Zika virus transmission by Brazilian Aedes aegypti and Aedes albopictus is virus dose and temperature-dependent.

BACKGROUND: Zika virus (ZIKV) emerged in the Pacific Ocean and subsequently caused a dramatic Pan-American epidemic after its first appearance in the Northeast region of Brazil in 2015. The virus is transmitted by Aedes mosquitoes. We evaluated the role of temperature and infectious doses of ZIKV in vector competence of Brazilian populations of Ae. aegypti and Ae. albopictus. METHODOLOGY/PRINCIPAL FINDINGS: Two Ae. aegypti (Rio de Janeiro and Natal) and two Ae. albopictus (Rio de Janeiro and Manaus) populations were orally challenged with five viral doses (102 to 106 PFU / ml) of a ZIKV strain (Asian genotype) isolated in Northeastern Brazil, and incubated for 14 and 21 days in temperatures mimicking the spring-summer (28°C) and winter-autumn (22°C) mean values in Brazil. Detection of viral particles in the body, head and saliva samples was done by plaque assays in cell culture for determining the infection, dissemination and transmission rates, respectively. Compared with 28°C, at 22°C, transmission rates were significantly lower for both Ae. aegypti populations, and Ae. albopictus were not able to transmit the virus. Ae. albopictus showed low transmission rates even when challenged with the highest viral dose, while both Ae. aegypti populations presented higher of infection, dissemination and transmission rates than Ae. albopictus. Ae. aegypti showed higher transmission efficiency when taking virus doses of 105 and 106 PFU/mL following incubation at 28°C; both Ae. aegypti and Ae. albopictus were unable to transmit ZIKV with virus doses of 102 and 103 PFU/mL, regardless the incubation temperature. CONCLUSIONS/SIGNIFICANCE: The ingested viral dose and incubation temperature were significant predictors of the proportion of mosquito's biting becoming infectious. Ae. aegypti and Ae. albopictus have the ability to transmit ZIKV when incubated at 28°C. However Brazilian populations of Ae. aegypti exhibit a much higher transmission potential for ZIKV than Ae. albopictus regardless the combination of infection dose and incubation temperature.

The inability of a dengue NS1 ELISA to detect Zika infections.

The presence of dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) in Brazil, may result in a difficult diagnosis due to the signs and symptoms shared by those. Moreover, as DENV and ZIKV belong to the same family, serological assays may show a high rate of cross-reactivity. Here, we evaluated a Dengue NS1 capture assay for early and differential diagnosis of dengue during the Zika epidemic occurred in Brazil in 2016. Samples (n = 227) from 218 patients included sera, plasma and urine from previously confirmed acute cases of Zika, dengue and Zika/dengue co-infections. Nine of those patients presented two specimens. The Dengue NS1 test was very specific for dengue diagnosis (99.32%), even in the co-circulation with ZIKV, and exhibited a high accuracy in not detecting acute Zika infections (92.43%). Our findings showed that the dengue NS1 capture test analyzed here was not able to recognize the ZIKV NS1 and its potential for cross-reaction.

DENV-1 Genotype V in Brazil: Spatiotemporal dispersion pattern reveals continuous co-circulation of distinct lineages until 2016.

In Brazil, DENV-1 introduced in the 80's, remained the prevalent serotype from 2012 to 2016. After its re-emergence in the country in 2009, the co-circulation of different viral lineages was identified, however, its transmission dynamics afterwards, was not fully characterized. In this study, we performed the continuous molecular surveillance after the reemergence period (2012 to 2016), covering the 30 years of circulation of DENV-1 in Brazil. Phylogenetic analysis allowed confirmation of the continued presence of genotype V, as well as three distinct co-circulating lineages. The molecular characterization of the E gene presented two new amino acid substitutions previously unidentified in the country. Phylogeographic analysis has shown that a large flow of migrations has occurred between Brazil and Argentina in the last 10 years.

Following in the Footsteps of the Chikungunya Virus in Brazil: The First Autochthonous Cases in Amapá in 2014 and Its Emergence in Rio de Janeiro during 2016.

Currently, Brazil lives a triple arboviruses epidemic (DENV, ZIKV and CHIKV) making the differential diagnosis difficult for health professionals. Here, we aimed to investigate chikungunya cases and the possible occurrence of co-infections during the epidemic in Amapá (AP) that started in 2014 when the first autochthonous cases were reported and in Rio de Janeiro (RJ) in 2016. We further performed molecular characterization and genotyping of representative strains. In AP, 51.4% of the suspected cases were confirmed for CHIKV, 71.0% (76/107). Of those, 24 co-infections by CHIKV/DENV, two by CHIKV/DENV-1, and two by CHIKV/DENV-4 were observed. In RJ, 76.9% of the suspected cases were confirmed for CHIKV and co-infections by CHIKV/DENV (n = 8) and by CHIKV/ZIKV (n = 17) were observed. Overall, fever, arthralgia, myalgia, prostration, edema, exanthema, conjunctival hyperemia, lower back pain, dizziness, nausea, retroorbital pain, and anorexia were the predominating chikungunya clinical symptoms described. All strains analyzed from AP belonged to the Asian genotype and no amino acid changes were observed. In RJ, the East-Central-South-African genotype (ECSA) circulation was demonstrated and no E1-A226V mutation was observed. Despite this, an E1-V156A substitution was characterized in two samples and for the first time, the E1-K211T mutation was reported in all samples analyzed.

The impact of the age of first blood meal and Zika virus infection on Aedes aegypti egg production and longevity.

The impact of senescence and pathogen infection on Aedes aegypti life-history traits remains poorly understood. This laboratory study focused on the impact of Zika virus (ZIKV) infection and the age of first blood intake on blood meal and clutch sizes, and more importantly on the egg production ratio per µL of blood. Three groups of ZIKV-infected and uninfected Ae. aegypti females that received their first blood meal at 7 (young feeders), 14 (mature feeders) and 21 days old (old feeders) were monitored daily for survival and received a blood meal free of ZIKV once a week. The number of eggs laid per female were registered 3-4 days after blood feeding. Infection by ZIKV and age of feeding produced a strong negative impact on survival and oviposition success (e.g. likelihood of laying at least one egg per gonotrophic cycle). Interestingly, clutch size presented a dramatic reduction on uninfected mosquitoes, but raised from 36.5 in clutch1 to 55.1 eggs in clutch 3. Blood meal size remained stable in uninfected females, while a slight increase was observed for the infected counterparts. In uninfected Ae. aegypti, egg production was strongly affected by the age of feeding with younger females laying three times more eggs than when older. On the other hand, ZIKV-infected mosquitoes had a constant but low egg production. Overall, mosquito senescence and ZIKV infection had an impact on mosquito egg production by causing a sharp decrease in the number of eggs along the clutches for uninfected mosquitoes and a slight increase for infected mosquitoes. Despite some study limitations, our results contribute to a better understanding of the effects of mosquito aging and pathogen infection on the vectorial capacity of Ae. aegypti.

30 years of dengue fatal cases in Brazil: a laboratorial-based investigation of 1047 cases.

BACKGROUND: Dengue viruses (DENV) have emerged and reemerged in Brazil in the past 30 years causing explosive epidemics. The disease may range from clinically asymptomatic infections to severe and fatal outcomes. We aimed to describe the epidemiological, clinical and laboratorial aspects of the dengue fatal cases received by a Regional Reference Laboratory, Brazil in 30 years. METHODS: A total of 1047 suspected fatal dengue cases were received from 1986 to 2015 and analyzed in the Laboratory of Flavivirus, FIOCRUZ. Suspected cases were submitted to viral detection, serological and molecular methods for cases confirmation. Influence of gender, age, serotype and type of infection (primary/secondary) on death outcome, as well the interactions between serotype and age or infection and age and type of infection were also studied. RESULTS: A total of 359 cases (34.2%) were confirmed and DENV-1 (11.1%), DENV-2 (43.9%), DENV-3 (32.8%) and DENV-4 (13.7%) were detected. Overall, fatal cases occurred more often in primary infections (59.3%, p = 0.001). However, in 2008, fatal cases were mainly associated to secondary infections (p = 0.003). In 2008 and 2011, deaths were more frequent on children and those infected by DENV-2 presented a higher risk for fatal outcome. Moreover, children with secondary infections had a 4-fold higher risk for death. CONCLUSIONS: Dengue is a multifactorial disease and, factors such as viral strain/serotype, occurrence of secondary infections and co-morbidities may lead to a severe outcome. However, the high dengue incidence and transmission during epidemics, such as those observed in Brazil may overwhelm and collapse the public health services, potentially impacting on increased disease severity and mortality.

NS1 Antigenemia and Viraemia Load: Potential Markers of Progression to Dengue Fatal Outcome?

Dengue is a worldwide problem characterized by a multifactorial pathogenesis. Considering the viral components, it is known that high viremia or high levels of the secreted nonstructural protein 1 (NS1) may be associated with a more severe disease. We aimed to characterize the NS1 antigenemia and viremia in dengue fatal and non-fatal cases, as potential markers of progression to a fatal outcome. NS1 antigenemia and viremia were determined in Brazilian dengue fatal cases (n = 40) and non-fatal cases (n = 40), representative of the four dengue virus (DENV) serotypes. Overall, the fatal cases presented higher NS1 levels and viremia. Moreover, the fatal cases from secondary infections showed significantly higher NS1 levels than the non-fatal ones. Here, irrespective of the disease outcome, DENV-1 cases presented higher NS1 levels than the other serotypes. However, DENV-2 and DENV-4 fatal cases had higher NS1 antigenemia than the non-fatal cases with the same serotype. The viremia in the fatal cases was higher than in the non-fatal ones, with DENV-3 and DENV-4 presenting higher viral loads. Viral components, such as NS1 and viral RNA, may be factors influencing the disease outcome. However, the host immune status, comorbidities, and access to adequate medical support cannot be ruled out as interfering in the disease outcome.

Rapid, noninvasive detection of Zika virus in Aedes aegypti mosquitoes by near-infrared spectroscopy.

The accelerating global spread of arboviruses, such as Zika virus (ZIKV), highlights the need for more proactive mosquito surveillance. However, a major challenge during arbovirus outbreaks has been the lack of rapid and affordable tests for pathogen detection in mosquitoes. We show for the first time that near-infrared spectroscopy (NIRS) is a rapid, reagent-free, and cost-effective tool that can be used to noninvasively detect ZIKV in heads and thoraces of intact Aedes aegypti mosquitoes with prediction accuracies of 94.2 to 99.3% relative to quantitative reverse transcription polymerase chain reaction (RT-qPCR). NIRS involves simply shining a beam of light on a mosquito to collect a diagnostic spectrum. We estimated in this study that NIRS is 18 times faster and 110 times cheaper than RT-qPCR. We anticipate that NIRS will be expanded upon for identifying potential arbovirus hotspots to guide the spatial prioritization of vector control.

An overview of mosquito vectors of Zika virus.

The mosquito-borne arbovirus Zika virus (ZIKV, Flavivirus, Flaviviridae), has caused an outbreak impressive by its magnitude and rapid spread. First detected in Uganda in Africa in 1947, from where it spread to Asia in the 1960s, it emerged in 2007 on the Yap Island in Micronesia and hit most islands in the Pacific region in 2013. Subsequently, ZIKV was detected in the Caribbean, and Central and South America in 2015, and reached North America in 2016. Although ZIKV infections are in general asymptomatic or causing mild self-limiting illness, severe symptoms have been described including neurological disorders and microcephaly in newborns. To face such an alarming health situation, WHO has declared Zika as an emerging global health threat. This review summarizes the literature on the main vectors of ZIKV (sylvatic and urban) across all the five continents with special focus on vector competence studies.

Analysis of Clinical and Laboratory Alterations Related to Dengue Case Severity: Comparison between Serotypes 2 and 4 in Brazil.

The increase in severe dengue (SD) cases has caused great impact on public health and has concerned authorities of countries where the disease is endemic and epidemics reach high proportions. The recognition of progression signs of this severe disease during the initial febrile phase can be difficult, since the symptoms are often indistinguishable from other febrile diseases. The aim of this study was to evaluate the clinical manifestations and laboratory findings in patients from two dengue outbreaks and their association with the disease. The study was conducted in patients (n = 153) with signs and symptoms consistent with dengue occurred during two distinct epidemics, 2010 and 2013, in the city of Campos dos Goytacazes, Rio de Janeiro, Brazil. According to the 2009 World Health Organization criteria, patients were classified as dengue without warning signs ([DwoWS] 60.6%, 57/94), dengue with warning signs ([DwWS] 30.9%, 29/94), and SD (4.25%, 4/94). Patients with DwWS/SD presented lower platelet and leukocyte counts and higher transaminase levels when compared with the DwoWS ones. Interestingly, patients from the epidemic of 2010 caused by dengue virus 2 (DENV-2) had lower platelet counts than patients of the 2013 epidemic caused by DENV-4. Furthermore, plasma leakage, gastrointestinal bleeding, and pleural effusion, hallmarks for a more severe disease, were also more frequently observed in those cases. Although previous studies may have extensively reported the wide range of the clinical aspects of dengue, the characterization of DENV-4 is desirable considering the burden of the disease during epidemics, especially for the health units and hospitals performing patient's management.

Dengue type 4 in Rio de Janeiro, Brazil: case characterization following its introduction in an endemic region.

BACKGROUND: Due to the populations' susceptibility, DENV-4 introduction in 2010 led to the occurrence of explosive epidemics in the following years in Brazil. In 2011, DENV-4 was identified in Rio de Janeiro (RJ) and it was prevalent in 2012 and 2013. Here, we aimed to characterize clinical, epidemiological and laboratorial aspects of DENV-4 cases after this serotype introduction in an endemic scenario. METHODS: Dengue suspected cases (n = 3727) were received and analyzed from January 2011 to December 2013, during outbreaks occurred in RJ, Brazil. Samples were submitted to virological, serological and molecular methods for case confirmation. DENV-4 cases (n = 705) were characterized according to the type of infection, disease severity and, viremia levels and NS1 antigenemia were accessed. Representative strains were partial sequenced for genotyping. RESULTS: DENV-4 was identified in 44.2% (705/1593) of dengue positive cases, virus isolated in 48.7% of the cases. Anti-DENV IgM was detected in 39.4% of the cases, however an increased detection was observed in cases with ≥4 days of symptoms (57.0%). NS1 antigen was identified in 41.5% of DENV-4 cases however, after immune complexes dissociation, the detection significantly increased (87.6%). Females were more affected than males, so did children aged 11-15 years old. Primary cases were more frequently observed than secondary ones and most of them were classified as dengue. No differences on NS1 antigenemia and viraemia within the groups were observed. Despite the higher frequency of severe disease on individuals >65 years old, no differences were observed among the groups and type of infection. However, DENV-4 fatal cases were more frequent on secondary infections (57.1%). DENV-4 Genotype II was identified with a probable origin from Venezuela and Colombia. CONCLUSIONS: It has been shown that laboratorial diagnosis is still a reliable tool for the disease surveillance, detecting and confirming emerging epidemics. Despite the occurrence of secondary infections, most DENV-4 cases presented a mild disease. As RJ is endemic for dengue, high rates of secondary infections would be expected. Despite the existence of two genotypes, only Genotype II was identified in our study.

First Report of the East-Central South African Genotype of Chikungunya Virus in Rio de Janeiro, Brazil.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus that causes an acute febrile syndrome with a severe and debilitating arthralgia. In Brazil, the Asian and East-Central South African (ECSA) genotypes are circulating in the north and northeast of the country, respectively. In 2015, the first autochthonous cases in Rio de Janeiro, Brazil were reported but until now the circulating strains have not been characterized. Therefore, we aimed here to perform the molecular characterization and phylogenetic analysis of CHIKV strains circulating in the 2016 outbreak occurred in the municipality of Rio de Janeiro. METHODS: The cases analyzed in this study were collected at a private Hospital, from April 2016 to May 2016, during the chikungunya outbreak in Rio de Janeiro, Brazil. All cases were submitted to the Real Time RT-PCR for CHIKV genome detection and to anti-CHIKV IgM ELISA. Chikungunya infection was laboratorially confirmed by at least one diagnostic method and, randomly selected positive cases (n=10), were partially sequenced (CHIKV E1 gene) and analyzed. RESULTS: The results showed that all the samples grouped in ECSA genotype branch and the molecular characterization of the fragment did not reveal the A226V mutation in the Rio de Janeiro strains analyzed, but a K211T amino acid substitution was observed for the first time in all samples and a V156A substitution in two of ten samples. CONCLUSIONS: Phylogenetic analysis and molecular characterization reveals the circulation of the ECSA genotype of CHIKV in the city of Rio de Janeiro, Brazil and two amino acids substitutions (K211T and V156A) exclusive to the CHIKV strains obtained during the 2016 epidemic, were reported.

Differential Susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika Virus.

BACKGROUND: Since the major outbreak in 2007 in the Yap Island, Zika virus (ZIKV) causing dengue-like syndromes has affected multiple islands of the South Pacific region. In May 2015, the virus was detected in Brazil and then spread through South and Central America. In December 2015, ZIKV was detected in French Guiana and Martinique. The aim of the study was to evaluate the vector competence of the mosquito spp. Aedes aegypti and Aedes albopictus from the Caribbean (Martinique, Guadeloupe), North America (southern United States), South America (Brazil, French Guiana) for the currently circulating Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia. METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were orally exposed to an Asian genotype of ZIKV (NC-2014-5132). Upon exposure, engorged mosquitoes were maintained at 28° ± 1 °C, a 16h:8h light:dark cycle and 80% humidity. 25-30 mosquitoes were processed at 4, 7 and 14 days post-infection (dpi). Mosquito bodies (thorax and abdomen), heads and saliva were analyzed to measure infection, dissemination and transmission, respectively. High infection but lower disseminated infection and transmission rates were observed for both Ae. aegypti and Ae. albopictus. Ae. aegypti populations from Guadeloupe and French Guiana exhibited a higher dissemination of ZIKV than the other Ae. aegypti populations examined. Transmission of ZIKV was observed in both mosquito species at 14 dpi but at a low level. CONCLUSIONS/SIGNIFICANCE: This study suggests that although susceptible to infection, Ae. aegypti and Ae. albopictus were unexpectedly low competent vectors for ZIKV. This may suggest that other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak.

Dengue epidemics in two distinct periods reveal distinct epidemiological, laboratorial and clinical aspects in a same scenario: analysis of the 2010 and 2013 epidemics in Mato Grosso do Sul, Brazil.

BACKGROUND: Dengue is a major problem in Brazil. Epidemiological and clinical aspects were characterized in patients from two epidemics which occurred in Mato Grosso do Sul, Brazil. METHODS: Dengue cases were classified according to the 2009 WHO criteria, tested by serological and molecular biology tests and analysed for nonstructural protein 1 (NS1) antigenemia. RESULTS: Dengue was confirmed in 78.7% (48/61) and 75.6% (118/156) of the cases studied in 2010 and 2013, respectively. DENV-1 and DENV-2 were the serotypes involved in the 2010 epidemic and DENV-4 in the 2013 one. Most of the cases were classified as dengue without warning; however, severe dengue was observed in 18.7% (9/48) of the cases in 2010 and less observed in DENV-4 cases. NS1 levels were higher in patients with dengue with warning signs and severe dengue in 2010. Circulating aspartate aminotransferase (AST) and alanine transferase (ALT) were altered in all groups, independently of the infecting serotype or epidemic. Patients with DENV-1 and DENV-2 presented significant lower monocyte counts when compared to patients with DENV-4. An inverse correlation was found between platelet count, leucocytes, monocytes and NS1 levels. CONCLUSIONS: Epidemics caused by the prevalence of distinct DENV serotypes had different impacts and clinical characteristics in a same scenario and, despite the occurrence of secondary infections, the DENV-4 emergence was not associated with severe cases.