Márcio Roberto Teixeira Nunes

Zika virus in the Americas: Early epidemiological and genetic findings

Zika virus genomes from Brazil The Zika virus outbreak is a major cause for concern in Brazil, where it has been linked with increased reports of otherwise rare birth defects and neuropathology. In a phylogenetic analysis, Faria et al. infer a single introduction of Zika to the Americas and estimated the introduction date to be about May to December 2013—some 12 months earlier than the virus was reported. This timing correlates with major events in the Brazilian cultural calendar associated with increased traveler numbers from areas where Zika virus has been circulating. A correlation was also observed between incidences of microcephaly and week 17 of pregnancy. Science, this issue p. 345 Virus sequencing indicates that Zika arrived in Brazil during the middle of 2013, coincident with a surge in air travelers. Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Evaluation of an Enzyme Immunoassay for Detection of Dengue Virus NS1 Antigen in Human Serum

ABSTRACT We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Duplex Reverse Transcription-PCR Followed by Nested PCR Assays for Detection and Identification of Brazilian Alphaviruses and Flaviviruses

ABSTRACT A new approach was developed for the rapid detection and identification of Brazilian alphaviruses and flaviviruses. The methodology involves the genus-specific detection of Alphavirus and Flavivirus by a duplex reverse transcription-PCR (D-RT-PCR), followed by multiplex nested PCR (M-N-PCR) or nested PCR (N-PCR) assays for species-specific identification. By this protocol, 25 arboviruses were specifically detected and identified. Detection levels between 101.3 and 103.5 50% tissue culture infective doses (TCID50)/ml of Flavivirus and Alphavirus strains were achieved by D-RT-PCR, and levels of <1 TCID50/ml were achieved by M-N-PCR assays. To assess the suitability and clinical application of this methodology, a total of 101 human or animal stored samples were analyzed. Results obtained suggest that this technique could be applied as a rapid diagnostic tool in clinical samples in which arbovirus infection is suspected and differential diagnosis is required, avoiding the need to test specimens by separate PCR methods.

Persistent West Nile Virus Infection in the Golden Hamster: Studies on Its Mechanism and Possible Implications for Other Flavivirus Infections

Golden hamsters (Mesocricetus auratus) experimentally infected with West Nile virus (WNV) developed chronic renal infection and persistent shedding of virus in urine for up to 8 months, despite initial rapid clearance of virus from blood and the timely appearance of high levels of specific neutralizing antibodies. Infectious WNV could be recovered by direct culture of their urine and by cocultivation of kidney tissue for up to 247 days after initial infection. Only moderate histopathologic changes were observed in the kidneys or brain of the chronically infected hamsters, although WNV antigen was readily detected by immunohistochemistry within epithelium, interstitial cells, and macrophages in the distal renal tubules. Comparison of WNV isolates from serial urine samples from individual hamsters over several months indicated that the virus underwent both genetic and phenotypic changes during persistent infection. These findings are similar to previous reports of persistent infection with tickborne encephalitis and Modoc viruses.

Phylogeography of dengue virus serotype 4, Brazil, 2010-2011.

Multiple origins indicate this serotype was introduced in several episodes.

Mayaro Fever Virus, Brazilian Amazon

In February 2008, a Mayaro fever virus (MAYV) outbreak occurred in a settlement in Santa Barbara municipality, northern Brazil. Patients had rash, fever, and severe arthralgia lasting up to 7 days. Immunoglobulin M against MAYV was detected by ELISA in 36 persons; 3 MAYV isolates sequenced were characterized as genotype D.

Genetic ancestry and income are associated with dengue hemorrhagic fever in a highly admixed population

To test whether African ancestry is protective for severe dengue, we genotyped 49 hospitalized cases of dengue hemorrhagic fever (DHF) as well as 293 neighborhood cases of dengue fever and 294 asymptomatic controls in Salvador, Bahia, Brazil. Ancestry-informative markers and 282 unlinked SNPs not associated with the clinical presentation of dengue were used to estimate ancestry. After controlling for income, both self-defined Afro-Brazilian ethnicity and African ancestry were protective for DHF (P=0.02, OR=0.28 and P=0.02, OR=0.13, respectively). Income or an index of income indicators, however, was also independently associated with the diagnosis of DHF.

Molecular Epidemiology of Group C Viruses (Bunyaviridae, Orthobunyavirus) Isolated in the Americas

ABSTRACT To date, no molecular studies on group C viruses (Bunyaviridae, Orthobunyavirus) have been published. We determined the complete small RNA (SRNA) segment and partial medium RNA segment nucleotide sequences for 13 group C members. The full-length SRNA sequences ranged from 915 to 926 nucleotides in length, and revealed similar organization in comparison with other orthobunyaviruses. Based on the 705 nucleotides of the N gene, group C members were distributed into three major phylogenetic groups, with the exception of Madrid virus, which was placed outside of these three groups. Analysis of the Caraparu virus strain BeH 5546 revealed that it has an SRNA sequence nearly identical to that of Oriboca virus and is a natural reassortant virus. In addition, analysis of 345 nucleotides of the Gn gene for eight group C viruses and for strain BeH 5546 revealed a different phylogenetic topology, suggesting a reassortment pattern among them. These findings represent the first evidence for natural reassortment among the group C viruses, which include several human pathogens. Furthermore, our genetic data corroborate previous relationships determined using serologic assays (complement fixation, hemagglutination inhibition, and neutralization tests) and suggest that a combination of informative molecular, serological, and ecological data is a helpful tool to understand the molecular epidemiology of arboviruses.

Nucleotide sequences and phylogeny of the nucleocapsid gene of Oropouche virus

The nucleotide sequence of the S RNA segment of the Oropouche (ORO) virus prototype strain TRVL 9760 was determined and found to be 754 nucleotides in length. In the virion-complementary orientation, the RNA contained two overlapping open reading frames of 693 and 273 nucleotides that were predicted to encode proteins of 231 and 91 amino acids, respectively. Subsequently, the nucleotide sequences of the nucleocapsid genes of 27 additional ORO virus strains, representing a 42 year interval and a wide geographical range in South America, were determined. Phylogenetic analyses revealed that all the ORO virus strains formed a monophyletic group that comprised three distinct lineages. Lineage I contained the prototype strain from Trinidad and most of the Brazilian strains, lineage II contained six Peruvian strains isolated between 1992 and 1998, and two strains from western Brazil isolated in 1991, while lineage III comprised four strains isolated in Panama during 1989.

Air Travel Is Associated with Intracontinental Spread of Dengue Virus Serotypes 1-3 in Brazil

Dengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, with the largest disease burden in tropical and subtropical regions. We investigated the spatial dynamics of DENV-1, DENV-2 and DENV-3 in Brazil by applying a statistical framework to complete genome sequences. For all three serotypes, we estimated that the introduction of new lineages occurred within 7 to 10-year intervals. New lineages were most likely to be imported from the Caribbean region to the North and Northeast regions of Brazil, and then to disperse at a rate of approximately 0.5 km/day. Joint statistical analysis of evolutionary, epidemiological and ecological data indicates that aerial transportation of humans and/or vector mosquitoes, rather than Aedes aegypti infestation rates or geographical distances, determine dengue virus spread in Brazil.