Co-circulation of two Alphaviruses in Burkina Faso: Chikungunya and O'nyong nyong viruses.

BACKGROUND: Chikungunya virus (CHIKV) and O'nyong nyong virus (ONNV) are phylogenetically related alphaviruses in the Semliki Forest Virus (SFV) antigenic complex of the Togaviridae family. There are limited data on the circulation of these two viruses in Burkina Faso. The aim of our study was to assess their circulation in the country by determining seroprevalence to each of the viruses in blood donor samples and by retrospective molecular and serological testing of samples collected as part of national measles and rubella surveillance. METHODOLOGY/PRINCIPAL FINDINGS: All blood donor samples were analyzed on the Luminex platform using CHIKV and ONNV E2 antigens. Patient samples collected during national measles-rubella surveillance were screened by an initial ELISA for CHIKV IgM (CHIKjj Detect IgM ELISA) at the national laboratory. The positive samples were then analyzed by a second ELISA test for CHIKV IgM (CDC MAC-ELISA) at the reference laboratory. Finally, samples that had IgM positive results for both ELISA tests and had sufficient residual volume were tested by plaque reduction neutralization testing (PRNT) for CHIKV and ONNV. These same patient samples were also analyzed by rRT-PCR for CHIKV. Among the blood donor specimens, 55.49% of the samples were positive for alphaviruses including both CHIKV and ONNV positive samples. Among patient samples collected as part of national measles and rubella surveillance, 3.09% were IgM positive for CHIKV, including 2.5% confirmed by PRNT. PRNT failed to demonstrate any ONNV infections in these samples. No samples tested by RT-qPCR. had detectable CHIKV RNA. CONCLUSIONS/SIGNIFICANCE: Our results suggest that CHIKV and ONNV have been circulating in the population of Burkina Faso and may have been confused with malaria, dengue fever or other febrile diseases such as measles or rubella. Our study underscores the necessity to enhance arbovirus surveillance systems in Burkina Faso.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.

Detection and genome characterization of Middelburg virus strains isolated from CSF and whole blood samples of humans with neurological manifestations in South Africa.

BACKGROUND: The Old world Alphavirus, Middelburg virus (MIDV), is not well known and although a few cases associated with animal illness have previously been described from Southern Africa, there has been no investigation into the association of the virus with human illness. The current study aimed to investigate possible association of MIDV infection with febrile or neurological manifestations in hospitalized or symptomatic patients fromGauteng, South Africa. METHODS: This study is a descriptive retrospective and prospective laboratory based study. Archived cerebrospinal fluid (CSF) samples submitted to the National Health Laboratory Service (NHLS), Tshwane Academic division for viral investigation from public sector hospitals in Gauteng as well as EDTA (ethylenediaminetetraacetic acid) whole blood samples from ad hoc cases of veterinary students, presenting with neurological and febrile illness, were selected and screened for the presence of alphaviruses using real-time reverse transcription(rtRT) PCR.Virus isolations from rtRT-PCR positive samples were conducted in Vero cell culture and used to obtain full genome sequences. Basic descriptive statistical analysis was conducted using EpiInfo. RESULTS: MIDV was detected by rtRT-PCR in 3/187 retrospective CSF specimens obtained from the NHLS from hospitalised patients in the Tshwane region of Gauteng and 1/2 EDTA samples submitted in the same year (2017) from ad hoc query arbovirus cases from veterinary students from the Faculty of Veterinary Science University of Pretoria.Full genome sequences were obtained for virus isolates from two cases; one from an EDTA whole blood sample (ad hoc case) and another from a CSF sample (NHLS sample).Two of the four Middelburg virus positive cases,for which clinical information was available, had other comorbidities or infections at the time of infection. CONCLUSION: Detection of MIDV in CSF of patients with neurological manifestations suggests that the virus should be investigated as a human pathogen with the potential of causing or contributing to neurological signs in children and adults.

Development of a Sensitive Detection Method for Alphaviruses and Its Use as a Virus Neutralization Assay.

Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.

No Evidence of O'nyong-nyong Viremia among Children with Febrile Illness in Kenya (2015-2018).

O'nyong-nyong virus (ONNV) is a little-known arbovirus causing intermittent, yet explosive, outbreaks in Africa. It is closely related to chikungunya virus, an emerging infectious disease. O'nyong-nyong virus causes a self-limited illness characterized by bilateral polyarthritis, rash, low-grade fever, and lymphadenopathy. In 1959, an extensive outbreak of ONNV occurred in East Africa, and decades later, another large outbreak was documented in Uganda in 1996. Limited evidence for interepidemic transmission is available, although serologic studies indicate a high prevalence of exposure. 1,045 febrile child participants in western and coastal Kenya were tested for the presence of ONNV using a multiplexed real-time reverse transcriptase-PCR assay. More than half of the participants had malaria parasitemia, and there was no evidence of active ONNV viremia in these participants. Further work is required to better understand the interepidemic circulation of ONNV and to reconcile evidence of high serologic exposure to ONNV among individuals in East Africa.

Species Traits and Hotspots Associated with Ross River Virus Infection in Nonhuman Vertebrates in South East Queensland.

Ross River virus (RRV) is a mosquito-borne zoonotic arbovirus associated with high public health and economic burdens across Australia, but particularly in South East Queensland (SEQ). Despite this high burden, humans are considered incidental hosts. Transmission of RRV is maintained among mosquitoes and many nonhuman vertebrate reservoir hosts, although the relative contributions of each of these hosts are unclear. To clarify the importance of a range of vertebrates in RRV transmission in SEQ, a total of 595 serum samples from 31 species were examined for RRV exposure using a gold-standard plaque reduction neutralization test. Data were analyzed statistically using generalized linear models and a coefficient inference tree, and spatially. RRV exposure was highly variable between and within species groups. Critically, species group ("placental mammal," "marsupial," and "bird"), which has previously been used as a proxy for reservoir hosts, was a poor correlate for exposure. Instead, we found that generalized "diet" and greater "body mass" were most strongly correlated with seropositivity. We also identified significant differences in seropositivity between the two major possum species (ringtail possums and brushtail possums), which are ecologically and taxonomically different. Finally, we identified distinct hotspots and coldspots of seropositivity in nonhuman vertebrates, which correlated with human notification data. This is the largest diversity of species tested for RRV in a single study to date. The analysis methods within this study provide a framework for analyzing serological data in combination with species traits for other zoonotic disease, but more specifically for RRV highlight areas to target further public health research and surveillance effort.

Sindbis Virus Strains of Divergent Origin Isolated from Humans and Mosquitoes During a Recent Outbreak in Finland.

Sindbis virus (SINV) is a mosquito-borne avian hosted virus that is widely distributed in Europe, Africa, Asia, and Oceania. Disease in humans is documented mainly from Northern Europe and South Africa and associated with genotype I. In 2018 under extremely warm climatic conditions, a small outbreak of 71 diagnosed SINV infections was recorded in Finland. We screened 52 mosquito pools (570 mosquitoes) and 223 human sera for SINV with real-time RT-PCR and the positive samples with virus isolation. One SINV strain was isolated from a pool (n = 13) of genus Ochlerotatus mosquitoes and three strains from patient serum samples. Complete genome analysis suggested all the isolates to be divergent from one another and related to previous Finnish, Swedish, and German strains. The study provides evidence of SINV strain transfer within Europe across regions with different epidemiological characteristics. Whether these are influenced by different mosquito genera involved in the transmission remains to be studied.

Reconstructing Mayaro virus circulation in French Guiana shows frequent spillovers.

Characterizing the circulation of Mayaro virus (MAYV), an emerging arbovirus threat, is essential for risk assessment but challenging due to cross-reactivity with other alphaviruses such as chikungunya virus (CHIKV). Here, we develop an analytical framework to jointly assess MAYV epidemiology and the extent of cross-reactivity with CHIKV from serological data collected throughout French Guiana (N = 2697). We find strong evidence of an important sylvatic cycle for MAYV with most infections occurring near the natural reservoir in rural areas and in individuals more likely to go to the forest (i.e., adult males) and with seroprevalences of up to 18% in some areas. These findings highlight the need to strengthen MAYV surveillance in the region and showcase how modeling can improve interpretation of cross-reacting assays.

Development of an enzyme-linked immunosorbent assay for Getah virus infection in horses using recombinant E2 protein as an antigen.

Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), for all horses experimentally infected with Getah virus (n = 7), optical density (OD) exceeded the cutoff value at 14 days post-infection. ODs in five of nine vaccinated horses also slightly exceeded the cutoff value after vaccination. Among naturally infected horses (n = 28), 24 were seronegative in the acute sera, which turned seropositive in the convalescent sera. For the four horses seropositive in the acute sera, an endpoint method with serial dilutions of paired sera detected a ≥4-fold increase in titer. In conclusion, we established rE2-ELISA that could detect horse antibodies against Getah virus after experimental and natural infections; this should be useful in the diagnosis and surveillance of Getah virus infection.

Ross River Virus Antibody Prevalence, Fiji Islands, 2013-2015.

A unique outbreak of Ross River virus (RRV) infection was reported in Fiji in 1979. In 2013, RRV seroprevalence among residents was 46.5% (362/778). Of the residents who were seronegative in 2013 and retested in 2015, 10.9% (21/192) had seroconverted to RRV, suggesting ongoing endemic circulation of RRV in Fiji.

A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus.

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.

Seroprevalence and associated risk factors of mosquito-borne alphaviruses in horses in northern Queensland.

OBJECTIVE: To investigate the seroprevalence and associated risk factors of alphaviruses (Ross River virus (RRV), Barmah Forest virus (BFV) and Whataroa virus (WHAV)) in northern Queensland horses. METHODS: A cross-sectional study of alphavirus antibodies in horses (n = 287) from 147 properties in northern Queensland from September 2013 to June 2014 was conducted. Owners of sampled horses were interviewed on potential risk factors. Data were analysed for associations using multivariable logistic regression. RESULTS: Antibody titres for RRV were demonstrated in samples from 134 properties (91%; 95% confidence interval (CI) 87-96%); 22 properties (15%) had BFV reactors (95% CI 12-18%) and 2 properties (1.4%) had WHAV reactors (95% CI -0.5-3.2%). The highest seroprevalence of RRV was in the Townsville-Burdekin region (93%; 95% CI 90-96%) followed by the Mackay-Whitsunday (90%; 95% CI 88-98%) and Far North Coast-Tableland (82%; 95% CI 74-90%) regions. No association (P ≤ 0.05) could be shown between any of the viruses and age groups, sexes, annual average temperature, degree of rainfall or proximity to wet environments. An association with reported large numbers of mosquitoes was seen for RRV but not BFV. A significant association between properties in close proximity to poultry and pigs was shown for BFV. CONCLUSION: RRV is endemic within the horse population of northern Queensland, but horses exhibit few clinical signs and could play a role as amplifying hosts in the tropics. Exposure of horses to BFV is significant in northern Queensland and it should be considered a differential diagnosis for RRV. WHAV warrants further study.

Madariaga Virus: Identification of a Lineage III Strain in a Venezuelan Child With Acute Undifferentiated Febrile Illness, in the Setting of a Possible Equine Epizootic.

We report identification of Madariaga virus (MADV) in plasma and urine samples from a child with acute undifferentiated febrile illness in Venezuela. Our data document the occurrence of milder MADV infections (ie, without encephalitis), with a symptom complex that resembles that seen with other arboviral infections, including dengue and zika.

Ross River virus in Australian blood donors: possible implications for blood transfusion safety.

BACKGROUND: Emerging transfusion-transmissible pathogens, including arboviruses such as West Nile, Zika, dengue, and Ross River viruses, are potential threats to transfusion safety. The most prevalent arbovirus in humans in Australia is Ross River virus (RRV); however, prevalence varies substantially around the country. Modeling estimated a yearly risk of 8 to 11 potentially RRV-viremic fresh blood components nationwide. This study aimed to measure the occurrence of RRV viremia among donors who donated at Australian collection centers located in areas with significant RRV transmission during one peak season. STUDY DESIGN AND METHODS: Plasma samples were collected from donors (n = 7500) who donated at the selected collection centers during one peak season. Viral RNA was extracted from individual samples, and quantitative reverse transcription-polymerase chain reaction was performed. RESULTS: Regions with the highest rates of RRV transmission were not areas where donor centers were located. We did not detect RRV RNA among 7500 donations collected at the selected centers, resulting in a zero risk estimate with a one-sided 95% confidence interval of 0 to 1 in 2019 donations. CONCLUSION: Our results suggest that the yearly risk of collecting a RRV-infected blood donation in Australia is low and is at the lower range of previous risk modeling. The majority of Australian donor centers were not in areas known to be at the highest risk for RRV transmission, which was not taken into account in previous models based on notification data. Therefore, we believe that the risk of RRV transfusion transmission in Australia is acceptably low and appropriately managed through existing risk management, including donation restrictions and recall policies.

Investigation into High Barmah Forest Virus Disease Case Numbers Reported in the Northern Territory, Australia in 2012-2013.

Between October 2012 and October 2013, unprecedented high numbers of Barmah Forest virus (BFV) disease cases were reported in the Northern Territory (NT). An investigation was launched by the NT Department of Health in cooperation with the Department of Primary Industry and Fisheries and the Department of Land Resource Management to investigate possible causes for this phenomenon. The investigation included virus isolations from mosquitoes collected in Darwin urban areas, BFV antibody testing in peri-urban small mammals and a human BFV disease case series investigation of recent cases. No BFV was isolated from the 4641 mosquitoes tested, none of the mammals tested positive for BFV antibodies, and the high BFV disease case numbers did not correlate with the relatively low mosquito vector numbers trapped in 2012-2013. It was estimated that up to 89% of the 79 human cases investigated did not have an acute arboviral illness and therefore had tested falsely positive. An Alere PanBio BFV immunoglobulin M enzyme-linked immunosorbent assay test kit is generally used to test for BFV, with the BFV disease case definition based on immunoglobulin M positives only. Other jurisdictions in Australia also reported high numbers of BFV disease cases, with the majority of the cases suspected to be false positives. Therefore, current testing methods need to be revised to reflect the true numbers of BFV disease cases occurring in Australia and to provide correct diagnoses for patients.

Silent Circulation of Ross River Virus in French Polynesia.

OBJECTIVES: Ross River is an emerging mosquito-borne disease in the Western Pacific. Ross River virus (RRV) circulation has been sporadically reported in some Pacific Island Countries and Territories but never in French Polynesia. To determine if RRV has circulated locally among the French Polynesian population, we conducted a seroprevalence study on blood donors. METHODS: Sera of 593 blood donors were collected from July 2011 to October 2013 and tested by ELISA for the presence of RRV-specific Immunoglobulin G (IgG) antibodies. RESULTS: A total of 204 (34.40%) blood donors were found seropositive for RRV. Among the 132 blood donors that were born in French Polynesia and had never travelled abroad, 56 (42.42%) had RRV-specific IgGs. DISCUSSION: Our results support the existence of autochthonous RRV transmission and suggest that this pathogen has silently circulated in French Polynesia. These findings raise the question of possible undetected circulation of RRV in other Pacific Island Countries and Territories.

Seroepidemiology of selected alphaviruses and flaviviruses in bats in Trinidad.

A serosurvey of antibodies against selected flaviviruses and alphaviruses in 384 bats (representing 10 genera and 14 species) was conducted in the Caribbean island of Trinidad. Sera were analysed using epitope-blocking enzyme-linked immunosorbent assays (ELISAs) specific for antibodies against West Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), all of which are zoonotic viruses of public health significance in the region. Overall, the ELISAs resulted in the detection of VEEV-specific antibodies in 11 (2.9%) of 384 bats. Antibodies to WNV and EEEV were not detected in any sera. Of the 384 sera, 308 were also screened using hemagglutination inhibition assay (HIA) for antibodies to the aforementioned viruses as well as St. Louis encephalitis virus (SLEV; which also causes epidemic disease in humans), Rio Bravo virus (RBV), Tamana bat virus (TABV) and western equine encephalitis virus (WEEV). Using this approach, antibodies to TABV and RBV were detected in 47 (15.3%) and 3 (1.0%) bats, respectively. HIA results also suggest the presence of antibodies to an undetermined flavivirus(es) in 8 (2.6%) bats. Seropositivity for TABV was significantly (P<0.05; χ2) associated with bat species, location and feeding preference, and for VEEV with roost type and location. Differences in prevalence rates between urban and rural locations were statistically significant (P<0.05; χ2) for TABV only. None of the aforementioned factors was significantly associated with RBV seropositivity rates.

Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss.

Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (µCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.

Effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection.

OBJECTIVE: To evaluate whether chloroquine (CQ) is more effective than meloxicam for treating early musculoskeletal pain and arthritis following acute chikungunya (CHIK) virus infection. METHODS: During the 2006 CHIK epidemic, 509 rural community cases of acute CHIK virus infection were identified in the district of Sholapur in India. Seventy consenting adult patients (seropositive for IgM/IgG anti-CHIK antibody) with early persistent musculoskeletal pain and arthritis were randomized into a 24-week, 2-arm, parallel efficacy trial of CQ (250 mg/day) and meloxicam (7.5 mg/day). Assessors completed a rheumatology evaluation in a blinded manner and collected blood samples in the patients' homes, as per protocol. Laboratory parameters included serum cytokine assay (interleukin-6 [IL-6], interferon-γ [IFNγ], tumor necrosis factor α, CXCL10/IFNγ-inducible protein 10, and IL-13). Twenty-two patients who failed to meet the eligibility criteria (low pain cohort) were also followed up with similar evaluations. An intent-to-treat analysis was completed. At baseline, the 2 groups (38 patients randomized to receive CQ and 32 patients randomized to receive meloxicam) were well matched. RESULTS: There were no significant efficacy differences between the meloxicam group and the CQ group (mean changes in the visual analog scale score for pain -3.9 and -4.2, respectively). Patients improved significantly. Cytokine levels remained several-fold increased, were disproportionate to the clinical response, and were not different from those in the low pain cohort. Seven patients withdrew. Adverse events were mild and infrequent. CONCLUSION: This exploratory community intervention trial failed to identify an advantage of CQ over meloxicam to treat early musculoskeletal pain and arthritis following acute CHIK virus infection, but therapeutic efficacy of CQ was not ruled out. The inflammatory cytokine response was intense and was not consistent with clinical status.