Co-Circulation of Two Independent Clades and Persistence of CHIKV-ECSA Genotype during Epidemic Waves in Rio de Janeiro, Southeast Brazil.

The Chikungunya virus infection in Brazil has raised several concerns due to the rapid dissemination of the virus and its association with several clinical complications. Nevertheless, there is limited information about the genomic epidemiology of CHIKV circulating in Brazil from surveillance studies. Thus, to better understand its dispersion dynamics in Rio de Janeiro (RJ), one of the most affected states during the 2016-2019 epidemic waves, we generated 23 near-complete genomes of CHIKV isolates from two main cities located in the metropolitan mesoregion, obtained directly from clinical samples. Our phylogenetic reconstructions suggest the 2019-CHIKV-ECSA epidemic in RJ state was characterized by the co-circulation of multiple clade (clade A and B), highlighting that two independent introduction events of CHIKV-ECSA into RJ state have occurred between 2016-2019, both mediated from the northeastern region. Interestingly, we identified that the two-clade displaying eighteen characteristic amino acids changes among structural and non-structural proteins. Our findings reinforce that genomic data can provide information about virus genetic diversity and transmission dynamics, which might assist in the arbovirus epidemics establishing of an effective surveillance framework.

Yellow Fever Virus Reemergence and Spread in Southeast Brazil, 2016-2019.

The recent reemergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the rapid dissemination of the virus in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil, we generated 18 complete and nearly complete genomes from the peak of the epidemic curve from nonhuman primates (NHPs) and human infected cases across the Espírito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the estimated timing, source, and likely routes of yellow fever virus transmission and dispersion during one of the largest outbreaks ever registered in Brazil. We showed that during the recent epidemic, YFV was reintroduced from Minas Gerais to the Espírito Santo and Rio de Janeiro states multiple times between 2016 and 2019. The analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce the idea that continued genomic surveillance strategies can provide information on virus genetic diversity and transmission dynamics that might assist in understanding arbovirus epidemics.IMPORTANCE Arbovirus infections in Brazil, including yellow fever, dengue, zika, and chikungunya, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak by analyzing genomic data from areas in southeastern Brazil not covered by other previous studies. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from nonhuman primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control.

Dynamics of nonstructural glycoprotein-1 in dengue patients presenting with different clinical manifestations from 1986 to 2012 in Rio de Janeiro, Brazil.

The hyperendemicity and co-circulation of different dengue serotypes in Brazil have increased the number of severe dengue cases and the rate of hospitalization for dengue. Virological and individual factors are associated with the complexity of the disease. Antigenemia levels of nonstructural glycoprotein-1 (NS1) have been associated with severe dengue. Aiming to identify a severity marker during the acute phase (days 0 to 5 of disease), the association of NS1 antigenemia with clinical presentation, sex, age range, immune response, number of days of disease, and serotype RNA levels was evaluated in serum samples of patients from the state of Rio de Janeiro clinically classified as having dengue without warning signs (DWWS) or dengue with warning signs/severe dengue (DWWS/SD). The immune response was classified by in-house enzyme-linked immunosorbent assay, antigenemia was determined by quantification of NS1, and viremia was quantified by real-time PCR. Of the total number of patients, 36.6% (74 of 202) presented warning signs/severe dengue and 72.3% (146 of 202) were classified with primary infection. DENV-2 presented an association between clinical presentation and antigenemia (P = 0.02). DENV-3 had higher levels of NS1 (P < 0.0001). This study has shown that the infecting serotype influences circulating NS1 levels in the host, as well as NS1 antigenemia may vary as to the clinical presentation of the patient infected with DENV-2. However, the criterion used to screen patients for clinical presentation, in DWWS and DWWS/SD patients, was not a good marker for dengue severity in our study.

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.

Dengue severity associated with age and a new lineage of dengue virus-type 2 during an outbreak in Rio De Janeiro, Brazil.

Dengue virus-type 2 (DENV-2) caused three outbreaks, in the years 1990, 1998, and 2008, in Rio de Janeiro, Brazil. The 2008 outbreak was the most severe in reported cases, hospitalizations, and deaths. To investigate virological and epidemiological factors that may have contributed to the pathogenic profile of 2008 epidemic, 102 patients sera obtained during the epidemic and inter-epidemic periods of three outbreaks were analysed by qRT-PCR to estimate viremia levels and their correlation with the clinical, immunological, and demographic patient characteristics. DENV-2 isolates from the outbreaks were sequenced. Two DENV-2 lineages (I and II) of the American/Asian genotype were confirmed, each exclusive for 1990-2002 and 2007-2011, respectively. The mean viremia level in the 2008 samples was two orders of magnitude higher than that of the 1990-2002 samples. Severe dengue cases increased from 31% in 1990-2002 to 69% in 2007-2011; in patients aged ≤15 years, from 3% in 1990-2002 to 37% in 2007-2011. The DENV-2 lineage II and younger age significantly contributed to the pathogenic profile of 2008 epidemic in Rio de Janeiro.

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance.

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and Simplexa™ Dengue real-time RT-PCR confirmed DENV-4 infection in all cases. Additionally, DENV-4 was confirmed in a female Ae. aegypti with 1.08 x 10(3) copies/mL of virus, as determined by quantitative real-time RT-PCR. This is the first time the Simplexa™ Dengue real-time assay has been used for the classification of cases of infection and for entomological investigations. The use of these molecular techniques was shown to be important for the surveillance of dengue in humans and vectors.

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and SimplexaTM Dengue real-time RT-PCR confirmed DENV-4 infection in all cases. Additionally, DENV-4 was confirmed in a female Ae. aegypti with 1.08 x 10³ copies/mL of virus, as determined by quantitative real-time RT-PCR. This is the first time the SimplexaTM Dengue real-time assay has been used for the classification of cases of infection and for entomological investigations. The use of these molecular techniques was shown to be important for the surveillance of dengue in humans and vectors.