Development of genus-specific universal primers for the detection of flaviviruses.

BACKGROUND: Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. METHODS: We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. RESULTS: The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. CONCLUSION: This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

Identification of Three Novel Genes in Phenuiviridae Detected from Aedes Mosquitoes in Hokkaido, Japan.

Mosquitoes are important arthropod vectors of arboviruses. The family Phenuiviridae includes several medically important arboviruses, such as the Rift Valley fever phlebovirus and Toscana phlebovirus. Recent comprehensive genetic analyses have identified many novel mosquito-specific viruses that are phylogenetically related to Phenuiviridae. We collected mosquitoes from Hokkaido in northern Japan, and conducted reverse transcription polymerase chain reactions (RT-PCRs) targeting the RNA-dependent RNA polymerase (RdRp) gene of Phenuiviridae. A total of 285 pools, comprising 3,082 mosquitoes from 2 genera and 8 species, were collected. Partial RdRp sequences were detected in 97 pools, which allowed us to classify the viruses into 3 clusters provisionally designated as Etutanne virus (ETTV) 1, 2, and 3. The virus most closely related to ETTVs is Narangue virus (family Phenuiviridae, genus Mobuvirus), which was detected in Mansonia mosquitoes; the nucleotide and amino acid sequences of the Narangue virus are 58.4-66.2% and 64.7-86.7% similar, respectively, to those of ETTVs. PCR and RT-PCR using DNA and RNase digestion methods showed that the ETTVs are RNA viruses that do not form non-retroviral integrated RNA virus sequences in the mosquito genome.

Update on temporal and spatial abundance of dengue vectors in Penang, Malaysia.

It is important to obtain frequent measurements of the abundance, distribution, and seasonality of mosquito vectors to determine the risk of disease transmission. The number of cases of dengue infection has increased in recent years on Penang Island, Malaysia, with recurring epidemics. However, ongoing control attempts are being critically hampered by the lack of up-to-date information regarding the vectors. To overcome this problem, we examined the current situation and distribution of dengue vectors on the island. Residences throughout the urban, suburban, and rural areas were inspected through wet and dry seasons between February 2009 and February 2010. Two vectors were encountered in the survey, with Aedes aegypti present in especially high numbers mostly in urban areas. Similar observations were noted for Ae. albopictus in rural areas. The former species was more abundant in outdoor containers, while the latter showed almost equivalent abundance both outdoors and indoors. The dengue virus was active in both urban and rural areas, and the number of cases of infection was higher in areas where Ae. aegypti was predominant. The abundance of immature Ae. albopictus was positively correlated with rainfall (r2 = 0.461; P < 0.05), but this was not the case for Ae. aegypti. For both species, the size of immature populations tended to increase with increasing intensity of rain, but heavy rains resulted in population loss. In addition to updating data regarding the larval habitats and locations (outdoors and indoors), this study highlighted the importance of spatial vector control stratification, which has the potential to reduce costs in control programs.

Antioxidative systems defense against oxidative stress induced by blood meal in Aedes aegypti.

The release of iron from hemoglobin via the digestion of a blood meal in female mosquitoes can potentially induce oxidative damage and even death. These mosquitoes need an effective antioxidant to prevent this. We carried out this study to determine the antioxidant activities of ferritin, glutathione peroxidase (GPx), glutathione S-transferase (GST) and catalase, and glutathione (GSH). These enzymes had their greatest activity among 4 day old virgin female mosquitoes. Using a single blood feed model, groups of female mosquitoes were tested at 4, 7 and 20 days post-emergence. They were allowed to feed on a hamster for 1 hour. The engorged mosquitoes were collected at 48 and 72 hours after their blood meal. There were no changes in GSH, GPx, GST or catalase levels, but ferritin levels increased markedly (about 2-3 fold) by 48 hours post blood-feed in all mosquito age groups. On repeated blood-feed experiments, mosquitoes aged 4 days were blood fed, once every 3 days and were collected 48 hours after their most recent blood meal. A significant decrease in GSH and GPx activity and a further increase in ferritin, were detected. Ferritin levels were 0.19+/-0.03 and 0.14+/-0.02 ng/microg protein in the repeat and single blood-feed groups, respectively. These results suggest ferritin is an inducible, sensitive defense system protecting against oxidative stress caused by iron derived from blood meals in Aedes aegypti mosquitoes.

Zika Virus Potential Vectors among Aedes Mosquitoes from Hokkaido, Northern Japan: Implications for Potential Emergence of Zika Disease.

The Zika virus (ZIKV) is a rapidly expanding mosquito-borne virus that causes febrile illness in humans. Aedes aegypti and Ae. albopictus are the primary ZIKV vectors; however, the potential vector competence of other Aedes mosquitoes distributed in northern Japan (Palearctic ecozone) are not yet known. In this study, the susceptibility to Zika virus infection of three Aedes mosquitoes distributed in the main city of the northern Japan and their capacities as vectors for ZIKV were evaluated. Field-collected mosquitoes were fed ad libitum an infectious blood meal containing the ZIKV PRVABC59. The Zika virus was detected in the abdomen of Ae. galloisi and Ae. japonicus at 2-10 days post infection (PI), and from the thorax and head of Ae. galloisi at 10 days PI, resulting in 17.6% and 5.9% infection rates, respectively. The Zika virus was not detected from Ae. punctor at any time. Some northern Japanese Aedes could be suspected as vectors of ZIKV but the risk may be low when compared with major ZIKV vectors.

New Insights into the Biology of the Emerging Tembusu Virus.

Reported for the first time in 1955 in Malaysia, Tembusu virus (TMUV) remained, for a long time, in the shadow of flaviviruses with human health importance such as dengue virus or Japanese encephalitis virus. However, since 2010 and the first large epidemic in duck farms in China, the threat of its emergence on a large scale in Asia or even its spillover into the human population is becoming more and more significant. This review aims to report current knowledge on TMUV from viral particle organization to the development of specific vaccines and therapeutics, with a particular focus on host-virus interactions.

Vector Competence for Dengue-2 Viruses Isolated from Patients with Different Disease Severity.

Dynamics of dengue serotype 2 virus isolated from patients with different disease severity, namely flu-like classic dengue fever (DF) and dengue shock syndrome (DSS) were studied in its mosquito vector Aedes aegypti. We compared isolate infectivity and vector competence (VC) among thirty two A. aegypti-viral isolate pairs. Mosquito populations from high dengue incidence area exhibited overall greater VC than those from low dengue incidence area at 58.1% and 52.5%, respectively. On the other hand, the overall infection rates for the isolates ThNR2/772 (DF, 62.3%) and ThNR2/391 (DSS, 60.9%), were significantly higher than those for isolates ThNR2/406 (DF, 55.2%) and ThNR2/479 (DSS, 54.8%). These results suggest that the efficacy of dengue virus circulation was likely to vary according to the combination between the virus strains and origin of the mosquito strains, and this may have epidemiologic implications toward the incidence of flu-like classic dengue fever (DF) and dengue shock syndrome (DSS).

Geometric morphometrics approach towards discrimination of three member species of Maculatus group in Thailand.

Members of the Maculatus group are important malaria vectors in the border regions of Thailand. However, the role of each species in malaria transmission remains unclear because of their highly similar morphologies, making them difficult to be differentiated. Whereas An. pseudowillmori may be identified by the color pattern of some scales on abdomen and wings, the distinction between An. maculatus and An. sawadwongporni relies on the wings only. Scales are labile structures, as they may be accidentally removed during capture and transportation to the laboratory. To discriminate among the species of this group, we tested the suitability of geometric techniques. Shape variables were used as input for discriminant analyses and validated reclassification. Both landmark- and outline-based geometric techniques disclosed significant differences between the three species. For the delicate An. maculatus - An. sawadwongporni distinction, the outline-based approach appeared as the most promising, with validated reclassification scores reaching 93%, as compared to 77% obtained by landmark data. For An. pseudowillmori, reclassification scores were 100% and 94%, respectively. Geometric morphometrics may provide an alternative and useful complement for discriminating members of the Maculatus group.

Dengue Virus-Induced Reactive Oxygen Species Production in Rat Microglial Cells.

Encephalitis has been described worldwide as a severe complication in patients infected by dengue virus. Reactive oxygen species (ROS) production is a key mechanism involved in the neuronal damage caused by viral encephalitis. In the present study, the capability of dengue virus serotypes 2 (DENV2) and DENV4 to induce ROS production was investigated in a rat microglial cell line, HAPI cells. The cells were infected with DENV2 and DENV4 at a multiplicity of infection of 0.1 for a 2-h adsorption period. Japanese encephalitis virus (JEV) was used as the reference. DENV2- and DENV4-induced microglial activation and significantly increased ROS production corresponded to decreased cell viability. The activity of DENV4 was significantly higher than the activities of DENV2 and JEV at 48 and 72 h post infection. DENV4 partly induced ROS production via an iron-induced Fenton reaction, as demonstrated by the treatment with an iron chelator, deferiprone. Despite the induction of increased inducible nitric oxide synthase expression and nitric oxide (NO) production by JEV, DENV2, and DENV4 did not induce NO production, suggesting the activation of different pathways in response to infections by different viruses. In conclusion, DENV2 and DENV4 have the capability to induce ROS production and activate microglia, which have been reported as the key components of neuronal damage.

Effect of iron overload on furin expression in wild-type and ß-thalassemic mice.

Furin is a proprotein convertase enzyme. In the liver, it cleaves prohepcidin to form active hepcidin-25, which regulates systemic iron homeostasis. Hepcidin deficiency is a component of several iron overload disorders, including ß-thalassemia. Several studies have identified factors that repress hepcidin gene transcription in iron overload. However, the effect of iron overload on furin, a post-translational regulator of hepcidin, has never been evaluated. The present study aimed to investigate the changes in furin and related factors in parenteral iron-overloaded mice, including those with ß-thalassemia. Wild-type (WT) and ß-thalassemia intermedia (th3/+) C57BL/6 mice were intraperitoneally injected with 9 doses of iron dextran (1 g iron/kg body weight) over 2 weeks. In the iron overload condition, our data demonstrated a significant Furin mRNA reduction in WT and th3/+ mice. In addition, the liver furin protein level in iron-overloaded WT mice was significantly reduced by 70% compared to control WT mice. However, the liver furin protein in iron-overloaded th3/+ mice did not show a significant reduction compared to control th3/+ mice. The hepcidin gene (hepcidin antimicrobial peptide gene, Hamp1) expression was increased in iron-overloaded WT and th3/+ mice. Surprisingly, the liver hepcidin protein level and total serum hepcidin were not increased in both WT and th3/+ mice with iron overload, regardless of the increase in Hamp1 mRNA. In conclusion, we demonstrate furin downregulation in conjunction with Hamp1 mRNA-unrelated pattern of hepcidin protein expression in iron-overloaded mice, particularly the WT mice, suggesting that, not only the amount of hepcidin but also the furin-mediated physiological activity may be decreased in severe iron overload condition.

The phenetic structure of Aedes albopictus.

The large and recent migrations of the main vector of the Chikungunya virus, Aedes albopictus, raise questions about the epidemiological impact of possible microevolutionary changes in new territories. Quantitative traits are suitable to detect such changes as induced by environmental adaptations, local competition and founder effects. Using landmark-based geometric morphometrics, we compared the size and shape of 22 populations (1572 females) of Ae. albopictus. The collection sites covered five countries around the world, with special emphasis on Asia, which is assumed to be the origin of the vector. Some collections came from places where an important epidemic outbreak of Chikungunya has recently occurred. Quantitative comparisons were based on 18 anatomical landmarks on the wing. To contrast geographic and possible interspecific shape variation, close species were introduced in the sample, namely five Aedes riversi and nine Ae. flavopictus from Japan. The three species had similar size, but they were clearly separated by shape. Within Ae. albopictus, there was general agreement on size variation with the available climatic data. Shape variation was less related to climatic data than to either geography or a known itinerary of past territorial expansion. Thus, two main clusters were distinguished by wing shape variation: the first one composed of the Southeast Asian sample, the second one grouping Japan, Florida, Hawaii and the Reunion Island samples. The Southeast Asian countries, assumed to be at the origin of the geographic expansion of the mosquito, had similar wings and constituted a distinct group where localities clustered into northern and southern localities. Contrasting with this homogeneous group, very distant localities such as United States (US) and Japan shared a common shape pattern. The US Ae. albopictus samples (Hawaii and Florida) were indeed very similar to the Japanese samples, with Florida behaving exactly like a northern Japanese locality, close to Tokyo and Yokohama. Shape proximity among these distant areas could be explained by a common and relatively recent ancestor, as generally suggested in the literature. The three Reunion Island samples conformed a group external to the Southeast Asian countries, loosely connected to the remaining localities. Thus, areas where recent epidemics took place, the Reunion Island and the southernmost localities of Thailand, did not cluster together, but represented separate groups. In sum, the size of the wing was apparently under the influence of climatic factors, while its shape could contribute information on species and geographic differences. Apparent departures from expectations based on geography were explained by known past and present migratory routes. These patterns of shape variation were compatible with genetic drift, suggesting microevolutionary changes probably induced by the expansion of the Ae. albopictus mosquito.

Characterization of chikungunya virus infection of a human keratinocyte cell line: role of mosquito salivary gland protein in suppressing the host immune response.

The chikungunya virus (CHIKV) is a mosquito-borne virus that has recently re-emerged in several countries. On infection, the first vertebrate cells to come into contact with CHIKV are skin cells; mosquitoes inoculate the virus together with salivary gland protein into host skin while probing and feeding on blood. However, there is little known about the susceptibility of human skin cells to CHIKV infection. To clarify this, we investigated the kinetics of CHIKV in the human keratinocyte cell line, HaCaT. CHIKV actively replicated in HaCaT cells, with virus titers in the supernatant increasing to 2.8 × 10(4) plaque-forming units (PFU) ml(-1) 24h post infection. CHIKV infection suppressed production of interleukin-8 (IL-8) in HaCaT cells. The function of IL-8 is to recruit immune cells to virus-infected sites, a process known as chemotaxis. Furthermore, we assessed the role of mosquito salivary gland protein in CHIKV infections by comparing the levels of CHIKV gene expression and chemokine production in HaCaT cells with and without salivary gland extract (SGE). SGE enhanced both the expression of the CHIKV gene and the suppression effect of CHIKV on IL-8 production. Our data suggest that the HaCaT cell line represents an effective tool for investigating the mechanism of CHIKV transmission and spread in skin cells. At the mosquito bite site, CHIKV works together with SGE to ensure the virus replicates in skin cells and escapes the host immune system by suppression of IL-8 production.

Climate associated size and shape changes in Aedes aegypti (Diptera: Culicidae) populations from Thailand.

In spite of the adult body size variability of Aedes aegypti (Linnaeus) and its likely association with life history and vectorial capacity, the causes of size variation itself have been only partially identified. In particular, possible important factors such as climatic variation have not received much attention. The objective of this 2-year study was to describe from field collections the relationship of Ae. aegypti metric properties with available climatic data. The study took place in a dengue hyperendemic area of Thailand. Fourth instar larvae (L(4)) and pupae were collected from the same breeding places allowing the comparisons between seven successive collections, four in 2007 and three in 2008. Climatic data were relative humidity (RH) and temperature (T). They were considered for the periods covering either the pre-imaginal development or, assuming heritability of size, the previous generation. The pre-imaginal period was further subdivided into embryonic and larval phases of development. Size was estimated by traditional and geometric techniques, the latter based on 18 landmarks collected at the intersections of veins also allowing estimation of shape. The shape variation of the wing followed similar patterns as for size and was shown to be a passive allometric change. No significant correlation of size or shape could be disclosed with T. In contrast, significant correlation with RH was found during two periods of examination: (i) the period affecting the generation previous to the time of collection, suggesting possible selective mechanisms on genitors, and (ii) the one occurring during pre-imaginal development. The subdivision of the latter into embryonic and larval phases allowed to evidence a possible selecting effect on embryonic development. The selection would act through the resistance to water loss which is known to depend on the relative surface of the cuticle. In conclusion, our data highlight the importance of the emerged period of Ae. aegypti eggs as a critical time for the size of future adults, and point to the relative humidity as the likely selecting factor.