Zika 2014 Haiti Outbreak - Molecular & Clinical - PLOS NTD

[niman]

Zika Virus Outbreak in Haiti in 2014: Molecular and Clinical Data

http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0004687

 

Edited April 25, 2016 by niman

[niman]

• John Lednicky,
• Valery Madsen Beau De Rochars,
• Maha El Badry,
• Julia Loeb,
• Taina Telisma,
• Sonese Chavannes,
• Gina Anilis,
• Eleonora Cella,
• Massimo Ciccozzi,
• Mohammed Rashid,
• Bernard Okech,
• Marco Salemi,
• J. Glenn Morris Jr. 

 

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• Published: April 25, 2016
• http://dx.doi.org/10.1371/journal.pntd.0004687

• Author Contributions

  Conceived and designed the experiments: JLe VMBDR BO MS JGM. Performed the experiments: JLe MEB MR JLo. Analyzed the data: JLe JGM MS EC MC. Wrote the paper: JLe VMBDR BO MS JGM MEB MR JLo TT SC GA EC MC. Clinical patient management and data collection: TT SC GA. Had full access to all data and had final responsibility for the decision to submit for publication: JGM.

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Edited April 25, 2016 by niman

[niman]

Abstract

Background

Zika virus (ZIKV), first isolated in Uganda in 1947, is currently spreading rapidly through South America and the Caribbean. In Brazil, infection has been linked with microcephaly and other serious complications, leading to declaration of a public health emergency of international concern; however, there currently are only limited data on the virus (and its possible sources and manifestations) in the Caribbean.

Methods

From May, 2014-February, 2015, in conjunction with studies of chikungunya (CHIKV) and dengue (DENV) virus infections, blood samples were collected from children in the Gressier/Leogane region of Haiti who presented to a school clinic with undifferentiated febrile illness. Samples were initially screened by RT-PCR for CHIKV and DENV, with samples negative in these assays further screened by viral culture.

Findings

Of 177 samples screened, three were positive for ZIKV, confirmed by viral sequencing; DENV-1 was also identified in culture from one of the three positive case patients. Patients were from two different schools and 3 different towns, with all three cases occurring within a single week, consistent with the occurrence of an outbreak in the region. Phylogenetic analysis of known full genome viral sequences demonstrated a close relationship with ZIKV from Brazil; additional analysis of the NS5 gene, for which more sequences are currently available, showed the Haitian strains clustering within a monophyletic clade distinct from Brazilian, Puerto Rican and Guatemalan sequences, with all part of a larger clade including isolates from Easter Island. Phylogeography also clarified that at least three major African sub-lineages exist, and confirmed that the South American epidemic is most likely to have originated from an initial ZIKV introduction from French Polynesia into Easter Island, and then to the remainder of the Americas.

Conclusions

ZIKV epidemics in South America, as well as in Africa, show complex dissemination patterns. The virus appears to have been circulating in Haiti prior to the first reported cases in Brazil. Factors contributing to transmission and the possible linkage of this early Haitian outbreak with microcephaly remain to be determined.

[niman]

Author Summary

Zika virus is currently spreading rapidly through the Americas, including the Caribbean, where it has emerged as a major public health problem due to the linkage with birth defects, including microcephaly. We report the isolation of Zika virus from 3 children in rural Haiti in December, 2014, as part of a study of acute undifferentiated febrile illness that was being conducted by our research group; from one of these children, we also isolated dengue virus serotype 1. On analysis of nucleotide sequence data from these and Zika strains from other locales, the South American/Haitian sequences cluster within the Asian clade and clearly branch out from a sequence circulating in Easter Island, which originated, in turn, from French Polynesia. On further analysis of one specific gene sequence for which more data were available, there appeared to be slight separation of Haitian strains and the strains from Brazil, Suriname, Puerto Rico and Guatemala, with molecular clock analysis suggesting that Zika virus was present in Haiti as early as mid-2013. These findings raise questions about the origin of Zika virus in the Caribbean, and subsequent patterns of circulation of the virus within the Americas.

[niman]

Introduction

Zika is a mosquito-borne flavivirus initially isolated in the Zika forest of Uganda in 1947 [1]. There were periodic human cases reported from Africa and Asia in the intervening decades, but it was not until 2007 that a major epidemic was reported, on Yap Island, Federated States of Micronesia [2]. Zika infections were subsequently identified in other parts of Asia, with a shift toward the Americas presaged by an outbreak on Easter Island in May, 2014 [3]. In March, 2015, cases were identified in Bahia, Brazil [4], with subsequent rapid spread through multiple Brazilian states [1,5], and other countries in South America and the Caribbean [1,5]: as of January, 2016, locally-transmitted cases had been reported by the Pan American Health Organization in Puerto Rico and 19 countries/territories in the Americas.

Infection with Zika virus (ZIKV) has traditionally been associated with asymptomatic or mild illness. Clinical manifestations, when they occur, include acute onset of fever, headache, maculopapular rash, arthralgias, myalgias, and/or non-purulent conjunctivitis [1,2]. In an outbreak in French Polynesia in 2013–14, there were, for the first time, reports of neurological and auto-immune complications, such as Guillain-Barré syndrome, in the setting of co-circulating dengue (DENV) and chikungunya (CHIKV) viruses [6,7]. With the progression of the Brazilian outbreak in 2015, the Brazilian Ministry of Health noted a striking concurrent increase in the number of infants born with microcephaly in areas with ZIKV transmission. Multiple subsequent studies have provided further documentation of the link between ZIKV and microcephaly and other birth defects, as well as with Guillain-Barré syndrome [8–14]. Based on the “strongly suspected” causal link between Zika virus and the observed fetal brain abnormalities, WHO has declared the current Zika epidemic a “public health emergency of international concern” [15].

As a step in monitoring and understanding spread of the epidemic, we report here the isolation of ZIKV from three children in Haiti in December, 2014, before the first reported Brazilian cases.

[niman]

Materials and Methods

Our group has been involved in studies of CHIKV and DENV transmission in Haiti since May, 2014, when CHIKV swept across the island of Hispaniola. Work was done in collaboration with the Christianville Foundation, which operates 4 schools in the Gressier/Leogane region of Haiti (some 20 miles west of Port-au-Prince) with a total of approximately 1,250 students from pre-kindergarten to grade 12; students attending the school receive care at no cost in an outpatient school clinic staffed by a physician and two nurses [16]. As part of these studies, UF has protocols for collection of diagnostic blood samples from children presenting to the school clinic with acute undifferentiated febrile illness (i.e., febrile illness with no localizing signs, such as would be expected with pneumonia, urinary tract infections, etc.).

Blood samples were obtained from a total of one hundred seventy-seven (n = 177) Haitian children presenting with a history of acute undifferentiated febrile illnesses between May, 2014, and February, 2015. Blood smears were prepared for microscopic analyses for malaria parasites. To obtain plasma for virologic analysis, whole blood (5 mL) was collected into purple top (K2EDTA) tubes (Becton, Dickinson, and Company, Franklin Lakes, New Jersey), the collected blood centrifuged to pellet red and white blood cells, and the resulting plasma transferred to sterile screw-top vials and stored at -70°C pending tests.

For the initial CHIKV and DENV screens, vRNA was extracted from virions in the plasma using a QIAamp Viral RNA Mini Kit (Qiagen Inc., Valencia, CA). The extracted vRNAs were tested using primers described by Lanciotti et al [17] for CHIKV and Santiago et al [18] for DENV types 1–4. Many specimens were positive for CHIKV and DENV1 or DENV4 vRNA (data to be presented elsewhere). Those negative for CHIKV and DENV 1–4 vRNA were also tested with a universal primer system for flavivirus: RT-PCR system Flav100F-200R [19]. No virus-specific amplicons were generated by the latter approach. Samples negative or borderline in the above assays were screened in a variety of mammalian cell lines inoculated with aliquots of plasma; detailed methods are provided in supplemental material, as are methods for transmission microscopy.

Detection and sequencing of Zika virus RNA in spent cell media

As virus-specific CPE were observed in LLC-MK2 and Vero E6 cells inoculated with plasma, but the identity of the agent unknown, spent cell growth media was treated with cyanase nuclease to degrade nucleic acids external to that packaged (and thus protected) in virions using a Nucleic Acid Removal Kit (RiboSolutions, Inc., Cedar Creek, Texas), and vRNA once again extracted from the treated material using a QIAamp Viral RNA Mini Kit. A panel of PCR and RT-PCR tests were performed; for RT-PCR, first-strand synthesis was performed using random 9-mers and Accuscript High Fidelity 1st strand cDNA kit (Agilent Technologies, Santa Clara, CA). The presence of flavivirus RNA was detected using the Flav100F-200R, and Zika virus RNA effectively detected [20] using RT-PCR systems ZIKVF9027-ZIKVR9197c [21], 9271–9373 [22], and 835 – 911c [17]. For confirmation, PCR amplicons were purified and sequenced.

Sequencing of the complete Zika virus genome of one isolate (from the first patient), designated Haiti/1225/2014, was accomplished using a genome walking strategy with the PCR primers described in S1 Table. Briefly, targeted overlapping sequences (approximately 800 bp amplicons) were amplified using Accuscript High Fidelity reverse transcriptase in the presence of SUPERase-In RNase inhibitor (Ambion, Austin, TX), followed by PCR with Phusion Polymerase (New England Biolabs) with denaturation steps performed at 98°C. To obtain the 5′ and 3′ ends of the viral genome, a 5′ and 3′ system for the Rapid Amplification of cDNA Ends (RACE) was used per the manufacturer's protocols (Life Technologies, Carlsbad, CA, USA). PCR amplicons were purified, sequenced bidirectionally using Sanger Sequencing, and the sequences assembled with the aid of Sequencher DNA sequence analysis software v2.1 (Gene Codes, Ann Arbor, MI, USA). The GenBank accession number is KU509998.

Phylogenetic analysis

All available ZIKV nucleotide sequences were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/) and four data sets were assembled (S1 Table) using the following inclusion criteria: (1) sequences were published in peer-review journals; (2) known sampling time; (3) city/state was known and clearly established in the original publication. The first data set included all ZIKV complete genome sequences available in NCBI (23 sequences) and the Haiti complete genome sequence obtained in the present study. The second data set included 109 NS5 gene region reference sequences as well as NS5 sequences of the three Haitian isolates obtained in the present study. The third data set included 58 ENV gene region reference sequences as well as ENV sequences of the three Haitian isolates obtained in the present study. The fourth data set included 21 NS3 gene region reference sequences as well as the NS3 sequence of the Haitian isolate fully sequenced in the present study. Sequences in each dataset were aligned using ClustalW [23] followed by manual optimization using Bioedit [24]. The best fitting nucleotide substitution model for each data set was chosen in accordance with the results of the hierarchical likelihood ratio test (HLRT) implemented with the Modeltest software version 3.7 [25].

Detailed phylogenetic and phylodynamic methods are included in the supplemental material. In brief, the phylogenetic signal in each data set of aligned nucleotide sequences was investigated by likelihood mapping, which evaluates the tree-like signal in all possible groups of four sequences (quartets) [26];. The NS5 data set, which included the largest number of sequences and the largest number of phylogenetic informative sites (S1 Table), was used to investigate ZIKV phylogeographic patterns with the Bayesian coalescent framework implemented in Beast v 1.8 [27]. The maximum likelihood credibility (MCC) tree was chosen from the posterior distribution of trees with the TreeAnnotator program in the BEAST package. Statistical support for branching patterns in the MCC tree was obtained by calculating the posterior probability along each internal branch. The MCC tree with reconstructed ancestral states (ancestral locations inferred by Bayesian phylogeography) was manually edited in FigTree for display purposes.

Ethics statement

The protocol for sample collection was approved by the University of Florida IRB and the Haitian National IRB. Written parental informed consent was obtained from parents or guardians of all study participants.

[niman]

Results

Zika virus was identified in plasma from three students seen in the Christianville Foundation Schools clinic. Patient #1 (described below) appears to have been infected simultaneously with DENV-1. The three case patients were from two different schools within the four-school Christianville school system; all lived in different towns/neighborhoods, within a radius of approximately 20 miles. All case patients presented within a one-week period in December, 2014. Cases of DENV-1 had been identified among children in the school clinics in the weeks before occurrence of the ZIKV cases, which, in turn, were followed by a small cluster of DENV-4 cases.

The first patient was a 15 year-old boy who presented to the clinic on December 12, 2014, with a history of subjective fever, headache, and generalized arthralgias, myalgias and asthenia. When seen, temperature was 37 degrees C, with a pulse of 92 and respiratory rate of 24, weight 51.5 Kg. He had no rash, and physical exam was unremarkable. The second patient was a 7 year-old girl who was seen on December 15, 2014 at the clinic for subjective nocturnal fever, abdominal pain, anorexia, and cough. Temperature was 37 degrees C, pulse 116, RR 28, and weight 22.8 Kg. There was no rash, and physical exam was again unremarkable. The third patient was an asymptomatic 4 year-old boy who came in December 17, 2014 for follow up after being treated for tonsillitis on November 25, when he had presented with a fever of 39 degrees C. In none of the cases would it have been possible to have identified the illness as a ZIKV infection based on clinical presentation, rather than DENV or CHIKV (and, as indicated, one child was simultaneously infected with DENV). All patients received supportive care for reported symptoms, in keeping with standard practices within the clinic.

In tissue culture, viral agents from all three patients induced subtle CPE within 4–8 days post-inoculation of human (A549, HeLa, and MRC-5) and more pronounced CPE in simian (LLC-MK2 and Vero E6) cells incubated at either 33° and 37°C. Prior to cell death, the CPE consisted of perinuclear vacuoles (Fig 1). Electron microscopy revealed features typical of flavivirus-infected cells, such as the formation of paracrystalline arrays/convoluted membranes (Fig 2A), crystalline arrays of nascent virus cores in association with double-membrane vesicles (Fig 2B), multi-membraned “whorls” (autophagosomes), individual 55–59 nm vesicles containing 40 nm virus particles, and virus particles in packets. As mentioned (Materials and Methods), direct tests of the plasma sample using RT-PCR system Flav100F-200R yielded negative results. However, specific amplicons were obtained when vRNA from cyanase-treated spent media from LLC-MK2 or Vero cells were tested with the same primers. On sequence analysis, viral agents from all three patients were identified as ZIKV.

 

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Fig 1. Virus-specific CPE in simian kidney cell line LLC-MK2.

Non-inoculated cells (A) and cells inoculated with plasma specimen 1225/2014, 8 days post-inoculation (B). Perinuclear vacuoles are evident. Original images taken at 400x magnification; insets at approx. 800X.

 

http://dx.doi.org/10.1371/journal.pntd.0004687.g001

 

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Fig 2.

A) Transmission electron micrograph detail of a ZIKV-infected LLC-MK2 cell. The large arrow points out an area containing typical flavivirus-induced paracrystalline arrays/convoluted membranes in a ZIKV-infected LLC-MK2 cell. (B) Transmission electron micrograph detail of ZIKV-infected LLC-MK2 cell. Crystalline arrays of virus cores (large arrow) are shown in association with membrane vesicles.

 

http://dx.doi.org/10.1371/journal.pntd.0004687.g002

On phylogenetic analysis, likelihood mapping showed that all data sets (full genome alignment and gene-specific alignments) displayed relatively low phylogenetic noise (<20%, S2 Table) and no recombination signal was detected. The full genome alignment was, as expected, the one with the lowest phylogenetic noise (0.3%), while the NS5 alignment contained the highest number of informative sites, as well as the largest number of available sequences (S2 Table). Therefore, these two data sets were used to investigate further the phylogenetic and phylogeographic patterns of ZIKV. Maximum likelihood (ML) (Fig 3) and Neighbor-joining (NJ) (S1 Fig) trees inferred from full genome sequences consistently show two major ZIKV clades: one including African, the other one including Asian, South American and the Haitian strains. In the ML tree (Fig 3), the earliest lineage in the African clade leads to a Ugandan strain, in agreement with the scenario of ZIKV emergence in the Eastern African country [1]. Moreover, both ML and NJ trees show three highly supported monophyletic clades within the African lineage, indicating a somewhat more complex pattern than a split between West African and Nigerian strains, as recently described [17, 28]. Indeed, one clade includes Nigeria/Senegal sequences; a second one includes only Central Africa strains, while a third one includes two well-supported sub-clades, one with Ugandan and the other with Senegalese strains. South American/Haitian sequences cluster within the Asian clade and clearly branch out from a sequence circulating in Easter Island, which originated in turn from French Polynesia. The Haitian sequence clusters with a Brazilian sequence in a monophyletic clade related, in turn, to sequences from Suriname and the recently isolated strains from Guatemala and Puerto Rico [29] (Fig 3).

 

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Fig 3. Maximum-Likelihood tree of ZIKV complete genome sequences.

The tree was obtained using the best fitting nucleotide substitution model (TN93+G) selected by a hierarchical likelihood ratio test. Branches are drawn to scale in nucleotide substitutions per site according to the bar at the bottom of the tree. Percentage bootstrap (out of 1000 replicates) support values are given along branches. The Haiti sequence is in bold.

 

http://dx.doi.org/10.1371/journal.pntd.0004687.g003

The pattern is confirmed by the Bayesian phylogeographic analysis showing the Asian origin of the South American sequences (Figs 4 and S2), as well as the close phylogenetic relationship between Haitian, Brazilian, Suriname and Puerto Rican strains, clustering within a larger clade of isolates from Easter Island. While not statistically significant, this latter analysis, based on the NS5 region, does show slight separation of Haitian strains and the strains from Brazil, Suriname, Puerto Rico and Guatemala. The molecular clock calibration indeed shows that the most recent common ancestor (MRCA) of the Haitian clade existed at least one year earlier (mid-2013, 95% high posterior density interval December 2012, June 2013) than the other South American lineages with the exception of the Easter Island (Chile) strains, which appear to be the oldest (Fig 4). The MRCA of the Asian lineage dates back to 1956 (95% high posterior density interval 1954–1958), while ZIKV MRCA in Africa circulated, consistently with previous estimates [27], since at least the early 1900s (95% high posterior density interval 1890–1925).

 

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Fig 4. Maximum clade credibility (MCC) tree with Bayesian phylogeography reconstruction of ZIKV NS5 gene region.

Branches are scaled in time and colored according to the legend to the left where each color represents the geographic location of the sampled sequence (tip branches), as well as of the ancestral lineage (internal branches) inferred by Bayesian phylogeography. The molecular clock was calibrated by using ZIKV strains known sampling times and enforcing a relaxed molecular clock with a Bayesian skyline plot demographic prior (see Supplementary Methods). For further clarity, the country of origin of the main strains in the MCC tree is also indicated to the right of each major clade (the MCC tree with full names of each isolate is provided in S2 Fig). Significant posterior probability support (p≥ 0.9) is indicated by the number along the branch. The Haitian sequences are in bold.

 

http://dx.doi.org/10.1371/journal.pntd.0004687.g004

[niman]

Discussion

Our data are consistent with the occurrence of an outbreak of Zita virus infection in rural areas of Haiti west of Port-au-Prince in December of 2014. Virus was isolated from three students, coming from two different schools and different towns, suggesting that the infection was relatively widespread in the community. In keeping with prior descriptions of ZIKV infection [2], illness was mild. Two patients reported subjective fevers prior to presentation at the clinic, but were afebrile on exam (possibly due to use of local herbal antipyretics); the third patient had had a temperature of 39 degrees three weeks before (diagnosed as tonsillitis), but was asymptomatic at the time of blood collection. However, the outbreak was tightly bounded in time, with all cases occurring within a single week; we maintained similar surveillance methods across a 10 month period, and this one week was the only time that ZIKV was isolated. In keeping with reports from French Polynesia, cases occurred at a time when there was co-circulation of CHIKV and DENV, with cases immediately preceded by a cluster of DENV-1 cases (with both DENV-1 and ZIKV isolated from the first patient identified), and followed by DENV-4.

Officially, no cases of ZIKV infection were reported by the Haitian Ministry of Public Health and Population (MSPP) until January 6, 2016, when 5 cases were confirmed in patients in the metropolitan Port-au-Prince area, based on RT-PCR assays performed at the Caribbean Public Health Agency “CARPHA” Laboratory at Trinidad and Tobago. While it is difficult to assemble an accurate timeline for Zika in Haiti, given the close similarity in symptoms with DENV and CHIKV cases and their apparent co-circulation, we would hypothesize that there was an initial “wave” of ZIKV cases in the late fall of 2014 in the Leogane/Gressier region, possibly emanating from near-by Port-au-Prince. Case numbers may have been reduced by relatively low rainfall amounts at that time, with persistence in the population and, in the setting of heavy rains in the fall of 2015, occurrence of a larger epidemic in the fall of 2015/spring of 2016. Alternatively, there may have been a reintroduction of the virus in late 2015; analysis of additional sequence data, from Haiti as well as from other countries, will be necessary to reconstruct the geographic progression of strains.

Our phylogenetic analysis highlights the relative indolence of the global Zika epidemic prior to its introduction into Asia and the south Pacific in 2007. In agreement with previous reports, the virus probably emerged in Africa at the beginning of the 20th century [28], where it diversified in several regional sub epidemics that, according to our analysis, span the entire equatorial Africa from Uganda, to Central Africa to Senegal. ZIKV Asian lineages, on the other hand, are of more recent origin, dating back 50–60 years ago, and the recent epidemic outbreaks in South America are probably the result of a limited introduction from French Polynesia via Easter Island no more than 3–4 year ago. The factors responsible for the rapid spread of the virus, and it’s apparent trophism for neural tissue and ability to cause severe birth defects [10–12], remain to be determined. The close association of ZIKV with the regional CHIKV epidemic, and epidemics of DENV, as we observed in Haiti, raises questions about immunologic interactions among these viruses, and/or the possibility that co-infection facilitates viral transmission or severity. Our observations highlight the critical ongoing need for careful epidemiologic and basic science research to guide public health interventions in Haiti and elsewhere where ZIKV is now epidemic.

Supporting Information

 

S1 Text. Supplemental Material.

doi:10.1371/journal.pntd.0004687.s001

(DOCX)

S1 Fig. Neighbor joining tree of ZIKV complete genome sequences.

The tree was obtained using the best fitting nucleotide substitution model (TN93+G) selected by a hierarchical likelihood ratio test. Branches are drawn to scale in nucleotide substitutions per site according to the bar at the bottom of the tree. Significant posterior probability support (p≥ 0.9) is indicated by the number along the branch. The Haiti sequence is in bold.

doi:10.1371/journal.pntd.0004687.s002

(PDF)

S2 Fig. Maximum clade credibility (MCC) tree with Bayesian phylogeography reconstruction of ZIKV NS5 gene region and tips labeled according to strain names.

Month (when available) and year of isolation are also indicated for each strain.

doi:10.1371/journal.pntd.0004687.s003

(PDF)

S1 Table. Primers for sequencing of ZIKV Haiti/1/2014.

doi:10.1371/journal.pntd.0004687.s004

(DOCX)

S2 Table. ZIKV evolutionary model and phylogenetic signal in different genes.

doi:10.1371/journal.pntd.0004687.s005

(DOCX)

S3 Table. Marginal likelihood estimates and Bayes factors comparing molecular clock and demographic models inferred by Bayesian phylogenetics of ZIKV NS5 gene sequences.

doi:10.1371/journal.pntd.0004687.s006

(DOCX)

Author Contributions

Conceived and designed the experiments: JLe VMBDR BO MS JGM. Performed the experiments: JLe MEB MR JLo. Analyzed the data: JLe JGM MS EC MC. Wrote the paper: JLe VMBDR BO MS JGM MEB MR JLo TT SC GA EC MC. Clinical patient management and data collection: TT SC GA. Had full access to all data and had final responsibility for the decision to submit for publication: JGM.

[niman]

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[niman]

 

NEWS

SCIENCE & WELLNESS

Zika present in Americas longer than previously thought

APRIL 26, 2016

EVAN BARTON

 

• EPI

The Zika virus was present in Haiti several months before the first Zika cases were identified in Brazil, according to new research by infectious-disease specialists at the University of Florida.

This finding confirms that the Zika virus was present in the Americas prior to March 2015, when the virus was first identified in Brazil, and suggests that the spread of Zika virus in the Americas was likely more complicated than early theories presumed.

“We know that the virus was present in Haiti in December of 2014,” said Dr. Glenn Morris, M.D., M.P.H., a professor of medicine and the director of UF’s Emerging Pathogens Institute. “And, based on molecular studies, it may have been present in Haiti even before that date.”

Although the findings suggest that the Zika virus was circulating in the Americas prior to 2015, what remains unclear is exactly what confluence of factors caused the virus to take off in Brazil.

The findings were published Monday in PLOS Neglected Tropical Diseases. Researchers hope further inquiry will shed light on the factors that led to the proliferation of Zika virus in Brazil as well as the sharp rise in the number of birth defects in that nation in cases where pregnant women were infected with the then-uncommon flavivirus.

Scientists from UF’s environmental and global health department and the Emerging Pathogens Institute isolated the Zika virus from three patients while studying the transmission of dengue and chikungunya in Haiti in 2014. School children exhibiting febrile illness within the Gressier/Leogane region of Haiti were taken to a free outpatient clinic, where blood samples were drawn and screened for dengue, chikungunya and malaria.

Upon isolation, the viruses were first considered “mystery” viruses, as PCR-based tests indicated they were neither dengue nor chikungunya viruses, and little attention had been paid to the possibility that Zika virus might be present in the Caribbean. Using a sophisticated RT-PCR based method that potentially amplifies any RNA, the researchers produced PCR amplicons that were subsequently sequenced and identified as Zika virus sequences. The plasma samples that yielded Zika virus were taken three months before March 2015, when Brazilian scientists first confirmed via genetic analysis that Zika virus was present in Brazil and causing a significant disease burden in the South American nation.

The Zika virus was virtually unknown outside of public health circles prior to the 2007 outbreak in the Yap Islands, a small group of islands in Micronesia where an estimated 73 percent of residents 3 years of age and older were infected with the virus. Questions still remain regarding how it came to the Americas.

“The Brazilian and Haitian strains are genetically similar,” said John Lednicky, Ph.D., an associate professor in the UF College of Public Health and Health Professions’ department of environmental and global health and an associate researcher at the Emerging Pathogens Institute.

Lednicky designed the project’s virus isolation work and identified and sequenced the Haitian Zika virus isolates. Lednicky said the genetic sequences of the Haitian isolates from 2014 are more similar to those of the French Polynesian strains than to many of the Brazilian Zika virus strains. Lednicky thinks this may be because the Haitian 2014 strain is slightly older than the isolates from Brazil in 2015.

Morris echoed Lednicky’s suggestion that Zika virus had been in the Americas for a period of time before it began causing a noticeable level of illness.

“There is a possibility that this virus had been moving around the Caribbean before it hit the right combination of conditions in Brazil and took off,” Morris said. “By using the sophisticated culturing and sequencing capabilities that we have here at the Emerging Pathogens Institute, we were able to begin to fill in some of the unknown areas in the history of the Zika virus, leading us toward a better understanding of what caused this outbreak to suddenly occur at the magnitude that it did in Brazil.”

http://news.ufl.edu/articles/2016/04/zika-present-in-americas-longer-than-previously-thought.php

 

[niman]

Colombia Confirms More Birth Defects Linked to Zika

By EZRA KAPLAN and DONALD G. McNEIL Jr.APRIL 26, 2016

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BOGOTÁ, Colombia — Colombian health officials said on Tuesday that they had confirmed two more cases of babies born with brain damage to mothers who had Zika during pregnancy, but said overall cases of the mosquito-borne virus were decreasing in the country.

“We are absolutely certain that Zika is on the decrease in Colombia,” said Fernando Ruiz, Colombia’s vice minister of Health, during a briefing here.

At the briefing, officials said they had now confirmed a total of four cases ofmicrocephaly, a condition in which babies are born with unusally small heads and damaged brains, and that they expect to see many more over the next three months as women who contracted Zika during an outbreak this year begin to give birth. They said they were studying 22 more newborns with microcephaly to determine if Zika was the cause. They also announced a total of 304 cases of Guillain-Barré syndrome — a condition that can causeparalysis, usually temporary — that are suspected to have been caused by Zika

Separately this week, a new study published in the journal PLoS Neglected Tropical Diseases reported that the Zika virus has been circulating in Haiti at least since 2014, and possibly since 2013, well before an outbreak in Brazil raised the alarm of public health officials around the world.

Researchers at the University of Florida found Zika gene sequences in the blood of three children from different rural towns who had visited clinics in the Gressier-Léogâne region of Haiti in December 2015. Comparisons with Zika DNA from elsewhere suggested the virus could have been in Haiti for months.

The Zika virus was confirmed in Brazil only in May 2015, but the new finding does not mean that it arrived in Haiti first. Instead, the authors said, the virus probably went from French Polynesia to Easter Island, then around the Americas, circulating for far longer than has been realized.

In recent weeks, according to various state health departments, more than a dozen Americans in five states have returned from missionary work in Haiti with confirmed Zika infections.

http://www.nytimes.com/2016/04/27/health/zika-virus-haiti.html?_r=0

[niman]

UF study: Zika in Haiti before ID'd in Brazil

 

 

 

Staff report

Published: Tuesday, April 26, 2016 at 4:37 p.m.

Last Modified: Tuesday, April 26, 2016 at 4:37 p.m.

The Zika virus was present in Haiti several months before the first Zika cases were identified in Brazil, new research by infectious-disease specialists at the University of Florida shows.

The finding confirms that the virus was present in the Americas before March 2015, when the virus was first identified in Brazil, and suggests that the spread of Zika in the Americas was likely more complicated than early theories believed.

Although the findings suggest that the Zika virus was circulating in the Americas before 2015, it remains unclear exactly what caused the virus to take off in Brazil.

The findings were published Monday in Public Library of Science's Neglected Tropical Diseases.

Researchers hope to shed light on the factors that led to the spread of Zika in Brazil as well as the sharp rise in the number of birth defects in that nation in cases where pregnant women were infected with the then-uncommon virus.

Zika illness is generally mild with a rash, fever and joint pain. It is spread primarily through the bite of an infected mosquito, according to the Centers for Disease Control. Zika infection during pregnancy can cause a serious birth defect called microcephaly.

http://www.gainesville.com/article/20160426/ARTICLES/160429757

 

[niman]

Zika presence in Americas older than previously thought

 

 

Indo Asian News Service

IANS India Private Limited

April 27, 2016

New York, April 27 (IANS) The Zika virus was present in Haiti several months before the first Zika cases were identified in Brazil in March last year, new research has found.

"We know that the virus was present in Haiti in December of 2014," said one of the researchers Glenn Morris, professor of medicine at the University of Florida in the US.

"And, based on molecular studies, it may have been present in Haiti even before that date," Morris said.

Although the findings suggest that the Zika virus was circulating in the Americas prior to 2015, what remains unclear is exactly what confluence of factors caused the virus to take off in Brazil that has confirmed 91,387 cases of Zika so far this year, including 7,584 pregnant women at risk of having babies with birth defects.

The findings were published in the journal PLOS Neglected Tropical Diseases.

The Zika virus was virtually unknown outside of public health circles prior to the 2007 outbreak in the Yap Islands, a small group of islands in Micronesia where an estimated 73 percent of residents three years of age and older were infected with the virus.

Researchers hope further inquiry would shed light on the factors that led to the proliferation of Zika virus in Brazil as well as the sharp rise in the number of birth defects in that nation in cases where pregnant women were infected with the then-uncommon flavivirus, the genus of viruses that include Zika.

Scientists from University of Florida isolated the Zika virus from three patients while studying the transmission of dengue and chikungunya in Haiti in 2014.

School children exhibiting febrile illness within the Gressier/Leogane region of Haiti were taken to a free outpatient clinic, where blood samples were drawn and screened for dengue, chikungunya and malaria.

Upon isolation, the viruses were first considered "mystery" viruses, as tests indicated they were neither dengue nor chikungunya viruses, and little attention had been paid to the possibility that Zika virus might be present in the Caribbean.

Using a sophisticated method, the researchers subsequently sequenced and identified them as Zika virus sequences.

The plasma samples that yielded Zika virus were taken three months before March 2015, when Brazilian scientists first confirmed via genetic analysis that Zika virus was present in Brazil and causing a significant disease burden in the South American nation.

--IANS

https://in.news.yahoo.com/zika-presence-americas-older-previously-thought-110005149.html?utm_source=dlvr.it&utm_medium=twitter

 

gb/dg

[niman]

HEALTH

 

ZIKA VIRUS OUTBREAK

 

APR 27 2016, 5:59 PM ET

Zika Virus Was In Haiti Before It Was in Brazil. Study Finds

by MAGGIE FOX

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The Zika virus was circulating in Haiti in 2014, long before it became obvious that it was spreading fast in Brazil, a new study finds.

A team that went back and checked out three mysterious infections in Haiti found they were caused by the Zika virus. Their study raises questions about when and how Zika actually arrived in the Americas.

 

A military truck carries out fumigation in a neighborhood to stop the breeding of the dengue mosquito in Havana March 1, 2016. ENRIQUE DE LA OSA / Reuters

"We know that the virus was present in Haiti in December of 2014," said Dr. Glenn Morris, a professor of medicine and the director of the University of Florida's Emerging Pathogens Institute. "And, based on molecular studies, it may have been present in Haiti even before that date."

Zika's sweeping across the Americas and the Caribbean, as well as parts of the South Pacific. Once virtually ignored as a harmless and unremarkable virus, it is now alarming doctors and health officials because it's causing devastating birth defects and paralyzing neurological conditions.

Brazil sounded the alert last year and at first scientists thought it had arrived there in 2014.

Related: Zika Mysteries Stump Even the Experts

But earlier this year, a large international team of experts used a "genetic clock" to show Zika virus has changed about as much as would be expected if it had been carried into the country in 2013. And it very closely matches a strain that circulated in French Polynesia in 2013.

What's not clear is why it's now being seen to cause disease. Tests show it has mutated a bit, as all viruses do, but it's not yet clear if the mutations somehow make it more virulent. It's possible that because it's hit a very large population with no previous immunity to Zika, what would have been events so rare as to go unnoticed are now getting attention.

"WE KNOW THAT THE VIRUS WAS PRESENT IN HAITI IN DECEMBER OF 2014."

Zika is a close relative of dengue and chikungunya viruses, which have been spreading for longer. And it is spread by the same Aedes aegypti mosquito. But Zika is usually milder than dengue and even than chikungunya, not even causing noticeable symptoms in most people and usually causing little more than a rash, fever and body aches.

Blood tests often confuse the three viruses.

Writing in the Public Library of Sciences journal PLOS Neglected Tropical Diseases, the team said they went back and tested blood samples taken when chikungunya was sweeping across Haiti.

"Zika virus was identified in plasma from three students seen in the Christianville Foundation Schools clinic," the team wrote. They included a 15-year old boy, a 7-year-old girl and a 4-year-old boy.

None was very sick at all. None had the rash that so clearly characterizes Zika.

"In none of the cases would it have been possible to have identified the illness as a Zika virus infection based on clinical presentation, rather than dengue virus or chikungunya virus," they wrote. So at least some of the cases diagnosed as chikungunya may have been Zika instead.

"THERE IS A POSSIBILITY THAT THIS VIRUS HAD BEEN MOVING AROUND THE CARIBBEAN BEFORE IT HIT THE RIGHT COMBINATION OF CONDITIONS IN BRAZIL AND TOOK OFF."

Because the children came from different schools and different towns, this suggests Zika was already fairly widespread in the area.

"Officially, no cases of Zika virus infection were reported by the Haitian Ministry of Public Health and Population until January 6, 2016, when five cases were confirmed in patients in the metropolitan Port-au-Prince area," the researchers wrote.

Related: New Map Finds 2 Billion at Risk of Zika

But no one was really looking for Zika in the Caribbean until after the U.S. Centers for Disease Control and Prevention issued a travel alert in January of this year.

"There is a possibility that this virus had been moving around the Caribbean before it hit the right combination of conditions in Brazil and took off," Morris said.

The Aedes aegypti and Aedes albopictus mosquitoes that spread zika and chikungunya are found across the southern United States and as far north as New York.

The Pan American Health Organization says Mexican researchers have found Zika in A. albopictus. If this becomes common, it suggests even wider spread of Zika than predicted, because these cousins of Aedes aegypti live farther north and circulate for longer.

Haiti has almost the perfect conditions for the spread of mosquito-borne illnesses such as Zika. It's got a warm climate, people are poor and live in homes without screens or air conditioning, and there are plenty of places for mosquitoes to breed.

Experts expect much less spread in the U.S., where most people live behind closed doors, with screens and air conditioning

http://www.nbcnews.com/storyline/zika-virus-outbreak/zika-virus-was-haiti-it-was-brazil-study-finds-n563706