A Mouse Model of Zika Virus Pathogenesis.

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. Two of the most important malaria vectors in the world exploit human activity to proliferate. Immature forms of Anopheles gambiae mosquitoes in Africa and An. dirus mosquitoes in Southeast Asia thrive in the small puddles (water-filled footprints, tire ruts, borrow pits, and drainage gullies) created around villages. Best adapted of all are Aedes aegypti mosquitoes, the cosmopolitan vector of epidemic yellow fever, dengue, chikungunya, and Zika viruses. Their ecologic niche is nearly ours. These mosquitoes lay eggs in artifacts: water storage jars, roof gutters, flower pots, dog dishes, even upturned bottle caps. Their cognate species, Ae. albopictus, is only slightly less versatile, having an attraction to discarded tires. Evolution of blood-feeding arthropods to our changing environment and evolution of some zoonoses to exploit this advantage are major links in the emergence of obscure pathogens into epidemic threats and is a timely subject for this issue of Emerging Infectious Diseases. Persistence of human yellow fever, the seeming inexorable expansion of dengue, and the surprising, explosive spread and severity of first chikungunya virus and now Zika virus bear testament to the threat posed by habituated Aedes species. Since its arrival in the Western Hemisphere ≈1 year ago, Zika virus, which had previously been associated with a clinically mild and inconsequential illness, is now increasingly suspected of being the cause of an alarming epidemic of neurologic birth defects and Guillain-Barre syndrome in tropical regions. Zika virus, the subject of several articles in this issue, reminds us of some of the impediments to responding to emerging vectorborne pathogens. First, Zika virus belongs to the most prevalent class of emerging pathogens, the zoonotic single-stranded RNA viruses, which have mutation rates as high as 1 base/104 bases each replication. The chikungunya pandemic that began 10 years ago was fueled in part by a single, nonsynonymous base change that enabled that alphavirus to replicate more efficiently in Ae. albopictus mosquitoes (2). Second, conditions enabling transition from vectorborne animal-to-animal transmission to arthropod-mediated human-to-human transmission are poorly understood. Like dengue virus, another flavivirus, Zika virus was likely originally a pathogen of subhuman primates. Between its discovery in a sentinel macaque in Uganda in 1947 and the first recorded epidemic 60 years later in Yap, Federated States of Micronesia, only 14 human cases had been reported, all from Africa and Asia (3). Third, the pathogenicity and transmission dynamics of vectorborne zoonotic pathogens are much more complex than those of directly communicable pathogens. It is not yet known if Zika virus will find sustaining, nonhuman hosts in the Western Hemisphere, as has yellow fever virus, or how wide the range of vector species will be. Pathogenicity and transmission dynamics will be factors in determining where Zika virus will become endemic and what will be the most suitable methods of control. Fourth, accurate diagnosis is key to surveillance and response. It might seem as if Zika virus sprang from nowhere, but almost certainly it must have been infecting many more humans in Africa and Asia than we had been aware. Our ability to serologically diagnose infections with emerging arboviruses is often compromised by close antigenic relationships within virus families. Zika, dengue, West Nile, and yellow fever viruses can co-circulate, not only among themselves, but possibly with unidentified or poorly characterized flaviviruses. The limitations of current diagnostics are a primary reason why the association between Zika virus and birth defects remained speculative so long. Fifth, vector control is a force multiplier that can reduce the risk from many viruses that would require the development of individual vaccines. However, insecticide resistance and application problems greatly impede effective implementation. The best defense is preventing a problem from growing into a threat. Fewer than 20 of the 86 known pathogenic arboviruses can be considered major causes of human disease, and 3 of these, West Nile, chikungunya, and Zika viruses, have emerged from relative obscurity within only the past 20 years (4). At least another 200 cataloged arboviruses whose relationship to human disease is unknown have been isolated from arthropods or animals. The discovery of 3 highly pathogenic mosquitoborne viruses in China and the United States during the past 5 years (5–7) underscores how unrepresentative even that large number might be. It is unrealistic to characterize each of these viruses. Besides needing better methods of vector control, we need a strategy for preemptively identifying arboviruses with the potential for emergence and to devote resources to better understand their transmission dynamics, their endemicity, and accurate diagnosis.

BackgroundAnimal models are critical to understand disease and to develop countermeasures for the ongoing epidemic of Zika virus (ZIKV). Here we report that immunocompetent guinea pigs are susceptible to infection by a contemporary American strain of ZIKV.MethodsDunkin-Hartley guinea pigs were inoculated with 106 plaque-forming units of ZIKV via subcutaneous route and clinical signs were observed. Viremia, viral load in the tissues, anti-ZIKV neutralizing antibody titer, and protein levels of multiple cytokine and chemokines were analyzed using qRT-PCR, plaque assay, plaque reduction neutralization test (PRNT) and multiplex immunoassay.ResultsUpon subcutaneous inoculation with PRVABC59 strain of ZIKV, guinea pigs demonstrated clinical signs of infection characterized by fever, lethargy, hunched back, ruffled fur, and decrease in mobility. ZIKV was detected in the whole blood and serum using qRT-PCR and plaque assay. Anti-ZIKV neutralizing antibody was detected in the infected animals using PRNT. ZIKV infection resulted in a dramatic increase in protein levels of multiple cytokines, chemokines and growth factors in the serum. ZIKV replication was observed in spleen and brain, with the highest viral load in the brain. This data demonstrate that after subcutaneous inoculation, the contemporary ZIKV strain is neurotropic in guinea pigs.ConclusionThe guinea pig model described here recapitulates various clinical features and viral kinetics observed in ZIKV-infected patients, and therefore may serve as a model to study ZIKV pathogenesis, including pregnancy outcomes and for evaluation of vaccines and therapeutics.

BackgroundAdverse fetal outcomes and infant birth defects may develop following Zika virus (ZIKV) infection during pregnancy, especially if this occurs in the first trimester. The aim of this study was to assess the relationship between plasma ZIKV load at the time of acute symptoms and (1) the rate and severity of birth defects in neonates born to mothers who had presented with ZIKV infection during pregnancy, and (2) clinical severity of maternal ZIKV infection.MethodsWithin a cohort of pregnant women living in the French territories in the Americas and exposed to ZIKV during the 2016 outbreak, we analyzed the data of women who developed a symptomatic infection confirmed by a positive plasma ZIKV RT–PCR, using the RealStar Zika virus RT–PCR Kit (Altona Diagnostics, Hamburg, Germany). Plasma ZIKV load quantification was based on the number of cycle times (CT) at which ZIKV RNA was detected (lower CTs indicating a higher viral load). Variables indicating clinical severity of infection included the number of symptoms experienced and the severity of rash. Birth defects possibly linked to ZIKV infection were defined as microcephaly, brain imaging abnormalities, and central nervous system dysfunction. Multivariable logistic regression was used to examine whether potentially ZIKV-related abnormalities were linked to changes in CT, and multivariable linear regression was used to identify clinical correlates with CT value.ResultsOf the 277 live-born neonates who were born to mothers who met the selection criteria, 15 (5.4%) had abnormalities possibly linked to ZIKV infection. The median (IQR) ZIKV RT–PCR CT values were similar, with 31.4 (29.3–33.2) and 31.8 (30.0–33.0), in women delivering normal neonates and those delivering neonates with defects, respectively (OR: 1.04, P = 0.685). Plasma ZIKV load was lower with every day since first symptom onset, and higher with each additional symptom experienced, as indicated by changes in CT of 0.3 (95% CI: 0.2 ‒ 0.5, P < .001) and −0.3 (95% CI: −0.5 ‒ −0.1, P = 0.002) for each unit, respectively.ConclusionNo relationship was observed between plasma ZIKV load and abnormal pregnancy outcomes but higher plasma ZIKV load was associated with a more recent and severe maternal ZIKV infection.DisclosuresAll authors: No reported disclosures.

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5'-SLA and 5'-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

Until recently, Zika virus (ZIKV) was an obscure virus that rarely caused infections and was unknown to most. In 2015 and 2016, ZIKV came into the public spotlight as Brazil and other countries began to report large increases in infections with ZIKV and reported potential complications with developing fetuses and neurologic manifestations. In 2016, the state of Florida identified and responded to an outbreak of locally acquired ZIKV infections in Miami-Dade County. This dramatic increase in infections demonstrated both its importance as an emerging infectious disease and the paucity of knowledge surrounding ZIKV. This study seeks to utilize the data collected during the ZIKV pandemic to further characterize the virus and examine the efficacy of current diagnostic algorithms. First, a systematic review was conducted to pool data from the literature on existing cases of ZIKV infections. Markov chain Monte Carlo modeling was used to determine a median incubation time of 6.5 days for infections with ZIKV. Median time to viral RNA clearance varied significantly by specimen type. Vaginal specimens demonstrated the shortest time to viral RNA clearance (9.9 days); whereas blood specimens exhibited the longest (49.2 days). Second, specimens from 934 symptomatic, non-congenitally acquired cases of ZIKV infection were analyzed to identify factors that contribute to the progression of viral load, as represented by the detection of ZIKV RNA. ZIKV RNA was detected most often in urine specimens and also was found to have higher viral loads than serum and whole blood specimens. Viral load was observed to be lower in non-pregnant women than pregnant women. Last, an evaluation of the Centers for Disease Control and Prevention’s (CDC) 2017 and 2019 ZIKV testing algorithms was conducted using data from all confirmed and probable cases identified in Florida between 2016 and 2018 (n = 1,522). ZIKV RNA was detected most frequently in urine specimens. When testing required plaque reduction neutralization test (PRNT) to discern between ZIKV and dengue virus, the PRNT assay was only able to discriminate between viruses about half of the time. Reducing the specimen collection window in the 2019 CDC algorithm resulted in fewer conclusive results.

"What's called a difficult decision is a difficult decision because either way you go there are penalties."—Elia Kazan Infectious diseases potentially transmissible through blood transfusion continue to emerge or reemerge globally. Ten articles in this issue of TRANSFUSION focus on the most recent of these, Zika virus, the third major arbovirus within the past two decades to have been introduced to the Western Hemisphere. Zika virus follows the introduction of West Nile virus to New York City in 1999 and chikungunya virus to Saint Martin Island in 2013.1, 2 Factors contributing to their introduction and subsequent spread throughout the Western Hemisphere include causes well identified for other emerging diseases, such as increased travel and trade, urbanization, and population growth. These viruses have become transfusion safety threats despite their relatively short durations of viremia because of their high incidence of infection of the human population during outbreaks and because a proportion of those infected remain asymptomatic and donate blood or alternatively may donate blood before developing symptoms.3, 4 Despite certain similarities of these arbovirus infections, important differences in their biology, epidemiology, and clinical impact have distinct implications for transfusion medicine. Although the widely dispersed Culex species mosquitoes that transmit West Nile virus have permitted viral spread throughout much of the Americas, including all states of the contiguous United States, for unclear reasons large human outbreaks have occurred with variable seasonal intensity only in the United States and Canada.5 Humans develop insufficient viremia to efficiently infect mosquitoes and do not contribute to viral transmission, while many bird species produce high-level viremias and serve as reservoirs for West Nile virus.6 The relatively short life span and high turnover of avian reservoir species do not permit the development of long-standing herd immunity; thus, repeated outbreaks will continue indefinitely.5 Given this epidemiology, along with the propensity of West Nile virus to cause severe neuroinvasive disease, particularly among the elderly, blood donor screening has been proven to be of benefit to public health surveillance over a number of years.7 However, the future epidemiology and impact of chikungunya and Zika viruses are far less certain. Both viruses produce sufficient viremia in humans to efficiently infect Aedes aegypti mosquitoes, thus permitting a human-mosquito-human transmission cycle. The A. aegypti mosquito is ubiquitous in urban environments throughout the tropical and subtropical world, enabling both viruses to become established throughout the Western Hemisphere as a result of spread by human travel. However, A. aegypti is endemic only in the southernmost United States, with occasional seasonal introductions further north, thus limiting the geographic potential for autochthonous viral transmission.8, 9 This trend is similar to the observations with dengue, another arbovirus spread from human to human via A. aegypti mosquitoes, which has only caused transient focal outbreaks in southern Florida and Texas in recent decades, and that autochthonous chikungunya and Zika virus transmission has only been documented in these areas to date.10-13 While Aedes albopictus is a competent vector for dengue, chikungunya, and Zika viruses with wider distribution in the United States than A. aegypti, to date, only one case of autochthonous transmission of dengue in New York and none of chikungunya and Zika viruses has been convincingly shown to result from A. albopictus vectored transmission in the United States.14 Nevertheless, thousands of people with travel-associated chikungunya and Zika virus infection return to nearly all areas of the United States after travel to areas of ongoing transmission, thus presenting a potential risk of transfusion transmission in areas without autochthonous transmission.13 Concerns about transfusion transmission of chikungunya virus have been tempered by its usually short-lived illness without permanent sequelae and the likelihood that development of human herd immunity would greatly reduce transmission.3 The latter may have in fact occurred, with the halving of chikungunya cases reported to the Pan American Health Association each year since its introduction to the Western Hemisphere (http://www.paho.org/hq/index.php?option=com_topics&view=readall&cid=5927&Itemid=40931&lang=en).15 At first, Zika virus' introduction into the Western Hemisphere seemed to raise a level of concern similar to that for chikungunya virus. However, in the fall of 2015, several months after the recognition of mosquito-borne transmission of Zika virus in Brazil, investigators noted a sharp increase in the number of infants born with microcephaly, which prompted the World Health Organization to declare a Public Health Emergency of International Concern on February 1, 2016.16 Insufficient evidence existed at that time to make a causal link between maternal Zika virus infection and birth defects; considerable skepticism remained particularly since mosquito-borne viruses had never been linked definitively to human birth defects. Another confounding factor in February 2016 was reports of sexual transmission of Zika virus, particularly since sexual contact had never been associated with transmission of any other mosquito-borne virus.17 Accumulating case reports and a case series of infants with microcephaly began to strengthen the causal relationship between Zika virus and microcephaly and to define a specific phenotype among affected infants consisting of severe microcephaly, intracranial calcifications, redundant scalp skin, hypertonia/spasticity, clubfoot, and congenital joint contractures.18 These data, along with experimental data showing that Zika virus produced cell death and attenuated future growth of human neural progenitor cells, led the CDC to conclude in April 2016 that Zika virus caused microcephaly and other serious brain defects.19, 20 Subsequent animal models demonstrated that Zika virus is a teratogen, and a case-control study demonstrated a strong relationship between maternal infection and microcephaly.21-25 The full spectrum of adverse fetal outcomes and the risk associated with infection throughout pregnancy is currently unknown and remains an area of active investigation. What is now clear, however, is the potential for serious lifelong adverse impact to a child if Zika virus were to be transmitted by transfusion to a pregnant woman or her sexual partner. The articles focusing on Zika virus in this issue collectively describe a tremendous amount of knowledge gained over the relatively short time span of about a year. For a graphic illustration of the progression of Zika virus and progress in knowledge made regarding the virus in 2016, please see Oussayef and coworkers (https://www.cdc.gov/mmwr/volumes/65/wr/mm6552e1.htm?s_cid=mm6552e1_e).26 For further discussion, the articles in this issue are categorized into five themes. When the first case of Zika virus infection was reported in Puerto Rico by the CDC at the end of December 2015 (https://www.cdc.gov/media/releases/2015/s1231-zika.html), no commercial tests were available in the United States for its detection. However, work conducted about a decade prior on West Nile virus provided a directly relevant paradigm for the development of NAT for screening of the blood supply. In addition, NAT had previously been used for the detection of Zika virus in outbreak settings outside of the Western Hemisphere. Such prior work helped facilitate both test development and the implementation of screening. Several different noncommercial entities initiated the development of laboratory developed tests for diagnostic purposes, and two commercial sponsors experienced in NAT screening tests for blood engaged in the development of commercial assays to screen potential donors. The three articles in this issue relevant to the development of NAT screening of the blood supply are illustrative of the progress made in this field. Bielaire and colleagues27 describe the use of their Zika virus laboratory developed test that was used at the time of the 2013 to 2014 Zika virus outbreak in French Polynesia, which they had determined to have a limit of detection of approximately 100 copies/mL. For context, during the period when the samples screened were collected there was a 30-day deferral in place for symptoms of arbovirus infection at the French Polynesia blood bank in Tahiti. Given the relatively high asymptomatic infection rate with Zika virus, it is not too surprising that using minipools of 3 samples they found that 2.8% of donor samples were reactive for Zika virus RNA. No transfusion-transmitted cases of Zika virus were detected. With the development of more sensitive NAT assays and knowledge of the increased sensitivity provided by individual donor NAT described below, it is interesting to speculate that the actual percentage of reactive donor samples may have been higher than that described in French Polynesia, where it was estimated that approximately 11.5% of the population reported symptomatic infection. Stone and colleagues28 describe the evaluation of a 25-member panel of samples by 11 laboratories using 17 different assays. The comparison of the various assays provided the notable finding that enhanced sensitivity was associated with using a greater sample volume of plasma for the initial RNA extraction. Although not designed to compare the investigational commercial assays developed for use in screening the blood supply, the study found that both of these assays had 100% detection of standards at levels of down to 10 to 40 estimated copies/mL and LOD50 values of less than 5 copies/mL. Given that differences in the sensitivity of various assays are a well-recognized phenomenon with NAT, the development of a reference standard to harmonize results between different assays and laboratories was highly desirable. Once again emanating from global collaboration and using data provided by 21 different laboratories, Baylis and colleagues29 report the development of such a standard: IS 11468/16 for Zika virus RNA. The understanding of the critical variables for assay sensitivity, along with the availability of a reference standard, should greatly facilitate testing with appropriate sensitivity and allow comparison of results obtained across the globe. Although a publication has appeared describing the incidence of blood donations positive for Zika virus in Puerto Rico, to date there has not been a scientific publication regarding blood donations in the rest of the United States.30 The articles by Galel and colleagues31 and Williamson and colleagues32 represent the initial positive results from the commercial investigational blood screening tests of Roche Molecular Systems, Inc., and Hologic, Inc., respectively. The Roche test was the first to be implemented, and its use was allowed to proceed under an investigational new drug application in March 2016, just 3 months after the report of the first case in Puerto Rico.30, 31 The results of this study were highly informative regarding both the assay itself and the epidemiology of Zika virus in the United States outside of Puerto Rico. There were 23 initially reactive donations out of 358,786 samples tested using individual-donor NAT, and after follow-up testing, 14 of these were determined to represent true-positive donations. All of the positive donations identified were collected in Florida. Of note regarding the assay itself, using simulated minipools of 6 samples, only seven of these donations were identified. This finding is consistent with the Zika virus assay sensitivity characteristics described above. Ten of the 14 positive donations came from individuals who had risk factors for sexual transmission or who had traveled to areas with local transmission of Zika virus within the 90 days prior. However, three individuals had neither sexual nor travel risk factors identified. Roughly 6 months after the first case of Zika was reported in Puerto Rico, testing using the Hologic assay began in June 2016.32 The report by Williamson and colleagues provides complementary data to that above for regions of the United States outside of Florida and Puerto Rico. Of 466,834 donations screened with individual-donor NAT, five were found to be positive by supplemental testing. These donations were collected in Nevada, New York, Arizona, California, and Texas. One donor had donated platelets (PLTs) 1 week before the donation that was identified as being positive, and follow-up testing available did not suggest that Zika virus was transmitted to the recipient. Based on characteristics of the donors, the authors speculate that the individuals providing the donations were likely at the tail end of their RNA-positive period yet acknowledge that there is still uncertainty regarding how long transmission can occur after initial infection. The small number of positive donations identified with either of these assays in the United States outside of Puerto Rico stands in stark contrast to the average of 1% positive donations reported from Puerto Rico during the summer months of 2016. Pathogen reduction technology is currently approved in the United States for use with apheresis PLTs and plasma, and the ability of amotosalen and UV light pathogen inactivation technology to reduce Zika virus in plasma has been previously reported.33, 34 The article in this issue by Laughhunn and colleagues35 reporting on pathogen reduction of Zika virus in red blood cell (RBC) components is significant because of both its findings and its potential implications for safety of the blood supply in the future. In vitro use of an investigational technology for pathogen reduction of RBCs, amustaline (S-303) and glutathione, was associated with complete inactivation of more than 7.75 log genomic equivalents of Zika virus RNA using NAT and 5.99 log of infectivity relative to sham treatment using a cell culture assay. This work is significant in that it provides further evidence that after appropriate additional studies, pathogen reduction technologies may ultimately be effective in mitigating arboviral threats in addition to a variety of other pathogens in RBCs, PLTs, and plasma. Although inactivation of Zika, an enveloped virus, by solvent/detergent (S/D) treatment would be expected based on evidence obtained with other similar pathogens, formal documentation of this inactivation, as well as documentation of the effect of other viral clearance interventions, is welcome. The trio of articles by Blümel, Farcet, and Kühnel and their colleagues36-38 document that Zika is indeed inactivated by S/D treatment and by standard pasteurization technology (58 to 60°C for 2 hr for albumin). Indeed, Zika virus seems to be more sensitive to heat than other closely related viruses. In addition, nanofiltration with a pore size of 40 nm or less was found to remove all Zika virus infectious activity.36 All of these findings represent reassuring news regarding Zika virus and the safety of plasma derivatives. Given that Zika virus infection is frequently asymptomatic and that serious complications outside of pregnancy are uncommon, defining a population most at risk of complications from transfusion-transmitted Zika virus is a reasonable undertaking. Note that recent publications indicate the Zika virus is most likely to cause congenital malformations with infection during the first or early second trimesters.25, 39 Infection during that gestational period is estimated to be associated with rates of microcephaly of 11% to 13%.40, 41 The article by Murphy and colleagues42 reports on the number of women receiving blood transfusions in a large tertiary care hospital in Ottawa, Canada. They note that in their hospital only 0.04% of expectant mothers receive a transfusion during the first trimester. Although this information is a welcome addition to the literature, it must be interpreted with caution for two reasons. First, the data are not directly applicable to areas with higher rates of sickle cell disease and other hemoglobinopathies. Second, and perhaps of much greater relevance, is that the data must be interpreted in the context of the potential for sexual transmission of Zika virus.43 At this time, the potential for male-to-female sexual transmission is well documented, with presence of Zika virus RNA in semen reported for up to over 90 days after infection, and the rate of clearance has been reported to be variable.44 The mean duration during which semen is infectious is not yet known. Therefore, one can conclude that the issue is larger than just transfusing a pregnant woman—it is transfusing her male partner. Pending further data on the period of infectivity, an analysis is required that takes into consideration men receiving transfusions who might then have sexual contact in the next 90 days with women who are in the first or second trimester of pregnancy. Zika virus represents yet another in a series of emerging or reemerging threats to the blood supply.45, 46 Though a virus of known identity for several decades, the potential implications of the extensive Zika virus outbreak in the Western Hemisphere became more apparent with each month as the year 2016 progressed. Given the uncertainty regarding the spread of the epidemic and the broad range of the potential vector of Aedes mosquitos in the United States, a cautious approach led initially to implementation of donor deferrals for travel to areas with local Zika virus transmission and then to testing throughout the United States and its territories. As the articles in this issue illustrate, building on experience with prior emerging infectious diseases, much has been learned in the relatively brief period of a year both about the nature of the virus and its epidemiology. This knowledge is invaluable as we refine the response to this epidemic. However, in addition to uncertainty regarding whether Zika virus will spread further or become endemic in some areas, there is also much that remains unknown about the complications of infection itself. Clearly, universal screening of the blood supply was a significant undertaking in the United States, and concern regarding resource utilization is understandable.47 However, it is too early to tell whether or not such continued universal screening is necessary. This potential requirement should become clearer during the next year as we observe whether warmer months in the Northern Hemisphere are associated with a resurgence of spread of the virus. In the meantime, as noted by Galel and colleagues, in addition to helping to facilitate an adequate blood supply by removing regional deferrals and allowing collection to continue in places such as Puerto Rico and Florida, screening of the blood supply has had the benefit of leading to the prompt reporting of reactive donors, facilitating a rapid public health response to evaluate and address potential local transmission of Zika virus.31 With the combination of the rapidity of the Zika virus outbreak in the Western Hemisphere, the potential for adverse fetal and other outcomes, and the uncertainty involved, decision-making certainly was not easy. Indeed there are real costs associated with the course that was taken that must be balanced against the potential costs that could have been incurred with a different course of action and different potential outcomes. The articles in this issue fill important gaps in our knowledge as we continue to learn more about this arboviral pathogen. The authors have disclosed no conflicts of interest. The opinions expressed herein are those of the authors and do not represent those of the Centers for Disease Control and Prevention, the U.S. Food and Drug Administration, the Department of Health and Human Services, or the U.S. Government. Peter W. Marks, MD, PhD1 e-mail: [email protected] Lyle R. Petersen, MD, MPH2 1Center for Biologics Evaluation and Research U.S. Food and Drug Administration Silver Spring, MD 2Division of Vector-Borne Diseases National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA

Zika virus is a flavivirus transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes, and infection can be asymptomatic or result in an acute febrile illness with rash (1). Zika virus infection during pregnancy is a cause of microcephaly and other severe birth defects (2). Infection has also been associated with Guillain-Barré syndrome (GBS) (3) and severe thrombocytopenia (4,5). In December 2015, the Puerto Rico Department of Health (PRDH) reported the first locally acquired case of Zika virus infection. This report provides an update to the epidemiology of and public health response to ongoing Zika virus transmission in Puerto Rico (6,7). A confirmed case of Zika virus infection is defined as a positive result for Zika virus testing by reverse transcription-polymerase chain reaction (RT-PCR) for Zika virus in a blood or urine specimen. A presumptive case is defined as a positive result by Zika virus immunoglobulin M (IgM) enzyme-linked immunosorbent assay (MAC-ELISA)* and a negative result by dengue virus IgM ELISA, or a positive test result by Zika IgM MAC-ELISA in a pregnant woman. An unspecified flavivirus case is defined as positive or equivocal results for both Zika and dengue virus by IgM ELISA. During November 1, 2015-July 7, 2016, a total of 23,487 persons were evaluated by PRDH and CDC Dengue Branch for Zika virus infection, including asymptomatic pregnant women and persons with signs or symptoms consistent with Zika virus disease or suspected GBS; 5,582 (24%) confirmed and presumptive Zika virus cases were identified. Persons with Zika virus infection were residents of 77 (99%) of Puerto Rico's 78 municipalities. During 2016, the percentage of positive Zika virus infection cases among symptomatic males and nonpregnant females who were tested increased from 14% in February to 64% in June. Among 9,343 pregnant women tested, 672 had confirmed or presumptive Zika virus infection, including 441 (66%) symptomatic women and 231 (34%) asymptomatic women. One patient died after developing severe thrombocytopenia (4). Evidence of Zika virus infection or recent unspecified flavivirus infection was detected in 21 patients with confirmed GBS. The widespread outbreak and accelerating increase in the number of cases in Puerto Rico warrants intensified vector control and personal protective behaviors to prevent new infections, particularly among pregnant women.

Disclosure: A.C. Valim: None. V.M. Nascimento: None. C.B. Andrade: None. S.V. Coelho: None. L.B. Arruda: None. D. Pereira-Carvalho: None. T.M. Ortiga-Carvalho: None.The Zika virus (ZIKV) is known to cause severe complications in humans, including microcephaly in newborns of mothers infected during pregnancy. ZIKV infection of the placenta can compromise gestational development by affecting hormonal production, which is essential for maintaining pregnancy and maternal behavior postpartum. We investigated the effects of ZIKV infection during pregnancy in mice, focusing on changes in maternal behavior and neonatal survival and offspring development. We investigated the effects of ZIKV infection during pregnancy in mice, focusing on changes in maternal behavior, neonatal survival, and offspring development. Female C57BL/6 mice were infected with ZIKV (ZIKV-BRPE243 strain) on gestational day (GD) 12.5, divided into groups: High Dose ZIKV (ZIKV HD; 5x107 PFU; n=25) and Low Dose ZIKV (ZIKV LD; 103 PFU; n=27) and Control (CTR; noninfected medium; n=31). We assessed offspring survival and development, including behavioral tests. We analyzed the expression of placental genes related to maternal behavior and hormonal production (Phlda2, Prl7a2, Prl8a8, Prl3a1, Prl3b1), as well as serum prolactin levels on GD 18.5. Data were analyzed using One-way ANOVA with Tukey post-hoc test, Mann-Whitney test, Mixed ANOVA with Greenhouse-Geisser correction and Log-rank test. Infection with a high viral load of ZIKV resulted in 100% neonatal mortality (p=0.002). In the ZIKV LD group, 77.2% of females completed the cliff avoidance test, compared to 50% in the CTR group (p=0.038). ZIKV LD-infected mothers built higher-quality nests (p=0.008). No significant changes were observed in the expression of placental genes related to hormonal production or serum prolactin levels on GD 18.5. Our findings suggest that low viral load activates compensatory mechanisms, possibly involving the hypothalamic-pituitary-adrenal (HPA) axis in mothers and female offspring. Conversely, high viral load appears to overwhelm mothers, preventing effective activation of these mechanisms and contributing to elevated mortality. Further investigation of HPA axis components in both mothers and offspring is warranted to validate this hypothesis. These results highlight the importance of understanding the endocrine alterations induced by ZIKV infection during gestation and their implications for maternal behavior and neonatal survival. Keywords: ZIKV; maternal behavior; placenta.Presentation: Saturday, July 12, 2025

In the past few years, and with COVID-19 most recently, we have been reminded of the importance of robust public health monitoring programs as they relate to birth defects surveillance, research, and prevention. With the never-ending appearance of new exposures—such as novel medications, infections, vaccines, chemicals, herbal products, and substances of abuse— it is vital that public health agencies are sufficiently prepared to evaluate the impact of these exposures on the occurrence of birth defects and to evaluate the presence of new birth defect syndromes. A multidisciplinary approach is needed to not only conduct birth defects surveillance but also to mitigate the impact of birth defects. As an example, in this editorial we propose a framework for a stronger collaboration between National Birth Defect Prevention Network (NBDPN) and the Organization of Teratology Information Specialists (OTIS) with the goal of addressing the impact of novel and existing exposures on the occurrence of birth defects. COVID-19 is not the first, nor the last, illness that will be monitored for its effects on pregnancy and birth outcomes. When Zika Virus (ZV) emerged in the Americas, the NBDPN and OTIS, including its local MotherToBaby affiliates, responded alongside other international partners such as the U.S. Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO). During the early stages of the pandemic, given the news reports and early scientific publications, birth defects and teratology experts suspected the teratogenic effects of ZV. Pregnancies and newborn health outcomes were subsequently monitored. Early on, and without clear scientific evidence, public health messaging urged pregnant women to avoid contracting ZV. Reports of microcephaly and other brain anomalies in Zika-exposed infants appeared in the clinical literature and in the media. Suspicions grew that ZV could cause birth defects. In a relatively short period of time, sufficient peer-reviewed evidence was gathered for experts to clearly state “that a causal relationship exists between prenatal Zika virus infection and microcephaly and other serious brain anomalies” and to urge movement “toward the prevention of adverse outcomes caused by congenital Zika virus infection” (Rasmussen, Jamieson, Honein, & Petersen, 2016). Following this statement, organizations such as OTIS and NBDPN worked together to craft more specific public health messages and engage in prevention efforts. Although ZV was not widespread in the United States (US), the lessons learned from ZV (Harris-Sagaribay et al., 2020) apply to our current threat of COVID-19. Strategic and proactive collaboration among government agencies, professional societies, and healthcare organizations builds the intangible infrastructure needed to protect mothers and babies from ongoing threats and helps promote healthy birth outcomes. When faced with a pandemic or other public health crisis, timely collaboration among scientific organizations contributes to successful disease response. NBDPN and OTIS are two organizations working to establish such a collaboration. These organizations fulfill different but complementary roles in identifying, educating about, and preventing adverse birth outcomes within and outside the context of pandemics. The NBDPN works with programs in all states, Puerto Rico, and the District of Columbia to conduct surveillance of birth defects, associated risk factors, and outcomes. It is comprised of individuals involved in birth defects surveillance, research, and prevention. Created to establish and maintain a national network of state and population-based programs for birth defects surveillance and research, NBDPN assesses the impact of birth defects upon children, families, and health care; identifies factors that can be used to develop primary prevention strategies; and assists families and their providers in secondary disabilities prevention. NBDPN analyses of surveillance data identify birth defects outcomes and associated demographic, genetic, and environmental risk factors through multi-site surveillance and research. NBDPN members and their colleagues have developed expertise in identifying and reporting on birth defects and developmental delays. While specifics vary, some local programs collect information from medical records of births, interviews with parents, and/or DNA tests; many have clinical geneticists review cases to ensure correct classification; and records are linked to vital statistics for population-based analysis. Pooling data in annual reports and collaborative studies help to ensure surveillance truly is population-based and nationally representative. More information about NBDPN can be found at www.nbdpn.org. OTIS works with the fourteen teratogen information services (MotherToBaby affiliates) across the US and covers all states and territories to influence research and provide health education to the public and clinicians. OTIS's members, teratogen information specialists, span the globe beyond those affiliates within the US. Some of the services in the US have operated for over 35 years, joining forces to establish OTIS as their professional organization (Leen-Mitchell, Martinez, Gallegos, Robertson, & Carey, 2000). OTIS's defining role has been providing teratogen information to the public, through its service arm MotherToBaby which serves women potentially exposed to teratogens during pregnancy as well as providers who care for those pregnant women and require information about potential risk of specific exposures, prescriptions or recommended medications (Clementi & Ornoy, 2004). OTIS/MotherToBaby answers thousands of questions each year about a wide range of exposures including cleaning products, cosmetics, occupational exposures, vaccines, diseases, substances of abuse, and medications (Campbell, Kast, Kamyar, Robertson, & Sherwin, 2016). MotherToBaby specialists counter the inaccurate information about teratogens often found online by patients and clinicians. Furthermore, providers and the public often lack the time and training to interpret large amounts of research and have inaccurate perceptions of risks from exposures (Conover & Polifka, 2011). Teratogen information specialists are experienced in reviewing studies, comparing outcomes, and translating health information for consumers. More information about OTIS/MotherToBaby can be found at www.mothertobaby.org. NBDPN and OTIS primarily intersect in five areas which impact disease response: Research, networking, health education, policy development, and workforce development. Given the gaps in knowledge about exposures, OTIS conducts and supports research (Clementi & Ornoy, 2004), and sometimes identifies research needs through routine operations. As local teratogen information services respond to questions from women and their providers, their staff develop a sense of where research and health education is needed. For example, daily concerns brought to OTIS about antidepressants and about supplements such as melatonin highlighted the need for research and education on some new and existing medications. These emerging trends in questions are used to inform future research studies. For most OTIS-led studies, a three-group prospective cohort design follows women with a condition and medication, women with the condition without a medication, and control groups of women who do not have the disease to help determine if the untreated condition increases risks rather than the medication, such as the risks associated with an asthma medication compared with risks of poorly controlled asthma (Chambers et al., 2001). The studies also collect data from maternal interviews and physical examinations of infants by trained dysmorphologists to supplement the data collection. The different research designs, large database studies, prospective cohorts, and surveillance studies, contribute different strengths to piecing together the puzzle of what may increase risks for birth defects, functional problems, or developmental delays. In contrast to the signals OTIS may receive through interaction with individuals, NBDPN members identify research opportunities through population-based birth defects surveillance. In the United States, NBDPN coordinates the collection of birth defects surveillance data and provides guidance to state programs to improve consistency in surveillance. Local birth defects surveillance programs sometimes employ clinicians such as geneticists and dysmorphologists to classify and review cases based on medical records. As clinicians review cases of birth defects in children, they are poised to notice trends in birth defects and exposures. Although each organization currently conducts its own research program, many researchers are involved in both organizations (Rasmussen, Erickson, Reef, & Ross, 2009). NBDPN and OTIS are working to identify more timely feedback mechanisms for sharing signals and events of interest to both organizations. Data, health education messaging, and research collaborations are strategically shared between both organizations. Both organizations engage teratogen experts and share mutually beneficial contacts. Both organizations aspire to provide coordinated, timely, and concurrent messaging on birth defects prevention and findings. OTIS utilizes research from NBDPN and other groups to inform its messages. OTIS/MotherToBaby provides individualized information through client contacts by phone, text, email, and chat in addition to providing traditional and social media (Facebook, Twitter, Instagram, blogs, podcasts, videos, etc.) messaging. NBDPN uses social media and research platforms to disseminate surveillance data and health education. Although the two organizations do not currently collaborate to influence policy, there is ample opportunity to do so. The two organizations can provide a unified voice as experts in the areas of birth defects surveillance and prevention to create white papers, policy proposals, and legislative briefs. Second, the organizations can work together under the larger umbrella of organizations interested in birth defects and developmental delays to further accelerate the creation and advocate for useful policies. Professional development opportunities among both organizations refine the skills of those committed to birth defects prevention. OTIS/MotherToBaby provides professional development seminars to improve the skills of teratogen information specialists and to update risk statements. The seminars typically review a research article about an exposure in pregnancy or breastfeeding. Local OTIS affiliates, pharmacists, teratogen database administrators, NBDPN members, clinicians, public health professionals, and others from multiple countries around the world add expertise and clinical experience to the discussion. Other professionals can join OTIS or the seminars by contacting OTIS. Annual meetings and webinars provide topics of interest and continuing education to both groups. OTIS/MotherToBaby collaborates with the Society for Birth Defects Research and Prevention (SBDRP) (formerly the Teratology Society) and the Developmental Neurotoxicology Society (DNTS) to hold joint, annual educational meetings. The three societies recognize the advantages of combining efforts to provide professional development and networking opportunities for members. While NBDPN as an organization has not collaborated often in these meetings, multiple NBDPN members attend joint meetings as members of the other three organizations, contributing to conference planning in various ways. In March 2020, shortly before the nationwide shutdown for the COVID-19 pandemic, OTIS/MotherToBaby held its Mid-Year Research Meeting co-located with NBDPN's Annual Meeting. This allowed for joint sessions as both organizations sought to foster a stronger partnership and working relationship. Joint, co-located and virtual meetings will continue providing opportunities to support professional development. Cross-training of professionals in these areas of birth defect surveillance and providing teratogen information is essential. Training is time-consuming and expensive, taking months to prepare individuals to begin to work in these fields, and years to become proficient. Understanding the complexities of birth defects and explaining teratogenic exposures to the public are specialized skills. As pandemics rise and fall, there may be more critical, hopefully relatively temporary, need for professionals on the tracking side than on the educating side. After a pandemic subsides, there may be a greater need for interpreting studies, providing public messages, and educating individuals and providers. Complementary and collaborative efforts between health agencies and OTIS have been ongoing: The ZV pandemic demonstrated the flexible but unique and collaborative roles of major players in disease response. CDC, NBDPN, OTIS/MotherToBaby, state birth defects programs, and local MotherToBaby affiliates collaborated to provide consistent messaging nationally and locally to help women avoid exposure to the virus (Harris-Sagaribay et al., 2020). Women who contracted the virus during travel were referred for testing, follow-up, and to the national pregnancy registry. Consistent messages were provided on websites and social media, and in press interviews and printed materials. CDC studied the virus and related outcomes, and provided public health messages. NBDPN and local birth defects registries identified and followed cases. OTIS and local MotherToBaby affiliates answered individual questions from families and their providers and referred women to the registries. With their different roles, the organizations were able to focus their expertise during the pandemic when resources were stretched thin. No single organization performs all of these roles. Having local programs work with national organizations allowed for rapid communication in both directions when challenges arose, and when the government needed data and input on adapting messaging and strategies. At the present time, evidence concerning COVID-19 does not suggest increased risks for structural malformations but other possible adverse outcomes are being monitored. However, the current pandemic provides an opportunity to collaborate and prepare for developments in knowledge about possibly teratogenic viruses (Ludorf, Salemi, Kirby, Tanner, & Agopian, 2020). The population-based surveillance data provided by CDC and programs in NBDPN remains crucial during the pandemic and its aftermath. Population-based surveillance enables tracking structural malformations and developmental disabilities in an organized manner. Now and in the future, clinicians associated with NBDPN programs can be tapped to identify emerging trends from case review. They can also propose research, suggest surveillance ideas, and encourage further research on potentially teratogenic exposures. Even if public health data and messaging are available on teratogens and birth defects, public health professionals rarely interact with individuals in the public. Private medical visits are not a quick source of information. OTIS is uniquely positioned to serve as a bridge between government agencies providing health messaging to individuals and complements the role of medical providers (Kaye et al., 2001). Many providers may be unfamiliar with the nuances of responding to questions about teratogenic exposures or addressing inaccurate information in outdated references or on the internet. The differentiation of roles allows clinical providers to be experts in treating conditions while allowing teratogen information specialists to be experts in explaining the potential teratogenicity of any exposure, including new medications and diseases. In this sense, the work of OTIS can greatly benefit both the public health and private healthcare communities in efficiently communicating health messages. Given the need for the specialized services provided by birth defects surveillance programs and teratogen information services, states and municipalities should consider investing in both programs. Additional funding, potentially from multiple sources including national and state funds, healthcare organizations, pharmaceutical industries, and other organizations, is needed to keep these national organizations and local programs operating. Research has demonstrated that these programs save millions of dollars a year in addition to improving public health by dispelling myths about medications and preventing untreated maternal conditions due to inaccurate information (Luca et al., 2020). Although budgets have been strained and cut across government agencies, OTIS and NBDPN continue to function, often behind-the scenes, ensuring that birth defects data are reviewed and education disseminated. The strategic partnership between NBDPN and OTIS provides an example of how public health infrastructure can be strengthened despite shifting priorities and uncertain funding. Participation from other professional societies and non-governmental organizations (for example, the March of Dimes Foundation, the Society for Birth Defects Research and Prevention, the American College of Obstetricians and Gyneologists, and the American Academy of Pediatrics) can enhance and amplify these collaborative efforts. The current and future work of these organizations will continue to protect maternal and infant health in the United States. In this time of (a) increasing data and information, (b) novel exposures and pathogens, and (c) uncertainty surrounding these issues—it is vital that public health professionals from various backgrounds unite to interpret these data and develop evidence-based recommendations and interventions. Through these efforts, we hope to reduce the impact of new and existing exposures on adverse outcomes—including birth defects. We propose that a first and important step is strengthening the collaboration between the NBDPN—an organization focused on birth defects surveillance, research, and prevention—and OTIS—a society of professionals dedicated to evaluating risks to pregnancy from medications and other exposures. Through this expanded collaboration, we anticipate that effective responses to emerging threats can be implemented in real time, thereby reducing the prevalence and impact of birth defects. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

1 year after acute Zika virus infection in men

Objective To establish a method for the isolation of Zika virus and to gather experiences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples positive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nucleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The sequences of NS5 gene were analyzed by using MEGA6.0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle threshold (Ct) values of 24.4 and 25.3, respectively. The second generation of Zika virus was identified in suckling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus (GDZ16002 strain) belonged to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage. Key words: Zika virus; Viral isolation; Arbovirus; Suckling mice

The recent outbreak of Zika virus (ZIKV) has emerged as a global health concern. ZIKV can persist in human semen and be transmitted by sexual contact, as well as by mosquitoes, as seen for classical arboviruses. We along with others have previously demonstrated that ZIKV infection leads to testis damage and infertility in mouse models. So far, no prophylactics or therapeutics are available; therefore, vaccine development is urgently demanded. Recombinant chimpanzee adenovirus has been explored as the preferred vaccine vector for many pathogens due to the low preexisting immunity against the vector among the human population. Here, we developed a ZIKV vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins. A single vaccination of AdC7-M/E was sufficient to elicit potent neutralizing antibodies and protective immunity against ZIKV in both immunocompetent and immunodeficient mice. Moreover, vaccinated mice rapidly developed neutralizing antibody with high titers within 1 week postvaccination, and the elicited antiserum could cross-neutralize heterologous ZIKV strains. Additionally, ZIKV M- and E-specific T cell responses were robustly induced by AdC7-M/E. Moreover, one-dose inoculation of AdC7-M/E conferred mouse sterilizing immunity to eliminate viremia and viral burden in tissues against ZIKV challenge. Further investigations showed that vaccination with AdC7-M/E completely protected against ZIKV-induced testicular damage. These data demonstrate that AdC7-M/E is highly effective and represents a promising vaccine candidate for ZIKV control.IMPORTANCE Zika virus (ZIKV) is a pathogenic flavivirus that causes severe clinical consequences, including congenital malformations in fetuses and Guillain-Barré syndrome in adults. Vaccine development is a high priority for ZIKV control. In this study, to avoid preexisting anti-vector immunity in humans, a rare serotype chimpanzee adenovirus (AdC7) expressing the ZIKV M/E glycoproteins was used for ZIKV vaccine development. Impressively, AdC7-M/E exhibited exceptional performance as a ZIKV vaccine, as follows: (i) protective efficacy by a single vaccination, (ii) rapid development of a robust humoral response, (iii) durable immune responses, (iv) robust T cell responses, and (v) sterilizing immunity achieved by a single vaccination. These advantages of AdC7-M/E strongly support its potential application as a promising ZIKV vaccine in the clinic.

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.

ZIKV Strains' Different Phenotypes in Human Neural Cells Could be a Hint for the Emergence of the New Clinical Neurological Outcomes

Effect of acute Zika virus infection on sperm and virus clearance in body fluids: a prospective observational study