, such as the agents of West Nile encephalitis and plague, are maintained in nature by animal reservoirs and transmitted to humans by arthropod vectors. Efforts to reduce disease incidence usually rely on vector control or immunization of humans. Lyme disease, for which no human vaccine is currently available, is a commonly reported vector-borne disease in North America and Europe. In a recently developed, ecological approach to disease prevention, we intervened in the natural cycle of the Lyme disease agent (Borrelia burgdorferi) by immunizing wild white-footed mice (Peromyscus leucopus), a reservoir host species, with either a recombinant antigen of the pathogen, outer surface protein A, or a negative control antigen in a repeated field experiment with paired experimental and control grids stratified by site. Outer surface protein A vaccination significantly reduced the prevalence of B. burgdorferi in nymphal blacklegged ticks (Ixodes scapularis) collected at the sites the following year in both experiments. The magnitude of the vaccine's effect at a given site correlated with the tick infection prevalence found on the control grid, which in turn correlated with mouse density. These data, as well as differences in the population structures of B. burgdorferi in sympatric ticks and mice, indicated that nonmouse hosts contributed more to infecting ticks than previously expected. Thus, where nonmouse hosts play a large role in infection dynamics, vaccination should be directed at additional species.     

Ecology of West Nile Virus in North America

The introduction, dispersal and establishment of West Nile virus in North America were reviewed, focusing on factors that may have enhanced receptivity and enabled the invasion process. The overwintering persistence of this tropical virus within temperate latitudes was unexpected, but was key in the transition from invasion to endemic establishment. The cascade of temporal events allowing sporadic amplification to outbreak levels was discussed within a future perspective.     

Crimean-Congo Hemorrhagic Fever Virus: Current Advances and Future Prospects of Antiviral Strategies

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread, tick-borne pathogen that causes Crimean-Congo hemorrhagic fever (CCHF) with high morbidity and mortality. CCHFV is transmitted to humans through tick bites or direct contact with patients or infected animals with viremia. Currently, climate change and globalization have increased the transmission risk of this biosafety level (BSL)-4 virus. The treatment options of CCHFV infection remain limited and there is no FDA-approved vaccine or specific antivirals, which urges the identification of potential therapeutic targets and the design of CCHF therapies with greater effort. In this article, we discuss the current progress and some future directions in the development of antiviral strategies against CCHFV.     

Immunological Responses to Seoul Orthohantavirus in Experimentally and Naturally Infected Brown Rats (Rattus norvegicus)

To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts.     

Interactions between pork consumption,CagA status and IL-1B-31 genotypes in gastric cancer

AIM: To explore potential interactions among Helicobacter pylori (H. pylori), CagA status, interleukin (IL)-1B-31 genotypes, and non-cardiac gastric cancer (GC) risk.METHODS: A case-control study of non-cardia GC was performed at 3 hospitals located in Xi’an, China, between September 2008 and July 2010. We included 171 patients with histologically diagnosed primary non-cardia GC and 367 population based controls (matched by sex, age and city of residence). A standardized questionnaire was used to obtain information regarding potential risk factors, including pork consumption. H. pylori CagA status was assessed by enzyme-linked immunosorbent assay, and IL-1B-31 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. Multivariate unconditional logistic regression was used to explore potential interactions among the factors.RESULTS: The CagA appeared to confer an increased risk of GC (OR = 1.81, 95%CI: 1.25-2.61). The main associations with IL-1B-31C allele here were 0.98 (95%CI: 0.59-1.63) for CC vs TT and 0.99 (95%CI: 0.64-1.51) for C Carriers vs TT. However, no associations were observed for CagA or IL-1B-31 genotype status among subjects who reported low pork consumption (P for interaction = 0.11). In contrast, high pork consumption and IL-1B-31C genotypes appeared to synergistically increase GC risk (P for interaction = 0.048) after adjusting for confounding factors, particularly among subjects with CagA (OR = 3.07, 95%CI: 1.17-10.79). We did not observe effect modification of pork consumption by H. pylori CagA status, or between H. pylori CagA status and IL-1B-31 genotypes after adjustment for pork consumption and other factors.CONCLUSION: These interaction relationships among CagA, IL-1B-31 and pork consumption may have implications for development of the preventive strategies for the early detection of non-cardiac GC.     

Q fever in Quebec (1989�C93): Report of 14 cases

Q fever, a zoonosis acquired by inhalation of the rickettsia Coxiella burnetii, is rarely diagnosed in Canada. The world incidence has been increasing since 1960, because of progressive dissemination of this microorganism in animal populations, particularly domestic ruminants. Some recent outbreaks were caused by cats. Of 14 cases reported in Quebec between 1989 and the beginning of 1993, nine occurred successively in an 18-month period in the rural region surrounding Trois-Rivières, after contact with livestock or cats. These cases are reported here, with the results of serological screening of the workers of an abattoir where one of the cases worked. Five additional cases reported in Quebec during the same period are briefly reviewed.     

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.     

Asymptomatic humans transmit dengue virus to mosquitoes

Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.With 3.97 billion people living in 128 countries currently at risk for infection, dengue viruses (DENV-1 to -4) cause more human morbidity and mortality worldwide than any other arthropod-borne virus (1, 2). Aedes aegypti mosquitoes are the primary vectors of DENV throughout the tropics (3). Dengue prevention relies on the control of Ae. aegypti populations, which is failing in most parts of the world due to lack of resources, lack of political will, and/or ineffective implementation (4).Virus transmission from infected humans to mosquitoes is a critical step in dengue epidemiology, but due to logistical constraints it has been directly examined only in a handful of studies to date (5). In initial experimental infections of human volunteers during the 1920s (6, 7), the onset of clinical symptoms occurred 4–9 d after virus inoculation by mosquito bite (8). DENV-infected humans were infectious to mosquitoes from 2 d before to 2 d after the onset of symptoms, and Ae. aegypti fed on viremic people were able to transmit virus to another person after at least 11 d of extrinsic incubation (8). Results from later studies indicated that, for naturally infected people with clinically apparent dengue, the duration of detectable viremia was on average 4–5 d after the onset of symptoms, but could range from 2 to 12 d (9, 10). Investigators in Vietnam fed Ae. aegypti directly on 208 symptomatic, hospitalized dengue patients and reported that the probability of successful human-to-mosquito DENV transmission was coincident with the kinetics of viremia (11). Dengue patients were infectious up to 5 d after the onset of symptoms, which generally corresponded with “defervescence” (11).All previous studies on human-to-mosquito DENV transmission were limited to people with overt illness and did not consider subclinical infections. An estimated 300 million of the total 390 million DENV infections per year are clinically inapparent or mildly symptomatic, i.e., no illness that disrupted a person’s daily routine (1). Following Grange et al. (12), we use “inapparent” or “subclinical” interchangeably to denote infections confirmed by virus detection or seroconversion, but with insufficient symptoms to be detected by existing surveillance systems and health care providers. “Asymptomatic” refers to a confirmed DENV infection in the complete absence of reported or detected symptoms. Inapparent human DENV infections are a potentially important component of the overall burden of dengue because they can serve as a previously unrecognized source of mosquito infection (12). Epidemic transmission of DENV associated with low viremia levels and mild illness has been reported (13). It has long been assumed, but not empirically verified, that people with inapparent infections fail to infect mosquitoes because they do not reach sufficiently high viremia levels (5). This assumption is based on the observation that disease severity is positively correlated with the magnitude of DENV viremia (10, 11, 14). To our knowledge, the only study that quantified viral RNA levels in a limited number of asymptomatic DENV infections in humans did not detect a significantly lower viremia (15), but infectiousness to mosquitoes was not evaluated. The aim of the present study was to document variation in DENV infectiousness of naturally infected humans across the spectrum of disease manifestations, including fully asymptomatic infections, and to verify the assumption that people with inapparent infections are not infectious to mosquitoes.