Nyong Virus Research Articles

A Field-Deployable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of the Chikungunya Virus.

BackgroundChikungunya virus (CHIKV) is a mosquito-borne virus currently transmitted in about 60 countries. CHIKV causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. Detection of the infection is mostly done using RT-RCR or ELISA, which are not suitable for point-of-care diagnosis.Methodology/Principal FindingsIn this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of the CHIKV was developed. The assay sensitivity, specificity, and cross-reactivity were tested. CHIKV RT-RPA assay detected down to 80 genome copies/reaction in a maximum of 15 minutes. It successfully identified 18 isolates representing the three CHIKV genotypes. No cross-reactivity was detected to other alphaviruses and arboviruses except O'nyong'nyong virus, which could be differentiated by a modified RPA primer pair. Seventy-eight samples were screened both by RT-RPA and real-time RT-PCR. The diagnostic sensitivity and specificity of the CHIKV RT-RPA assay were determined at 100%.Conclusions/SignificanceThe developed RT-RPA assay represents a promising method for the molecular detection of CHIKV at point of need.

Alphavirus protease inhibitors from natural sources: A homology modeling and molecular docking investigation

Alphaviruses such as Chikungunya virus (CHIKV), O’Nyong–Nyong virus (ONNV), Ross River virus (RRV), Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV), are mosquito-transmitted viruses that can cause fevers, rash, and rheumatic diseases (CHIKV, ONNV, RRV) or potentially fatal encephalitis (EEEV, VEEV, WEEV) in humans. These diseases are considered neglected tropical diseases for which there are no current antiviral therapies or vaccines available. The alphavirus non-structural protein 2 (nsP2) contains a papain-like protease, which is considered to be a promising target for antiviral drug discovery. In this work, molecular docking analyses have been carried out on a library of 2174 plant-derived natural products (290 alkaloids, 664 terpenoids, 1060 polyphenolics, and 160 miscellaneous phytochemicals) with the nsP2 proteases of CHIKV, ONNV, RRV, EEEV, VEEV, WEEV, as well as Aura virus (AURV), Barmah Forest Virus (BFV), Semliki Forest virus (SFV), and Sindbis virus (SINV) in order to identity structural scaffolds for inhibitor design or discovery. Of the 2174 phytochemicals examined, a total of 127 showed promising docking affinities and poses to one or more of the nsP2 proteases, and this knowledge can be used to guide experimental investigation of potential inhibitors.

Interferon-stimulated gene LY6E enhances entry of diverse RNA viruses

Abstract The type I interferon (IFN) response is the primary cellular defense mechanism against viruses. IFN protects against viral infection by activating the expression of hundreds of interferon-stimulated genes (ISGs), some of which have potent antiviral effector activity. Our recent screening efforts to identify novel antiviral ISGs unexpectedly uncovered a small subset of genes that enhanced viral infectivity. The goals of this project are to characterize the mechanism of LY6E, an ISG that enhanced unrelated viruses from multiple families. Here we show that ectopic expression of LY6E enhances cellular infection by a subset of enveloped RNA viruses from diverse families, including yellow fever virus, influenza A virus, vesicular stomatitis virus, and O’nyong’nyong virus. The enhancement phenotype is dependent on cell lineage, with the strongest effects seen in fibroblast and monocytic cell lines. Ablation of endogenous LY6E by CRISPR/Cas9 reduces but does not abrogate viral infectivity, indicating that LY6E is important for optimal infection. Loss of LY6E does not alter antiviral protection by IFN, suggesting that the enhancing effects may be direct. Mechanistic studies reveal that LY6E enhances infectivity at an early step in the viral life cycle, after viral binding but prior to the establishment of replication. Visualization of single-cell infections by ImageStream confirms that LY6E enhances viral entry. We conclude that LY6E enhances viral uptake at the population level, which may be important for initiation of the cell-mediated antiviral immune response. Understanding the mechanism and significance of LY6E to the antiviral interferon response may aid in the development of novel antiviral therapeutics.

Preparing for the Next Epidemic with Basic Virology.

Preparing for the Next Epidemic with Basic Virology.

Acquired Neonatal Chikungunya Encephalopathy

To the Editor:Re-emergence of Chikungunya has been reported worldwide. We report 3 neonates with postnatally acquired Chikungunya encephalopathy. Neonate A, a 19-d-old girl presented with tonic seizures and poor feeding. Her cerebrospinal fluid analysis, serum sodium (140 mmol/dl) and calcium (8.5 mg/dl) were normal. Neonate B, a 23-d-old boy presented with fever andmultifocal clonic seizures. He had CSF pleocytosis (total count 70neutrophils: 13 %, lymphocytes: 60 %, monocytes: 27 %, sugar: 53 mg%, protein: 87 mg/dl, culturesterile) and normal electrolytes (sodium 133 mmol/dl, calcium 8.8 mg/dl). Neonate C, a 25-d-old girl presented with poor feeding and multifocal clonic seizures. She had normal electrolytes (sodium 135 mmol/dl, calcium 8.5 mg/dl) and cerebrospinal fluid analysis showed total counts: 12 (neutrophils 82 %), protein: 115 mg/dl, sugar: 61 mg%. All the three neonates developed a blistering hyperpigmented rash involving the face, limbs, trunk and tip of the nose on the 3rd day of illness (Fig. 1). Chikungunya IgM performed using the OnSite Chikungunya IgM Rapid Test, a chromatographic immunoassay (sensitivity 90.3 % and relative specificity 100 %) were found to be positive [1]. The diagnosis of Chikungunya was based on the typical clinical presentation and positive Chikungunya IgM results. However RT-PCR assay or paired acute and convalescent sera to compare anti-Chikungunya antibody titers was not performed. The differentials of Herpes virus, O’nyong’nyong virus and Semliki Forest virus were considered unlikely, based on

High rates of o'nyong nyong and Chikungunya virus transmission in coastal Kenya.

BackgroundChikungunya virus (CHIKV) and o’nyong nyong virus (ONNV) are mosquito-borne alphaviruses endemic in East Africa that cause acute febrile illness and arthritis. The objectives of this study were to measure the seroprevalence of CHIKV and ONNV in coastal Kenya and link it to demographics and other risk factors.MethodologyDemographic and exposure questionnaires were administered to 1,848 participants recruited from two village clusters (Milalani-Nganja and Vuga) in 2009. Sera were tested for alphavirus exposure using standardized CHIKV IgG ELISA protocols and confirmed with plaque reduction neutralization tests (PRNT). Logistic regression models were used to determine the variables associated with seropositivity. Weighted K test for global clustering of houses with alphavirus positive participants was performed for distance ranges of 50–1,000 meters, and G* statistic and kernel density mapping were used to identify locations of higher seroprevalence.Principal Findings486 (26%) participants were seropositive by IgG ELISA. Of 443 PRNT confirmed positives, 25 samples (6%) were CHIKV+, 250 samples (56%) were ONNV+, and 168 samples (38%) had high titers for both. Age was significantly associated with seropositivity (OR 1.01 per year, 95% C.I. 1.00–1.01); however, younger adults were more likely to be seropositive than older adults. Males were less likely to be seropositive (p<0.05; OR 0.79, 95% C.I. 0.64–0.97). Adults who owned a bicycle (p<0.05; OR 1.37, 95% C.I. 1.00–1.85) or motor vehicle (p<0.05; OR 4.64, 95% C.I. 1.19–18.05) were more likely to be seropositive. Spatial analysis demonstrated hotspots of transmission within each village and clustering among local households in Milalani-Nganja, peaking at the 200–500m range.Conclusions/SignificanceAlphavirus exposure, particularly ONNV exposure, is common in coastal Kenya with ongoing interepidemic transmission of both ONNV and CHIKV. Women and adults were more likely to be seropositive. Household location may be a defining factor for the ecology of alphaviral transmission in this region.

Global emergence of Alphaviruses that cause arthritis in humans

Arthropod-borne viruses (arboviruses) may cause severe emerging and re-emerging infectious diseases, which pose a significant threat to human and animal health in the world today. These infectious diseases range from mild febrile illnesses, arthritis, and encephalitis to haemorrhagic fevers. It is postulated that certain environmental factors, vector competence, and host susceptibility have a major impact on the ecology of arboviral diseases. Presently, there is a great interest in the emergence of Alphaviruses because these viruses, including Chikungunya virus, O'nyong'nyong virus, Sindbis virus, Ross River virus, and Mayaro virus, have caused outbreaks in Africa, Asia, Australia, Europe, and America. Some of these viruses are more common in the tropics, whereas others are also found in temperate regions, but the actual factors driving Alphavirus emergence and re-emergence remain unresolved. Furthermore, little is known about the transmission dynamics, pathophysiology, genetic diversity, and evolution of circulating viral strains. In addition, the clinical presentation of Alphaviruses may be similar to other diseases such as dengue, malaria, and typhoid, hence leading to misdiagnosis. However, the typical presence of arthritis may distinguish between Alphaviruses and other differential diagnoses. The absence of validated diagnostic kits for Alphaviruses makes even routine surveillance less feasible. For that purpose, this review describes the occurrence, genetic diversity, clinical characteristics, and the mechanisms involving Alphaviruses causing arthritis in humans. This information may serve as a basis for better awareness and detection of Alphavirus-caused diseases during outbreaks and in establishing appropriate prevention and control measures.

Seroepidemiology of selected arboviruses in febrile patients visiting selected health facilities in the lake/river basin areas of Lake Baringo, Lake Naivasha, and Tana River, Kenya.

Introduction: Arboviruses cause emerging and re-emerging infections affecting humans and animals. They are spread primarily by blood-sucking insects such as mosquitoes, ticks, midges, and sandflies. Changes in climate, ecology, demographic, land-use patterns, and increasing global travel have been linked to an upsurge in arboviral disease. Outbreaks occur periodically followed by persistent low-level circulation.Aim: This study was undertaken to determine the seroepidemiology of selected arboviruses among febrile patients in selected lake/river basins of Kenya.Methods: Using a hospital-based cross-sectional descriptive survey, febrile patients were recruited and their serum samples tested for exposure to immunoglobulin M (IgM) and IgG antibodies against Crimean–Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), West Nile virus (WNV), and chikungunya virus (CHIKV). Samples positive for CHIKV and WNV were further confirmed by the plaque reduction neutralization test (PRNT).Results: Of the 379 samples examined, 176 were IgG positive for at least one of these arboviruses (46.4%, 95% confidence interval [CI] 41.4–51.5%). Virus-specific prevalence for CCHF, RVF, WN, and CHIK was 25.6%, 19.5%, 12.4%, and 2.6%, respectively. These prevalences varied significantly with geographical site (p<0.001), with Tana recording the highest overall arboviral seropositivity. PRNT results for Alphaviruses confirmed that the actual viruses circulating in Baringo were Semliki Forest virus (SFV) and CHIKV, o'nyong nyong virus (ONNV) in Naivasha, and SFV and Sindbis virus (SINDV) in Tana delta. Among the flaviviruses tested, WNV was circulating in all the three sites.Conclusion: There is a high burden of febrile illness in humans due to CCHFV, RVFV, WNV, and CHIKV infection in the river/lake basin regions of Kenya.

High Rates of O’Nyong Nyong and Chikungunya Virus Transmission in Coastal Kenya

High Rates of O’Nyong Nyong and Chikungunya Virus Transmission in Coastal Kenya

Chikungunya Virus: A Major Emerging Threat

Chikungunya Virus: A Major Emerging Threat

O'nyong nyong Virus Molecular Determinants of Unique Vector Specificity Reside in Non-Structural Protein 3

O'nyong nyong virus (ONNV) and Chikungunya virus (CHIKV) are two closely related alphaviruses with very different infection patterns in the mosquito, Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3), was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.

DETERMINANTS OF VECTOR SPECIFICITY OF O’NYONG NYONG AND CHIKUNGUNYA VIRUSES IN ANOPHELES AND AEDES MOSQUITOES

The alphaviruses o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) provide a unique system to study the viral genes involved in vector specificity. ONNV infects both anopheline and culicine mosquitoes, whereas CHIKV infects only culicine mosquitoes. In this study, chimeric viruses were constructed that contained genes from both ONNV and CHIKV. These chimeras and previously described full-length infectious clones of ONNV and CHIKV were evaluated in Anopheles gambiae and Aedes aegypti mosquitoes. Virus derived from the infectious clones of ONNV and CHIKV retained the vector specificity of the parental viruses. All six of the chimeras were found to infect Ae. aegypti mosquitoes at high rates but only the chimera containing viral genes encoding all of the structural proteins of ONNV was able to infect An. gambiae mosquitoes. These data indicate that all of the viral structural proteins are necessary for ONNV to infect An. gambiae mosquitoes.

Infection patterns of o’nyong nyong virus in the malaria‐transmitting mosquito, Anopheles gambiae

Arthropod-borne alphaviruses transmitted by mosquitoes almost exclusively use culicines; however, the alphavirus o'nyong-nyong (ONNV) has the unusual characteristic of being transmitted primarily by anopheline mosquitoes. This unusual attribute makes ONNV a valuable tool in the characterization of mosquito determinants of infection as well as a useful expression system in Anopheles species. We developed a series of recombinant alphaviruses, based upon the genome of ONNV, designed for the expression of heterologous genes. The backbone genome is a full-length infectious cDNA clone of ONNV from which wild-type virus can be rescued. Additional constructs are variants of the primary clone and contain the complete genome plus a duplicated subgenomic promoter element with a multiple cloning site for insertion of heterologous genes. We inserted a green fluorescent protein (GFP) gene downstream of this promoter and used it to characterize infection and dissemination patterns of ONNV within An. gambiae mosquitoes. These experiments allowed us to identify atypical sites of initial infection and dissemination patterns in this mosquito species not frequently observed in comparable culicine infections. The utility of these ONNVs for studies in anopheline mosquitoes includes the potential for identification of vector infection determinants and to serve as tools for antimalaria studies. Viruses that can express a heterologous gene in a vector and rapidly and efficiently infect numerous tissues in An. gambiae mosquitoes will be a valuable asset in parasite-mosquito interaction and interference research.

Antigenic relationship between chikungunya virus strains and o'nyong nyong virus using monoclonal antibodies

Chikungunya (CHIK) strains from Africa and Asia were shown to be antigenically closely related using a panel of monoclonal antibodies (mAb) prepared against strains H817 from Africa and PhH15483 from Asia. The one-way antigenic relationship between CHIK and o'nyong nyong (ONN) viruses was demonstrated at the epitope level using the immunofluorescence and haemagglutination inhibition techniques. Results of tests with mAb against ONN virus suggest that, while ONN virus has retained most of the CHIK antigenic sites, many of the ONN epitopes have undergone sufficient conformational change such that mAbs prepared against them do not recognize sites on CHIK virus.