Zika virus in the Americas: Early epidemiological and genetic findings

Abstract

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.

Fig. 1. Time series and cartography of reported Zika virus and microcephaly cases in Brazil.

(A) Number of suspected cases of ZIKV per week in 5596 municipalities in Brazil. The epidemic peaked from 12 to 18 July 2015 (n = 2791 cases). Letters indicate months. (B) Total incidence of ZIKV cases per 100,000 people in each federal state. Triangles indicate sampling locations of the sequences reported here; circles indicate locations of other genomes from Brazil [municipality of Natal in Rio Grande do Norte state (16) and an unknown municipality in Paraiba state (21)]. Red symbols indicate ZIKV genomes isolated from microcephaly cases. Federal states are indicated by 2-letter codes: PA: Para, MA: Maranhão, CE: Ceará, RN: Rio Grande do Norte, PB: Paraíba. Per capita incidences in each state were calculated using high-resolution gridded human population size datasets for Brazil (45). (C) Incidence of suspected microcephaly cases per 100,000 people in each federal state. Per capita incidences for each state were calculated as described for panel (B).

Fig. 2. Maximum likelihood phylogeny of ZIKV complete coding region sequences.

Bootstrap scores are shown next to well-supported nodes and the phylogeny was mid-point rooted. A fully annotated tree is provided in Fig. S2. The American ZIKV outbreak clade is drawn as a narrow white triangle and is shown in detail in Fig. 3. Asterisks highlight the four internal branches that are ancestral to the American ZIKV lineage (see main text and Fig. S3). Correlation between the sampling date of each sequence and the genetic distance of that sequence from the root of a maximum likelihood phylogeny of the Asian genotype (correlation coefficient R² = 0.997). A molecular clock phylogeny of this data is shown in Fig. 3. The Malaysian strain (HQ234499) sampled in 1966 is the oldest representative of the Asian genotype and falls on the regression line, indicating that it does not appear to be unusually divergent for its age. A similar analysis with the HQ234499 strain excluded is shown in fig. S5C.

Fig. 3. Timescale of the introduction of ZIKV to the Americas.

(A) Molecular clock phylogeny of the ZIKV outbreak lineage estimated from complete coding region sequences, plus 6 sequences (KJ634273, KU312315, KU312314, KU212313, KU646828, and KU646827) longer than 1500nt (available data as of 7th March 2016). For visual clarity, three basal sequences, HQ23499 (Malaysia, 1966), EU545988 (Micronesia, 2007) and JN860885 (Cambodia, 2010) are not displayed here (see Fig. S3). Gray horizontal bars represent 95% Bayesian credible intervals for divergence dates. A and B denote clades discussed in main text and numbers next to them denote posterior probabilities. Diamond sizes represent, at each node, the posterior probability support of that node. Taxa are labeled with accession number, sampling location, and sampling date. Names of sequences generated in this study are underlined. (B) Posterior distributions of the estimated ages (TMRCAs) of clades A and B, estimated in BEAST software using the best-fitting evolutionary model (table S2). The time and duration of the three events (i-iii) discussed in the main text are shown. (C) Number of airline passengers from specific countries arriving in Brazil per month versus number of suspected cases of ZIKV in French Polynesia. The blue curve (left y axis) shows a polynomial fitting of the number of travelers (blue points) from countries with recorded ZIKV outbreaks between 2012 and 2015 (French Polynesia, Thailand, Indonesia, Malaysia, Cambodia, and New Caledonia) (supplementary materials section 6), aggregated across 20 Brazilian national airports. The purple bars represent weekly numbers of suspected ZIKV cases (right y axis) in French Polynesia (FP) from 30 October 2013 to 14 February 2014 (4).