PREGNANT AWARENESS ABOUT ZIKA VIRUS INFECTION DURING PREGNANCY

To the Editor: Zika virus belongs to the genus Flavivirus, family Flaviviridae, and is transmitted by Aedes spp. mosquitoes. Clinical signs and symptoms of human infection include fever, headache, malaise, maculopapular rash, and conjunctivitis. Zika virus was first isolated in 1947 from the blood of a febrile sentinel rhesus monkey during a study of yellow fever in the Zika Forest of Uganda (1). During the next 20 years, Zika virus isolates were obtained primarily from East and West Africa during arbovirus surveillance studies in the absence of epidemics. During those 20 years, cases of Zika virus infection were detected sporadically; however, given the clinical similarity of Zika virus and dengue virus infections and the extensive cross-reactivity of Zika virus antibodies with dengue viruses, it is possible that Zika virus was associated with epidemics that were incorrectly attributed to dengue viruses. Beginning in 2007, substantial Zika virus outbreaks were reported first in Yap Island (Federated States of Micronesia), then in French Polynesia, and then in other Pacific Islands (2–4). Genetic studies have revealed that Zika virus has evolved into 3 distinct genotypes: West African (Nigerian cluster), East African (MR766 prototype cluster), and Asian. It has been postulated that the virus originated in East Africa and then spread into both West Africa and Asia ≈50–100 years ago (5). In early 2015, cases of Zika virus infection were detected in Rio Grande State, northern Brazil, and limited sequence analyses revealed that the virus was most closely related to a 2013 isolate from French Polynesia, within the Asian clade (6). In December 2015, the Centers for Disease Control and Prevention Arbovirus Diagnostic Laboratory detected Zika virus in serum specimens collected from persons in Guatemala and Puerto Rico. The complete nucleotide sequence of the virus was derived directly from 3 of these serum specimens by using next-generation sequencing on the Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) platform. The raw sequence reads were analyzed and assembled by using the CLC bio Genomics Workbench (CLC bio, Waltham, MA, USA) and Lasergene NextGen (DNAStar, Madison, WI, USA). The complete genome sequences were aligned by using ClustalW (http://www.megasoftware.net/) with all available full-length Zika virus sequences from GenBank representing the 3 genotypes. Nearly identical phylogenetic trees were generated by using several methods (minimum-evolution, maximum-likelihood, neighbor-joining), and a neighbor-joining tree was generated and analyzed with 1,000 replicates for bootstrap testing (Figure). GenBank accession numbers for ZIKV sequences presented in this article are {type:entrez-nucleotide,attrs:{text:KU501215,term_id:984874581,term_text:KU501215}}KU501215 (Puerto Rico PRVABC59), {type:entrez-nucleotide,attrs:{text:KU501216,term_id:984874583,term_text:KU501216}}KU501216 (Guatemala 8375), and {type:entrez-nucleotide,attrs:{text:KU501217,term_id:984874585,term_text:KU501217}}KU501217 (Guatemala 103344). Figure Phylogenetic tree of Zika virus isolates identified from Guatemala and Puerto Rico in December 2015 (indicated in boldface) compared with reference isolates obtained from GenBank. The isolates from Guatemala and Puerto Rico grouped with other Asian genotype ... In agreement with the initial sequencing of samples from Brazil conducted by Zanluca et al. (6), the 3 newly sequenced Zika viruses from Guatemala and Puerto Rico are all within the Asian genotype and most closely related to strains recently isolated from Brazil (2015) and French Polynesia (2013). The tree topology confirms previous findings and indicates that Asian genotype viruses have been gradually evolving and spreading geographically throughout Asia and the Pacific Islands since at least 1966; the tree suggests that the Malaysia 1966 isolate is representative of an ancestral genotype (7). The percent nucleotide identity among all the Western Hemisphere Zika viruses is >99%, and as a group, these Western Hemisphere viruses are ≈89% identical (96% aa) to viruses of the East African and West African genotypes. As reported by Musso et al. (8), the phylogeny and movement of Zika and chikungunya viruses are strikingly similar. Each virus is grouped into 3 genotypes of very similar geographic distribution: East Africa, West Africa, and Asia. For both viruses, it also seems that viruses from East Africa moved into Asia ≈50–100 years ago and evolved into a unique Asian genotype (9,10). In addition, the similarity with respect to the recent movement of these viruses from Asia into the Pacific Islands and then into the New World (9) is noteworthy. It seems that similar ecologic and/or human social factors might be responsible for the movement of chikungunya and ZIKV viruses into the New World at approximately the same time. Further studies might elucidate the exact mechanism of this transcontinental movement, leading to effective prevention strategies.

Zika virus in the female genital tract

The earliest members of genus Homo were surely bedeviled by blood-feeding arthropods, some of which doubtless carried zoonotic pathogens. However, the phenomenon of vectorborne human epidemic disease began only after humans began building settlements 15,000 years ago (1). Settlements offered pathogens not only host density but also opportunities for their vertebrate reservoirs and arthropod vectors to cohabit with us. Epidemic Yersinia pestis (the Medieval Black Death) was only possible because black rats (Rattus rattus), the host of the vector flea, had become extraordinarily successful at living off human garbage and nesting in our buildings.

Decision making in the face of uncertainty: the challenge of emerging infectious diseases.

Zika virus (ZIKV) and Dengue virus (DENV) are mosquito-borne viruses of the Flavivirus genus that could cause congenital microcephaly and hemorrhage, respectively, in humans, and thus present a risk to global public health. A preventive vaccine against ZIKV remains unavailable, and no specific antiviral drugs against ZIKV and DENV are licensed. Medicinal plants may be a source of natural antiviral drugs which mostly target viral entry. In this study, we evaluate the antiviral activity of Doratoxylum apetalum, an indigenous medicinal plant from the Mascarene Islands, against ZIKV and DENV infection. Our data indicated that D. apetalum exhibited potent antiviral activity against a contemporary epidemic strain of ZIKV and clinical isolates of four DENV serotypes at non-cytotoxic concentrations in human cells. Time-of-drug-addition assays revealed that D. apetalum extract acts on ZIKV entry by preventing the internalisation of virus particles into the host cells. Our data suggest that D. apetalum-mediated ZIKV inhibition relates to virus particle inactivation. We suggest that D. apetalum could be a promising natural source for the development of potential antivirals against medically important flaviviruses.

Estimating the numbers of pregnant women infected with Zika virus and infants with congenital microcephaly in Colombia, 2015–2017

To the Editor: Zika virus (ZIKV), a mosquito-borne flavivirus, causes Zika fever, a self-limiting febrile and exanthematic arthralgia syndrome closely resembling dengue fever. Most often, signs and symptoms are maculopapular rash, fever, arthralgia, myalgia, headache, and conjunctivitis; edema, sore throat, cough, and vomiting occur less frequently (1). The virus, which was initially isolated from a rhesus monkey (Macaca mulatta) in 1947 in Uganda, has come to attention recently after a large outbreak occurred in the western Pacific region, including French Polynesia, New Caledonia, Easter Island, and the Cook Islands (2). Travel-related imported infections have thus been increasingly reported from the western Pacific and sporadically also in travelers to other regions of the world, including Thailand, Indonesia, and Senegal (2,3). ZIKV is transmitted by different Aedes mosquito species, and nonhuman primates play a role as reservoirs (1). After the beginning of the ZIKV epidemic in late 2013, a 20-fold increase of Guillain-Barre syndrome incidence was noted in French Polynesia; 1 patient was infected a week before neurologic symptoms started (4). We report an acute ZIKV infection in a traveler returning from Malaysian Borneo who experienced bilateral hearing difficulties during the course of illness. On September 1, 2014, a 45-year-old woman was seen in an outpatient clinic in Heidelberg, Germany for fever of up to 39°C and maculopapular rash covering her trunk, arms, and legs. Fever had started on August 30, which was 6 days after she had returned from a 3-week vacation to peninsular Malaysia and Sabah, Malaysian Borneo. Laboratory analyses showed a slightly elevated C-reactive protein level of 5.2 mg/L (reference range <5.0), but liver function test and complete blood count results were within reference range. During the next 3 days, the fever subsided, but the patient experienced a sore throat, bilateral conjunctivitis, and a burning sensation of the palms and soles. These symptoms were accompanied by swelling of the hands and increasing arthralgia of the wrists, palms, and fingers. There was no lymphadenopathy. An indirect immunofluorescence assay for ZIKV (3) demonstrated an IgM titer of 1:640 and an IgG titer of 1:320 (cutoff <1:20) on day 6 of illness (Figure). An indirect immunofluorescence assay for dengue virus demonstrated an IgG titer of 1:80 and no IgM (cutoff <1:20). Figure Clinical course and laboratory results (reverse transcription-PCR [RT-PCR]) for a patient with Zika virus (ZIKV) infection acquired from Malaysian Borneo. Two days later, the patient experienced sudden bilateral dull and metallic hearing; in her left ear, she experienced a very short delay between a sound and her perception of the sound. Follow-up ZIKV serologic testing on day 11 of illness showed a decreased IgM titer of 1:160 and an increased IgG titer of 1:2,560 (Figure). Viral neutralization testing (3) of the same sample demonstrated the presence of ZIKV-specific neutralizing antibodies. Chikungunya virus serology results were negative. An archived serum sample from day 3 of illness studied by ZIKV serology and a ZIKV-specific real-time reverse transcription PCR (3) was negative (Figure). Hearing difficulties lasted for 10 days and resolved gradually (Figure). During her journey to several cities and villages in Sabah, Malaysian Borneo, the patient had noticed several mosquito bites even though she had used repellents. She had stayed in hotels, private homes, and remote church homes under various conditions (Technical Appendix). In Asia, Zika fever has been described sporadically in Cambodia, Thailand, and Indonesia (Java and Lombok) (1,3,5,6). On the basis of the incubation time of ≈6 days in returning travelers (2,3), we assumed that the patient became infected in Keningau or surrounding villages, in northern Borneo. Although ZIKV was detected in Ae. aegypti mosquitoes in peninsular Malaysia in 1969 (7) and antibodies against ZIKV were demonstrated in serum samples from 15 of 79 patients on peninsular Malaysia and 9 of 50 patients in Borneo in 1953 (8), Zika fever in peninsular Malaysia or Borneo has not been reported. In 2001, ZIKV seropositivity was demonstrated in a native Bornean, 2 migrants to Borneo, and 2 Bornean orangutans (Pongo pygmaeus) (9). A later study found an additional 8 Bornean orangutans to be seropositive for antibodies against ZIKV (10). Thus, in Borneo, either the virus only rarely infects humans or the disease is mistaken for dengue fever. Neurologic complications of ZIKV infections had previously been reported only as Guillain-Barre syndrome, and hearing difficulties in Zika fever patients have not been reported. Because this symptom resolved spontaneously, no audiometry or auditory brainstem response testing was performed, and the cause of the disorder remains unclear. Because of increasing travel and migration and heightened clinical and laboratory awareness, more ZIKV infections are likely to be diagnosed outside of epidemic events. Technical Appendix: Travel itinerary of a patient with Zika fever. Click here to view.(217K, pdf)

Virology and Virality: Confronting Zika in and Beyond the Emergency Department

Orthoflaviviruses are positive-sense single-stranded RNA viruses that hijack host proteins to promote their own replication. Zika virus (ZIKV) is infamous among orthoflaviviruses for its association with severe congenital birth defects, notably microcephaly. We previously mapped ZIKV-host protein interactions and identified the interaction between ZIKV non-structural protein 4A (NS4A) and host microcephaly protein ankyrin repeat and LEM domain-containing 2 (ANKLE2). Using a fruit fly model, we showed that NS4A induced microcephaly in an ANKLE2-dependent manner. Here, we explore the role of ANKLE2 in ZIKV replication to understand the biological significance of the interaction from a viral perspective. We observe that ANKLE2 localization is drastically shifted to sites of NS4A accumulation during infection and that knockout of ANKLE2 reduces ZIKV replication in multiple human cell lines. This decrease in virus replication is coupled with a moderate increase in innate immune activation. Using microscopy, we observe dysregulated formation of virus-induced endoplasmic reticulum rearrangements in ANKLE2 knockout cells. Knockdown of the ANKLE2 ortholog in Aedes aegypti cells also decreases virus replication, suggesting ANKLE2 is a beneficial replication factor across hosts. Finally, we show that NS4A from four other orthoflaviviruses physically interacts with ANKLE2 and is also beneficial to their replication. Thus, ANKLE2 likely promotes orthoflavivirus replication by regulating membrane rearrangements that serve to accelerate viral genome replication and protect viral dsRNA from immune detection. Taken together with our previous results, our findings indicate that ZIKV and other orthoflaviviruses hijack ANKLE2 for a conserved role in replication, and this drives unique pathogenesis for ZIKV since ANKLE2 has essential roles in developing tissues.IMPORTANCEZIKV is a major concern due to its association with birth defects, including microcephaly. We previously identified a physical interaction between ZIKV NS4A and host microcephaly protein ANKLE2. Mutations in ANKLE2 cause congenital microcephaly, and NS4A induces microcephaly in an ANKLE2-dependent manner. Here, we establish the role of ANKLE2 in ZIKV replication. Depletion of ANKLE2 from cells significantly reduces ZIKV replication and disrupts virus-induced membrane rearrangements. ANKLE2's ability to promote ZIKV replication is conserved in mosquito cells and for other related mosquito-borne orthoflaviviruses. Our data point to an overall model in which ANKLE2 regulates virus-induced membrane rearrangements to accelerate orthoflavivirus replication and avoid immune detection. However, ANKLE2's unique role in ZIKV NS4A-induced microcephaly is a consequence of ZIKV infection of important developing tissues in which ANKLE2 has essential roles.

Zika virus: Growing guilt from association☆

Microcephaly in Brazil: confidence builds in Zika connection

Brain Trust

Promoting collaborations to improve birth defects surveillance, research, and prevention: A joint editorial from the National Birth Defects Prevention Network and the Organization of Teratology Information Specialists.

Robust epidemiological and biological evidence supports a causal link between prenatal Zika Virus (ZIKV) infection and congenital brain abnormalities including microcephaly. However, it remains uncertain if ZIKV infection in pregnancy also increases the risk for other adverse fetal and birth outcomes. In a prospective cohort study we investigated the influence of ZIKV on the prevalence of prematurity, low birth weight, small-for-gestational-age, and fetal death as well as microcephaly (i.e., overall and disproportionate) in the offspring of women attending a high-risk pregnancy clinic during the recent ZIKV outbreak in Brazil. During the recruitment period (01 March 2016–23 August 2017), urine samples were tested for ZIKV by RT-PCR from all women attending the high-risk pregnancy clinic at Jundiaí University Hospital and from the neonates after delivery. Of the 574 women evaluated, 44 (7.7%) were ZIKV RT-PCR positive during pregnancy. Of the 409 neonates tested, 19 (4.6%) were ZIKV RT-PCR positive in the first 10 days of life. In this cohort, maternal ZIKV exposure was not associated with increased risks of prematurity, low birth weight, small-for-gestational-age, or fetal death. However, relative to ZIKV-negative neonates, ZIKV-positive infants had a five-fold increased risk of microcephaly overall (RR 5.1, 95% CI 1.2–22.5) and a ten-fold increased risk of disproportionate microcephaly (RR 10.3, 95% CI 2.0–52.6). Our findings provide new evidence that, in a high-risk pregnancy cohort, ZIKV RT-PCR positivity in the neonate at birth is strongly associated with microcephaly. However, ZIKV infection during pregnancy does not appear to influence the risks of prematurity, low birth weight, small-for-gestational-age or fetal death in women who already have gestational comorbidities. The results suggest disproportion between neonatal head circumference and weight may be a useful screening indicator for the detection of congenital microcephaly associated with ZIKV infection.

Zika virus and the nonmicrocephalic fetus: why we should still worry