Orthohantaviruses in Misiones Province, Northeastern Argentina.

Few cases of hantavirus pulmonary syndrome have been reported in northeastern Argentina. However, neighboring areas show a higher incidence, suggesting underreporting. We evaluated the presence of antibodies against orthohantavirus in small rodents throughout Misiones province. Infected Akodon affinis montensis and Oligoryzomys nigripes native rodents were found in protected areas of Misiones.

Anaplasmataceae presence in Amblyomma calcaratum associated with anteaters (Tamandua tetradactyla) in the rainforest ecoregion, Argentina.

Bacteria of the sister genera Ehrlichia and Anaplasma (Anaplasmataceae) are obligate intracellular Alphaproteobacteria that are transmitted mostly through arthropod vectors. These agents can infect different vertebrate cells, depending on the species involved, and can cause diseases in animals and humans. In this study, we evaluated the presence of Anaplasmataceae bacteria in Amblyomma calcaratum ticks collected from a road-killed Tamandua tetradactyla in the Rainforest ecoregion in Argentina. All samples were screened for Anaplasmataceae DNA using a real-time PCR assay targeting the 16S rRNA gene. Evidence of Anaplasmataceae DNA was detected in three out of thirty-nine Am. calcaratum ticks. Phylogenetic analysis of a portion of 16S rRNA gene positioned one sample (Ehrlichia sp. strain Ac124) with Ehrlichia sequences and the other two samples with Anaplasma sequences; Anaplasma sp. strain Ac145 close to Anaplasma odocoilei and Anaplasma sp. strain Ac152 in an ancestral position to most Anaplasma species. The groEL sequence obtained showed that Ehrlichia sp. strain Ac124 was phylogenetically related to Ehrlichia sp. strain Iberá reported infecting Amblyomma tigrinum from Iberá wetlands in Argentina. Phylogenetic analysis using the rpoB sequence positioned Anaplasma sp. strain Ac145 close to the canine pathogen Anaplasma platys, while Anaplasma sp. strain Ac152 was positioned close to the bovine pathogen Anaplasma marginale. In this study, three Anaplasmataceae agents were detected in adults of Am. calcaratum associated with a T. tetradactyla. These results suggest that the number of Anaplasmataceae species, as well as their distribution, is largely unknown.

Analyses of Early ZIKV Genomes Are Consistent with Viral Spread from Northeast Brazil to the Americas.

The Americas, particularly Brazil, were greatly impacted by the widespread Zika virus (ZIKV) outbreak in 2015 and 2016. Efforts were made to implement genomic surveillance of ZIKV as part of the public health responses. The accuracy of spatiotemporal reconstructions of the epidemic spread relies on the unbiased sampling of the transmission process. In the early stages of the outbreak, we recruited patients exhibiting clinical symptoms of arbovirus-like infection from Salvador and Campo Formoso, Bahia, in Northeast Brazil. Between May 2015 and June 2016, we identified 21 cases of acute ZIKV infection and subsequently recovered 14 near full-length sequences using the amplicon tiling multiplex approach with nanopore sequencing. We performed a time-calibrated discrete phylogeographic analysis to trace the spread and migration history of the ZIKV. Our phylogenetic analysis supports a consistent relationship between ZIKV migration from Northeast to Southeast Brazil and its subsequent dissemination beyond Brazil. Additionally, our analysis provides insights into the migration of ZIKV from Brazil to Haiti and the role Brazil played in the spread of ZIKV to other countries, such as Singapore, the USA, and the Dominican Republic. The data generated by this study enhances our understanding of ZIKV dynamics and supports the existing knowledge, which can aid in future surveillance efforts against the virus.

Acute-Phase Levels of CXCL8 as Risk Factor for Chronic Arthralgia Following Chikungunya Virus Infection.

The immunopathogenesis of chikungunya virus (CHIKV) infection and the role of acute-phase immune response on joint pain persistence is not fully understood. We investigated the profile of serum chemokine and cytokine in CHIKV-infected patients with acute disease, compared the levels of these biomarkers to those of patients with other acute febrile diseases (OAFD) and healthy controls (HC), and evaluated their role as predictors of chronic arthralgia development. Chemokines and cytokines were measured by flow Cytometric Bead Array. Patients with CHIKV infection were further categorized according to duration of arthralgia (≤ 3 months vs >3 months), presence of anti-CHIKV IgM at acute-phase sample, and number of days of symptoms at sample collection (1 vs 2-3 vs ≥4). Patients with acute CHIKV infection had significantly higher levels of CXCL8, CCL2, CXCL9, CCL5, CXCL10, IL-1ß, IL-6, IL-12, and IL-10 as compared to HC. CCL2, CCL5, and CXCL10 levels were also significantly higher in patients with CHIKV infection compared to patients with OAFD. Patients whose arthralgia lasted > 3 months had increased CXCL8 levels compared to patients whose arthralgia did not (p<0.05). Multivariable analyses further indicated that high levels of CXCL8 and female sex were associated with arthralgia lasting >3 months. Patients with chikungunya and OAFD had similar cytokine kinetics for IL-1ß, IL-12, TNF, IFN-γ, IL-2, and IL-4, although the levels were lower for CHIKV patients. This study suggests that chemokines may have an important role in the immunopathogenesis of chronic chikungunya-related arthralgia.

Risk of chronic arthralgia and impact of pain on daily activities in a cohort of patients with chikungunya virus infection from Brazil.

OBJECTIVES: To investigate risk factors for persistent arthralgia in patients with chikungunya, and describe its impact on daily activities. METHODS: From September 2014 to July 2016, a surveillance study enrolled patients with acute febrile illness in Salvador, Brazil, and detected those with chikungunya virus infection using IgM enzyme-linked immunosorbent assay or reverse transcriptase polymerase chain reaction. Telephone follow-ups were performed to ascertain the progression of disease. RESULTS: Of 153 followed cases, 65 (42.5%) reported chronic arthralgia that lasted >3 months, and 47 (30.7%) were still symptomatic at the time of the interview (approximately 1.5 years after symptom onset). Limitations in daily activities and mental distress were reported by 93.8% and 61.5% of those with chronic arthralgia, respectively. Female sex [risk ratio (RR) 1.79, 95% confidence interval (CI) 1.95-2.69] and age (RR 1.02 for each 1-year increase, 95% CI 1.01-1.03) were independent risk factors for chronic arthralgia. Chronic arthralgia was not associated with co-infection with dengue virus (RR 0.97, 95% CI 0.48-1.94) or chikungunya viral load at diagnosis (median chikungunya virus RNA of 5.60 and 5.52 log10 copies/µL for those with and without chronic arthralgia, respectively; P = 0.75). CONCLUSIONS: These findings reinforce the high frequency of chronic chikungunya arthralgia, and highlight the substantial disability associated with the persistence of pain. Development of novel strategies to mitigate the transmission of chikungunya virus and to provide long-term medical assistance for patients with chikungunya are needed urgently.

Autochthonous circulation of Saint Louis encephalitis and West Nile viruses in the Province of La Rioja, Argentina.

St. Louis encephalitis (SLEV) and West Nile (WNV) arboviruses, which circulate in Argentina, are maintained in enzootic transmission cycles involving Culex mosquitoes (vectors) and birds belonging to orders Passeriformes and Columbiformes (amplifier hosts). The objective of this work was to determine the circulation of both viruses among wild birds in a semiarid ecosystem in the Province of La Rioja through a serologic survey. During spring 2013 and fall 2014, a total of 326 wild birds belonging to 41 species were captured in areas close to the cities of La Rioja and Chilecito, in the Province of La Rioja. While exposure to SLEV and WNV was analyzed in birds' serum through neutralizing antibody detection, viral circulation was estimated through apparent seroprevalence of neutralizing antibodies. The exposure of the avian community to viruses was 3.02% for SLEV and 1.89% for WNV, while 1.19% corresponded to coinfections. Our study confirms for the first time the circulation of SLEV and WNV in wild birds in the Province of La Rioja. Moreover, it is the first study to register neutralizing antibodies for flavivirus in the species Leptotila verreauxi (White-tipped Dove) (WNV) and Melanerpes cactorum (White-fronted Woodpecker) (SLEV). These results suggest that in semiarid ecosystems from northwestern Argentina the requirements and conditions for amplification and enzootic maintenance of SLEV and WNV would be present.

Evaluation of two commercially available chikungunya virus IgM enzyme-linked immunoassays (ELISA) in a setting of concomitant transmission of chikungunya, dengue and Zika viruses.

OBJECTIVE: To evaluate the diagnostic performance of the Inbios (Seattle, US) and Euroimmun (Luebeck, Germany) chikungunya virus (CHIKV) IgM enzyme-linked immunoassays (ELISAs). METHODS: We evaluated the tests' accuracy on sera from 372 patients enrolled in an acute febrile illness surveillance study performed in Salvador, Brazil from Sept/2014 to Jul/2016, a period of simultaneous CHIKV, dengue (DENV), and Zika (ZIKV) virus transmission. We assessed the sensitivity on acute and paired convalescent sera from RT-PCR-confirmed CHIKV cases (collected at median one and 19 days post-onset of symptoms, respectively), and the specificity on sera of RT-PCR-confirmed DENV and ZIKV cases, and on negative patients. RESULTS: The Inbios and Euroimmun tests' sensitivities for acute samples were 4.0% and 10.3%, while for convalescent samples they were 92.4% and 96.9%, respectively. Overall, Inbios IgM ELISA specificities for acute and convalescent samples were 97.7% and 90.5%, respectively, and Euroimmun specificities were 88.5% and 83.9%, respectively. CONCLUSIONS: Both tests presented high sensitivity for convalescent samples. However, the Euroimmun test returned more equivocal results and presented a slightly lower specificity, which might result in a higher rate of false positives if the test is used in scenarios of low CHIKV transmission, when the chance of CHIKV infection is lower.

Molecular survey of flaviviruses and orthobunyaviruses in Amblyomma spp. ticks collected in Minas Gerais, Brazil / Investigação molecular de flavivírus e orthobunyaviruses em carrapatos do gênero Amblyomma spp. coletados em Minas Gerais, Brasil

Abstract Due to anthropic environmental changes, vector-borne diseases are emerging worldwide. Ticks are known vectors of several pathogens of concern among humans and animals. In recent decades, several examples of tick-borne emerging viral diseases have been reported (Crimean Congo hemorrhagic fever virus, Powassan virus, encephalitis virus, heartland virus, severe fever with thrombocytopenia syndrome virus). Unfortunately, few studies addressing the presence of viruses in wild ticks have been carried out in South America. With the aim of detecting flaviviruses and orthobunyaviruses in ticks, we carried out molecular detection in wild ticks collected in the state of Minas Gerais, Brazil. No Flavivirus-positive ticks were detected; however, we detected activity of Orthobunyavirus in 8 Amblyomma tick specimens. One of those individuals was positive for Bunyamwera orthobunyavirus, which represents the first report of this virus among ticks in South America. Further studies related to the ecology of zoonotic diseases are needed to increase knowledge of this topic, including attempts at viral isolation, full genome sequencing and biological characterization. In this way, we will obtain a better picture of the real risk of ticks as a vector for viral diseases for humans and animals on our continent, where no tick-borne viral disease is known to occur.

Resumo Alterações ambientais causadas pelo homem têm levado à emergência de doenças transmitidas por vetores no mundo. Carrapatos são vetores conhecidos de vários patógenos de importância médica e veterinária, tendo sido reportado nas últimas décadas um grande número de enfermidades virais emergentes transmitidas por eles (vírus da Febre Hemorrágica da Crimeia-Congo, vírus Powassan, vírus da Encefalite, vírus Heartland e vírus da Síndrome da Febre Trombocitopênica Severa). Infelizmente, poucos estudos envolvendo a pesquisa de vírus em carrapatos foram conduzidos na América do Sul até o momento, e nas últimas décadas um elevado número de enfermidades virais emergentes transmitidas por estes artrópodes foi relatado. Com o objetivo de investigar a presença de flavivírus e orthobunyavírus em carrapatos, foi conduzida uma análise molecular em espécimes coletados no estado de Minas Gerais, Brasil. Em nenhum carrapato foi detectada a presença de Flavivirus, no entanto, em 8 espécimes do gênero Amblyomma, foi detectada a presença de Orthobunyavirus, dos quais um espécime foi positivo para Bunyamwera orthobunyavirus. Novos estudos relacionados à ecologia de doenças zoonóticas, incluindo tentativas de isolamento viral, sequenciamento completo do genoma e caracterização biológica, são necessários. Desta forma, será possível ter uma base sobre os riscos da transmissão de vírus patogênicos por carrapatos em nosso continente, uma vez que até agora isso é desconhecido.

Molecular survey of flaviviruses and orthobunyaviruses in Amblyomma spp. ticks collected in Minas Gerais, Brazil.

Due to anthropic environmental changes, vector-borne diseases are emerging worldwide. Ticks are known vectors of several pathogens of concern among humans and animals. In recent decades, several examples of tick-borne emerging viral diseases have been reported (Crimean Congo hemorrhagic fever virus, Powassan virus, encephalitis virus, heartland virus, severe fever with thrombocytopenia syndrome virus). Unfortunately, few studies addressing the presence of viruses in wild ticks have been carried out in South America. With the aim of detecting flaviviruses and orthobunyaviruses in ticks, we carried out molecular detection in wild ticks collected in the state of Minas Gerais, Brazil. No Flavivirus-positive ticks were detected; however, we detected activity of Orthobunyavirus in 8 Amblyomma tick specimens. One of those individuals was positive for Bunyamwera orthobunyavirus, which represents the first report of this virus among ticks in South America. Further studies related to the ecology of zoonotic diseases are needed to increase knowledge of this topic, including attempts at viral isolation, full genome sequencing and biological characterization. In this way, we will obtain a better picture of the real risk of ticks as a vector for viral diseases for humans and animals on our continent, where no tick-borne viral disease is known to occur.

Genomic characterization of orthobunyavirus of veterinary importance in America.

During 2013, in Argentina, three new isolates of serogroup Bunyamwera virus (genus Orthobunyavirus, family Peribunyaviridae) were recovered from two horses with encephalitis, and from an aborted equine fetus. In the present study, we report the complete genome sequence, genetic characterization, and phylogenetic analysis of three new strains isolated in Argentina to clarifying their relationship within the Bunyamwera serogroup virus and to investigate the evolutionary history of viruses with segmented genomes.

A localized outbreak of Chikungunya virus in Salvador, Bahia, Brazil.

A localized Chikungunya virus (CHIKV; East/Central/South African genotype) outbreak (50 cases, 70% laboratory-confirmed; attack rate: 5.3 confirmed cases/100 people) occurred in a Salvador, Brazil neighborhood, between Apr-Jun/2017. Highly clustered cases in space and time, mostly along a single street, highlight an increased risk of CHIKV transmission among pockets of susceptible populations. This finding underscores the need for ongoing local level surveillance for arboviral outbreaks.

Accuracy of the SD BIOLINE Dengue Duo for rapid point-of-care diagnosis of dengue.

BACKGROUND: Rapid diagnosis tests (RDTs) are easy to carry out, provide fast results, and could potentially guide medical treatment decisions. We investigated the performance of a commercially available RDT, which simultaneously detects the non-structural 1 (NS1) dengue virus (DENV) antigen, and IgM and IgG DENV antibodies, using representative serum samples from individuals in a dengue endemic area in Salvador, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the accuracy of the SD BIOLINE Dengue Duo RDT (Abbott, Santa Clara, USA; former Alere Inc, Waltham, USA) in a random collection of sera. Samples included acute-phase sera from 246 laboratory-confirmed dengue cases and 108 non-dengue febrile patients enrolled in a surveillance study for dengue detection, 73 healthy controls living in the same surveillance community, and 73 blood donors. RDT accuracy was blindly assessed based on the combined results for the NS1 and the IgM test components. The RDT sensitivity was 46.8% (38.6% for the NS1 component and 13.8% for the IgM component). Sensitivity was greater for samples obtained from patients with secondary DENV infections (49.8%) compared to primary infections (31.1%) (P: 0.02) and was also influenced by the result in the confirmatory dengue diagnostic test, ranging from 39.7% for samples of cases confirmed by IgM-ELISA seroconversion between paired samples to 90.4% for samples of cases confirmed by a positive NS1-ELISA. The RDT specificity was 94.4% for non-dengue febrile patients, 87.7% for the community healthy controls, and 95.9% for the blood donors. CONCLUSIONS/SIGNIFICANCE: The SD BIOLINE Dengue Duo RDT showed good specificities, but low sensitivity, suggesting that it may be more useful to rule in than to rule out a dengue diagnosis in dengue endemic regions.

Concomitant Transmission of Dengue, Chikungunya, and Zika Viruses in Brazil: Clinical and Epidemiological Findings From Surveillance for Acute Febrile Illness.

BACKGROUND: Since their emergence in the Americas, chikungunya (CHIKV) and Zika (ZIKV) viruses co-circulate with dengue virus (DENV), hampering clinical diagnosis. We investigated clinical and epidemiological characteristics of arboviral infections during the introduction and spread of CHIKV and ZIKV through northeastern Brazil. METHODS: Surveillance for arboviral diseases among febrile patients was performed at an emergency health unit of Salvador, Brazil, between September 2014 and July 2016. We interviewed patients to collect data on symptoms, reviewed medical records to obtain the presumptive diagnoses, and performed molecular and serological testing to confirm DENV, CHIKV, ZIKV, or nonspecific flavivirus (FLAV) diagnosis. RESULTS: Of 948 participants, 247 (26.1%) had an acute infection, of which 224 (23.6%) were single infections (DENV, 32 [3.4%]; CHIKV, 159 [16.7%]; ZIKV, 13 [1.4%]; and FLAV, 20 [2.1%]) and 23 (2.4%) coinfections (DENV/CHIKV, 13 [1.4%]; CHIKV/FLAV, 9 [0.9%]; and DENV/ZIKV, 1 [0.1%]). An additional 133 (14.0%) patients had serological evidence for a recent arboviral infection. Patients with ZIKV presented with rash and pruritus (69.2% each) more frequently than those with DENV (37.5% and 31.2%, respectively) and CHIKV (22.9% and 14.7%, respectively) (P < .001 for both comparisons). Conversely, arthralgia was more common in CHIKV (94.9%) and FLAV/CHIKV (100.0%) than in DENV (59.4%) and ZIKV (53.8%) (P < .001). A correct presumptive clinical diagnosis was made for 9%-23% of the confirmed patients. CONCLUSIONS: Arboviral infections are frequent causes of febrile illness. Coinfections are not rare events during periods of intense, concomitant arboviral transmission. Given the challenge to clinically distinguish these infections, there is an urgent need for rapid, point-of-care, multiplex diagnostics.

A localized outbreak of Chikungunya virus in Salvador, Bahia, Brazil

A localized Chikungunya virus (CHIKV; East/Central/South African genotype) outbreak (50 cases, 70% laboratory-confirmed; attack rate: 5.3 confirmed cases/100 people) occurred in a Salvador, Brazil neighborhood, between Apr-Jun/2017. Highly clustered cases in space and time, mostly along a single street, highlight an increased risk of CHIKV transmission among pockets of susceptible populations. This finding underscores the need for ongoing local level surveillance for arboviral outbreaks.

Diagnostic performance of commercial IgM and IgG enzyme-linked immunoassays (ELISAs) for diagnosis of Zika virus infection.

BACKGROUND: Serologic detection of Zika virus (ZIKV) infections is challenging because of antigenic similarities among flaviviruses. OBJECTIVE: To evaluate the sensitivity and specificity of commercial ZIKV IgM and IgG enzyme-linked immunoassay (ELISA) kits. METHODS: We used sera from febrile patients with RT-PCR-confirmed ZIKV infection to determine sensitivity and sera from RT-PCR-confirmed dengue cases and blood donors, both of which were collected before ZIKV epidemics in Brazil (2009-2011 and 2013, respectively) to determine specificity. RESULTS: The ZIKV IgM-ELISA positivity among RT-PCR ZIKV confirmed cases was 0.0% (0/14) and 12.5% (1/8) for acute- and convalescent-phase sera, respectively, while its specificity was 100.0% (58/58) and 98.3% (58/59) for acute- and convalescent-phase sera of dengue patients, and 100.0% (23/23) for blood donors. The ZIKV IgG-ELISA sensitivity was 100.0% (6/6) on convalescent-phase sera from RT-PCR confirmed ZIKV patients, while its specificity was 27.3% (15/55) on convalescent-phase sera from dengue patients and 45.0% (9/20) on blood donors' sera. The ZIKV IgG-ELISA specificity among dengue confirmed cases was much greater among patients with primary dengue (92.3%; 12/13), compared to secondary dengue (7.1%; 3/42). CONCLUSIONS: In a setting of endemic dengue transmission, the ZIKV IgM-ELISA had high specificity, but poor sensitivity. In contrast, the ZIKV IgG-ELISA showed low specificity, particularly for patients previously exposed to dengue infections. This suggests that this ZIKV IgM-ELISA is not useful in confirming a diagnosis of ZIKV infection in suspected patients, whereas the IgG-ELISA is more suitable for ZIKV diagnosis among travelers, who reside in areas free of flavivirus transmission, rather than for serosurveys in dengue-endemic areas.

Neuronal Degeneration in Mice Induced by an Epidemic Strain of Saint Louis Encephalitis Virus Isolated in Argentina.

Saint Louis encephalitis virus (SLEV) is a neglected flavivirus that causes severe neurological disorders. The epidemic strain of SLEV, CbaAr-4005, isolated during an outbreak in Córdoba city (Argentina), causes meningitis and encephalitis associated with neurological symptoms in a murine experimental model. Here, we identified the affected brain areas and the damage triggered by this neurotropic arbovirus. We performed a detailed analysis of brain neurodegeneration associated with CbaAr-4005 SLEV infection in mice. The motor cortex, corpus striatum and cerebellum were the most affected structures. Neurodegeneration was also found in the olfactory bulb, thalamus, hypothalamus, hippocampus, and hindbrain. SLEV infection triggered brain cell apoptosis as well as somatodendritic and terminal degeneration. In addition, we observed massive excitotoxic-like degeneration in many cortical structures. Apoptosis was also detected in the neuroblastoma cell line N2a cultured with SLEV. The results evidenced that SLEV CbaAr-4005 infection induced severe degenerative alterations within the central nervous system of infected mice, providing new information about the targets of this flavivirus infection.