Molecular Detection of Dengue Virus, Zika Virus, and Chikungunya Virus Arboviruses in Neotropical Bats.

Background: The dengue, Zika, and Chikungunya arboviruses have spread in America in the past year, thus becoming global health issues. These viruses are maintained in nature in two transmission cycles: an urban cycle, transmitted from hematophagous mosquitoes to humans, and a wild cycle, recorded only in Africa and Asia, involving mosquitoes and nonhuman primates as natural hosts. The evidence shows that these arboviruses infect other wild mammals in America, such as rodents, marsupials, and bats. This study aimed to determine the potential natural infection of arboviruses in bats captured in contrasting sites (tropical forests, urban areas, and caves) in Oaxaca, Mexico. Materials and Methods: Liver samples were collected from some bats and tested for RNA from dengue, Zika, and Chikungunya with the quantitative real-time PCR assay. We analyzed 162 samples that encompassed 23 bat species. Results: No natural infection with any of the three arboviruses was detected in any sample tested. Conclusion: The existence of a wild cycle of the three arboviruses in the American continent is not ruled out. However, owing to the low or zero prevalence recorded in other studies and the present study, bats are likely involved in the arbovirus transmission cycle as accidental hosts.

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.

Adaptation of a Human Diagnostic Kit to Detect Dengue, Zika, and Chikungunya Viruses in Mosquito Samples (Aedes aegypti and Aedes albopictus): A Contribution to Public Health in the International Triple Border (Brazil, Paraguay, and Argentina).

Objective: The objective of this work was to adapt a diagnostic kit developed for humans to identify Dengue (DENV1, DENV2, DENV3, DENV4), Zika (ZIKV) and Chikungunya virus (CHIKV) in females of Aedes aegypti and Aedes albopictus and to verify if the occurrence of mosquitoes infected with these three arboviruses are being found in regions with high occurrence of these diseases in humans. Materials and Methods: For this purpose, live mosquitoes were captured between January and June 2020 using 3,476 traps permanently installed in the field were used. After capture, the species were identified, then the females were placed in a pool of 2 to 10 specimens and sent to the laboratory for detection of DENV1, DENV2, DENV3, DENV4, ZIKV and CHIKV by RT-PCR using a commercial human kit for arboviruses. Results: Of the 76 mosquito pools collected, six (7.9%) pools tested positive for the DENV2 virus. The DENV-positive mosquitoes were collected in regions with a high incidence of reported cases of Dengue or in adjacent areas. Conclusion: The absence of kits for the detection of these arboviruses in Aedes is a limiting factor and the adequacy of commercial kits, already used for the diagnosis of arboviruses in humans, the results presented demonstrate that it is possible to identify the presence of DENV2 in mosquitoes with the respective kit, reinforcing the use of RT-qPCR as a robust diagnostic tool for epidemiological surveillance allowing managers to receive timely results for decision-making regarding prevention and control actions.

Seroepidemiological survey on chikungunya in endemic zones for arboviruses in Brazil, 2019.

This study aimed to identify the seroprevalence of chikungunya and its associated factors in the city of Quixadá, Ceará, Brazil. We also aimed to identify the spatial distribution patterns of positive cases. A cross-sectional survey was conducted with a questionnaire about clinical symptoms, socioeconomic and demographic factors, and a 10 ml blood sample was collected and analysed by ELISA. For the bivariate analysis, we use the chi-square test, a prevalence ratio and its 95% confidence interval. A robust Poisson hierarchical regression was used to adjust for confounders. The Kernel density was performed for the spatial analysis. A total of 409 samples were analysed; of them, 70.7% were seropositive for previous exposure to chikungunya virus (CHIKV). High seropositivity for CHIKV was higher in female participants (75.5%; PR = 1.23; 95% CI: 1.06-1.43), those aged 31 years or more (74.3%; PR = 1.62; 95% CI: 1.04-2.52), and those with elementary education level (75.0%; PR = 1.30; 95% CI: 1.06-1.60). There were also high seroprevalence in those with less than a minimum wage per month (89.5%; PR = 1.59; 95% CI: 1.11-2.30), housewives (87.5%; PR = 1.64; 95% CI: 1.24-2.18) and unemployed (80.0%; PR = 1.50; 95% CI: 1.10-2.06). After adjusting for age, morning stiffness was the only chikungunya symptom that remained associated (PR = 1.20; 95% CI: 1.06-1.37; p < .001). There was an area of high density of cases in the downtown and two areas of medium density in nearby regions. Otherwise, the higher seroprevalence rates were in the peripherical neighbourhoods. There is a hyperendemicity of CHIKV in Quixadá, and most cases are spatially contiguous. The main associated clinical sign is morning stiffness, but other factors such as low income and spending a longer time at home were significantly associated with higher seroprevalence.

Evaluating livestock farmers knowledge, beliefs, and management of arboviral diseases in Kenya: A multivariate fractional probit approach.

Globally, arthropod-borne virus (arbovirus) infections continue to pose substantial threats to public health and economic development, especially in developing countries. In Kenya, although arboviral diseases (ADs) are largely endemic, little is known about the factors influencing livestock farmers' knowledge, beliefs, and management (KBM) of the three major ADs: Rift Valley fever (RVF), dengue fever and chikungunya fever. This study evaluates the drivers of livestock farmers' KBM of ADs from a sample of 629 respondents selected using a three-stage sampling procedure in Kenya's three hotspot counties of Baringo, Kwale, and Kilifi. A multivariate fractional probit model was used to assess the factors influencing the intensity of KBM. Only a quarter of the farmers had any knowledge of ADs while over four-fifths of them could not manage any of the three diseases. Access to information (experience and awareness), income, education, religion, and distance to a health facility considerably influenced the intensity of farmers' KBM of ADs in Kenya. Thus, initiatives geared towards improving access to information through massive awareness campaigns are necessary to mitigate behavioral barriers in ADs management among rural communities in Kenya.

High throughput estimates of Wolbachia, Zika and chikungunya infection in Aedes aegypti by near-infrared spectroscopy to improve arbovirus surveillance.

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2-4 days old and 5-7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions.

Current Status and a Perspective of Mosquito-Borne Diseases in the Republic of Korea.

Background: Mosquito-borne diseases in the Republic of Korea have a unique epidemiology due to the rapid improvement in hygiene and economic status, occurrence of four distinct seasons, and separation from North Korea owing to the political situation. Therefore, we aimed to analyze and review the epidemiology of mosquito-borne diseases in Korea. Methods: The incidence and geographical distribution of malaria, Japanese encephalitis (JE), Zika virus infection, chikungunya fever, and dengue fever were investigated using data from the Korean Centers for Disease Control and Prevention. Lymphatic filariasis and West Nile fever, which have rarely been reported in Korea, have also been discussed in this literature review. Results and Conclusions: Malaria disappeared from Korea in 1979, but since its re-emergence in 1993 there has been constant occurrence with local transmission. In Korea, vivax malaria is the only prevailing disease, and the clinically problematic chloroquine resistance has not been reported. The incidence of JE has greatly reduced since the introduction of the national vaccination program for children in 1985. However, the incidence of JE has been increasing recently, especially in adults >40 years of age. Filariasis, which was previously endemic to Jeju Island and the southern coastal area, has not been reported since 2002. Although there are numerous imported cases with increasing overseas travel, there are still no indigenous cases of Zika, chikungunya, and dengue fever reported in Korea. The West Nile virus was isolated from migratory birds, but there has been only one imported human case to date.

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies.

BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

Adverse event following live attenuated chikungunya vaccine in a cynomolgus macaque with pre-existing chronic hydrocephalus.

A cynomolgus macaque (Macaca fascicularis) with a pre-existing, undiagnosed, subclinical but severe cerebral hydrocephalus was enrolled in a study of long-term immunogenicity of the IRES/CHIK vaccine. The animal began showing signs of neurological dysfunction post-vaccination, which progressed and ultimately resulted in euthanasia. The underlying brain abnormality was revealed at necropsy and was subsequently investigated with gross and microscopic examination. This becomes the first reported case of an adverse event following administration of a live attenuated vaccine and suggests the possibility of an increased susceptibility risk of unwanted adverse outcome associated with vaccination in populations with pre-existing conditions such as hydrocephalus.

Engineering and evaluation of amyloid assemblies as a nanovaccine against the Chikungunya virus.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform. Chimeric peptides, comprising the peptide antigen attached to the amyloid core by a short flexible linker, were prepared by solid phase synthesis. As observed using atomic force microscopy, these polypeptides self-assembled into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. A quaternary conformation rich in cross-ß-sheets characterized these assemblies, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. ELISA assays and transmission electronic microscopy of immunogold labeled-fibrils revealed a high density of the Chikungunya virus E2 glycoprotein derived epitope exposed on the fibril surface. These amyloid fibrils were cytocompatible and were efficiently uptaken by macrophages. Mice immunization revealed a robust IgG response against the E2EP3 epitope, which was dependent on self-assembly and did not require co-injection of the Alhydrogel adjuvant. These results indicate that cross-ß-sheet amyloid assemblies constitute suitable synthetic self-adjuvanted assemblies to anchor antigenic determinants and to increase the immunogenicity of peptide epitopes.

Hematologic Parameters and Viral Status for Zika, Chikungunya, Bluetongue, and Epizootic Hemorrhagic Disease in White-tailed Deer ( Odocoileus virginianus) on St. John, US Virgin Islands.

A population of white-tailed deer ( Odocoileus virginianus) resides throughout the island of St. John, US Virgin Islands, predominately in the Virgin Islands National Park. Adult deer ( n=23), ranging from 1 yr to 8 yr old, were assessed to characterize body condition and health. Serologic samples were screened for important viral pathogens in the area, including Zika, chikungunya, bluetongue, and epizootic hemorrhagic disease viruses. Samples were collected in July 2016; males were in velvet and all females were in diestrus. Deer had recovered from a severe drought the previous year but were generally healthy, with a low-level but high incidence of tick parasitism. Marked statistically significant changes in hematocrit and hemoglobin levels were associated with the effects of the anesthetic mixture used for capture. No other statistically significant differences were observed. Serum from four deer induced reduction in Zika virus plaques, suggesting possible exposure. No serum was reactive for chikungunya virus. Bluetongue and epizootic hemorrhagic disease antibodies were present in 50% of the sampled deer, but no clinical signs associated with disease were observed during the study period. These data will be valuable for future dynamic health assessment and may help assess changes to the population, such as those induced by climate change, infectious disease, or other demographic events.

Reptiles and Amphibians as Potential Reservoir Hosts of Chikungunya Virus.

Chikungunya virus is an emerging arbovirus of significant human-health concern. Little is known about its sylvatic cycle, including whether ectothermic vertebrates are permissive to infection. In this study, individuals from ten species of reptiles and amphibians were inoculated with chikungunya virus and samples of blood were tested to characterize viremia and seroconversion. Viremia was not detected in cane toads, house geckos, or American alligators, but most of the green iguanas, red-eared sliders, ball and Burmese pythons, leopard frogs, Texas toads, and garter snakes developed viremia. Peak virus titers in serum of up to 4.5, 4.7, and 5.1 log10 plaque-forming units per milliliter were observed for garter snakes, ball pythons, and Texas toads, respectively. These results add to those of other studies that have suggested a possible role for ectothermic vertebrates in the ecology of arbovirus maintenance and transmission in nature.

Enzootic Circulation of Chikungunya Virus in East Africa: Serological Evidence in Non-human Kenyan Primates.

Chikungunya virus (CHIKV) is a globally emerging pathogen causing debilitating arthralgia and fever in humans. First identified in Tanzania (1953), this mosquito-borne alphavirus received little further attention until a 2004 re-emergence in Kenya from an unknown source. This outbreak subsequently spread to the Indian Ocean, with adaptation for transmission by a new urban vector. Under the hypothesis that sylvatic progenitor cycles of CHIKV exist in Kenya (as reported in West Africa, between non-human primates (NHPs) and arboreal Aedes spp. mosquitoes), we pursued evidence of enzootic transmission and human spillover events. We initially screened 252 archived NHP sera from Kenya using plaque reduction neutralization tests. Given an overall CHIKV seroprevalence of 13.1% (marginally higher in western Kenya), we sought more recent NHP samples during 2014 from sites in Kakamega County, sampling wild blue monkeys, olive baboons, and red-tailed monkeys (N = 33). We also sampled 34 yellow baboons near Kwale, coastal Kenya. Overall, CHIKV seropositivity in 2014 was 13.4% (9/67). Antibodies reactive against closely related o'nyong-nyong virus (ONNV) occurred; however, neutralization titers were too low to conclude ONNV exposure. Seroprevalence for the flavivirus dengue was also detected (28%), mostly near Kwale, suggesting possible spillback from humans to baboons. CHIKV antibodies in some juvenile and subadult NHPs suggested recent circulation. We conclude that CHIKV is circulating in western Kenya, despite the 2004 human outbreaks only being reported coastally. Further work to understand the enzootic ecology of CHIKV in east Africa is needed to identify sites of human spillover contact where urban transmission may be initiated.

Viremia in North American Mammals and Birds After Experimental Infection with Chikungunya Viruses.

Chikungunya virus (CHIKV) is an arthropod-borne virus, which is known to cause severe disease only in humans. To investigate its potential zoonotic host range and evaluate reservoir competence among these hosts, experimental infections were performed on individuals from nine avian and 12 mammalian species representing both domestic and wild animals common to North America. Hamsters and inbred mice have previously been shown to develop viremia after inoculation with CHIKV and were used as positive controls for infection. Aside from big brown bats (Eptesicus fuscus), none of the mammals or birds developed detectable viremia or overt clinical disease. However, most mammals and a smaller proportion of birds developed neutralizing antibody responses to CHIKV. On the basis of these results, it seems unlikely that CHIKV poses a significant health threat to most domestic animals or wildlife and that the species examined do not likely contribute to natural transmission cycles. Additional studies should further evaluate bats and wild rodents as potential reservoir hosts for CHIKV transmission during human epidemics.