Emergence Of Zika Virus Research Articles

Linking nutrient stoichiometry to Zika virus transmission in a mosquito.

Food quality and quantity serve as the basis for cycling of key chemical elements in trophic interactions; yet the role of nutrient stoichiometry in shaping host-pathogen interactions is under appreciated. Most of the emergent mosquito-borne viruses affecting human health are transmitted by mosquitoes that inhabit container systems during their immature stages, where allochthonous input of detritus serves as the basal nutrients. Quantity and type of detritus (animal and plant) were manipulated in microcosms containing newly hatched Aedes aegypti mosquito larvae. Adult mosquitoes derived from these microcosms were allowed to ingest Zika virus-infected blood and then tested for disseminated infection, transmission, and total nutrients (percent carbon, percent nitrogen, ratio of carbon to nitrogen). Treatments lacking high-quality animal (insect) detritus significantly delayed development. Survivorship to adulthood was closely associated with the amount of insect detritus present. Insect detritus was positively correlated with percent nitrogen, which affected Zika virus infection. Disseminated infection and transmission decreased with increasing insect detritus and percent nitrogen. We provide the first definitive evidence linking nutrient stoichiometry to arbovirus infection and transmission in a mosquito using a model system of invasive Ae. aegypti and emergent Zika virus.

Duration of seminal Zika viral RNA shedding in immunocompetent mice inoculated with Asian and African genotype viruses

Prior to the emergence of Asian genotype Zika virus (ZIKV) in the Western hemisphere, sexual transmission in humans was documented. Sexual transmission by African genotype ZIKVs has not been assessed in laboratory animal models, due to rapid and high mortality rates of immunodeficient mice following inoculation. To overcome these limitations, immunocompetent C57Bl/6 mice were used to longitudinally assess Asian and African genotype ZIKV sexual transmission potential. Furthermore, to determine if enhanced pathogenesis of African genotype ZIKVs is due to structural determinants, PRVABC59 prM/E was replaced with African MR766 prM/E (chimeric ZIKV). The African genotype and chimeric ZIKV elicited greater pathogenic effects in the male reproductive tract and generated higher viremias. Yet, the duration, magnitude and efficiency of seminal shedding of infectious virus and viral RNA were similar between chimeric-, African and Asian genotype ZIKV-inoculated mice. These data show that increased male reproductive tract pathology does not increase sexual transmission potential.

Epidemiological and clinical suspicion of congenital Zika virus infection: Serological findings in mothers and children from Brazil.

The emergence of Zika virus in the Americas has caused an increase of babies born with microcephaly or other neurological malformations. The differential diagnosis of Zika infection, particularly serological diagnosis, is an important but complex issue. In this study, we describe clinical manifestations of 94 suspected cases of congenital Zika from Bahia state, Brazil, and the results of serological tests performed on children and/or their mothers at an average of 71 days after birth. Anti‐Zika immunoglobulin M (IgM) antibodies were detected in 44.4% and in 7.1% of samples from mothers and children, respectively. Nearly all the IgM, and 92% of immunoglobulin G positive results were confirmed by neutralization test. Zika specific neutralizing antibodies were detected in as much as 90.4% of the cases. Moreover, dengue specific neutralizing antibodies were detected in 79.0% of Zika seropositive mothers. In conclusion, Zika IgM negative results should be considered with caution, due to a possible rapid loss of sensitivity after birth, while the NS1‐based Zika IgM enzyme‐linked immunosorbent assay test we have used has demonstrated to be highly specific. In a high percentage of cases, Zika specific neutralizing antibodies were detected, which are indicative of a past Zika infection, probably occurred during pregnancy in this population.

Identifying Protective Health Behaviors on Twitter: Observational Study of Travel Advisories and Zika Virus.

BackgroundAn estimated 3.9 billion individuals live in a location endemic for common mosquito-borne diseases. The emergence of Zika virus in South America in 2015 marked the largest known Zika outbreak and caused hundreds of thousands of infections. Internet data have shown promise in identifying human behaviors relevant for tracking and understanding other diseases.ObjectiveUsing Twitter posts regarding the 2015-16 Zika virus outbreak, we sought to identify and describe considerations and self-disclosures of a specific behavior change relevant to the spread of disease—travel cancellation. If this type of behavior is identifiable in Twitter, this approach may provide an additional source of data for disease modeling.MethodsWe combined keyword filtering and machine learning classification to identify first-person reactions to Zika in 29,386 English-language tweets in the context of travel, including considerations and reports of travel cancellation. We further explored demographic, network, and linguistic characteristics of users who change their behavior compared with control groups.ResultsWe found differences in the demographics, social networks, and linguistic patterns of 1567 individuals identified as changing or considering changing travel behavior in response to Zika as compared with a control sample of Twitter users. We found significant differences between geographic areas in the United States, significantly more discussion by women than men, and some evidence of differences in levels of exposure to Zika-related information.ConclusionsOur findings have implications for informing the ways in which public health organizations communicate with the public on social media, and the findings contribute to our understanding of the ways in which the public perceives and acts on risks of emerging infectious diseases.

Zika virus during pregnancy: From maternal exposure to congenital Zika virus syndrome.

What is already known about the topic?The Zika virus (ZIKV) emergence and its consequences during pregnancy led to an increasing number of data about the maternal and fetal consequences of ZIKV infections.What does this study add?This review evaluates risks of maternal ZIKV infection, maternal‐fetal transmission, congenital Zika syndrome (CZS), other adverse perinatal outcomes, and long‐term sequelae among pregnancies exposed to ZIKV. This review also provides contemporary criteria and guidelines for the follow‐up of exposed fetuses, based on prenatal and postnatal features of CZS.

Vector-borne transmission and evolution of Zika virus.

Zika virus (ZIKV), discovered in the Zika Forest of Uganda in 1947, is a mosquito-borne flavivirus related to yellow fever, dengue and West Nile viruses. From its discovery until 2007, only sporadic ZIKV cases were reported, with mild clinical manifestations in patients. Therefore, little attention was given to this virus before epidemics in the South Pacific and the Americas that began in 2013. Despite a growing number of ZIKV studies in the past three years, many aspects of the virus remain poorly characterized, particularly the spectrum of species involved in its transmission cycles. Here, we review the mosquito and vertebrate host species potentially involved in ZIKV vector-borne transmission worldwide. We also provide an evidence-supported analysis regarding the possibility of ZIKV spillback from an urban cycle to a zoonotic cycle outside Africa, and we review hypotheses regarding recent emergence and evolution of ZIKV. Finally, we identify critical remaining gaps in the current knowledge of ZIKV vector-borne transmission.

Identification of small molecule inhibitors targeting the Zika virus envelope protein

The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses.

Surveillance of Aedes aegypti indoors and outdoors using Autocidal Gravid Ovitraps in South Texas during local transmission of Zika virus, 2016 to 2018

The yellow fever mosquito, Aedes aegypti, has facilitated the re-emergence of dengue virus (DENV) and emergence of chikungunya virus (CHIKV) and Zika virus (ZIKV) in the Americas and the Caribbean. The recent transmission of these arboviruses in the continental United States has been limited, to date, to South Florida and South Texas despite Ae. aegypti occurring over a much larger geographical region within the country. The main goal of our study was to provide the first long term longitudinal study of Ae. aegypti and enhance the knowledge about the indoor and outdoor relative abundance of Ae. aegypti as a proxy for mosquito-human contact in South Texas, a region of the United States that is at high risk for mosquito-borne virus transmission. Here, the relative abundance of indoors and outdoors mosquitoes of households in eight different communities was described. Surveillance was done weekly from September 2016 to April 2018 using the CDC Autocidal Gravid Ovitraps in low- and middle-income communities. A total of 69 houses were included in this survey among which 36 were in the low-income communities (n = 11 for Donna, n = 15 for Progresso, n = 5 for Mesquite, n = 5 for Chapa) and 33 in middle-income communities (n = 9 for La Feria, n = 8 for Weslaco, n = 11 for McAllen, and n = 5 for Rio Rico). Overall, Ae. aegypti was the dominant species (59.2% of collections, n = 7255) followed by Culex spp. mosquitoes (27.3% of collections, n = 3350). Furthermore, we demonstrated for Ae. aegypti that 1) outdoor relative abundance was higher compared to indoor relative abundance, 2) low-income communities were associated with an increase in mosquito relative abundance indoors when compared to middle-income communities, 3) no difference was observed in the number of mosquitoes collected outdoors between low-income and middle-income communities, and 4) warmer months were positively correlated with outdoor relative abundance whereas no seasonality was observed in the relative abundance of mosquitoes indoors. Additionally, Ae. aegypti mosquitoes collected in South Texas were tested using a specific ZIKV/CHIKV multiplex real-time PCR assay, however, none of the mosquitoes tested positive. Our data highlights the occurrence of mosquitoes indoors in the continental United States and that adults are collected nearly every week of the calendar year. These mosquito data, obtained concurrently with local ZIKV transmission of 10 locally acquired cases in nearby communities, represent a baseline for future studies in the Lower Rio Grande Valley (LRGV) including vector control interventions relying on the oviposition behavior to reduce mosquito populations and pathogen transmission.

Zika dynamics in South America

Epidemiology The infection dynamics of Zika virus (ZIKV) are difficult to characterize. Many ZIKV infections are asymptomatic, and the clinical presentation of ZIKV is nonspecific. Rodriguez-Barraquer et al. took advantage of a long-term health study under way in Salvador, Brazil, the epicenter of the recent outbreak in the Americas. They used multiple serological assays, from before and after the emergence of ZIKV in October 2015, to distinguish ZIKV immune responses from those against Dengue virus (DENV). About 73% of the population was attacked by ZIKV. The presence of preexisting antibodies to DENV was associated with less risk of ZIKV infection and fewer symptoms. Science , this issue p. [607][1] [1]: /lookup/doi/10.1126/science.aav6618

Impact of preexisting dengue immunity on Zika virus emergence in a dengue endemic region.

The clinical outcomes associated with Zika virus (ZIKV) in the Americas have been well documented, but other aspects of the pandemic, such as attack rates and risk factors, are poorly understood. We prospectively followed a cohort of 1453 urban residents in Salvador, Brazil, and, using an assay that measured immunoglobulin G3 (IgG3) responses against ZIKV NS1 antigen, we estimated that 73% of individuals were infected during the 2015 outbreak. Attack rates were spatially heterogeneous, varying by a factor of 3 within a community spanning 0.17 square kilometers. Preexisting high antibody titers to dengue virus were associated with reduced risk of ZIKV infection and symptoms. The landscape of ZIKV immunity that now exists may affect the risk for future transmission.

RNA Helicase A Is an Important Host Factor Involved in Dengue Virus Replication.

Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents.IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

Experimental Zika virus infection of Jamaican fruit bats (Artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells.

The emergence of Zika virus (ZIKV) in the New World has led to more than 200,000 human infections. Perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with Guillain-Barré syndrome (GBS). ZIKV is transmitted to humans by Aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal Aedes sp. mosquitos and non-human primates. In the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to ZIKV with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. Because of ZIKV emergence in the Americas, we sought to determine susceptibility of Jamaican fruit bats (Artibeus jamaicensis), one of the most common bats in the New World. Bats were inoculated with ZIKV PRVABC59 but did not show signs of disease. Bats held to 28 days post-inoculation (PI) had detectable antibody by ELISA and viral RNA was detected by qRT-PCR in the brain, saliva and urine in some of the bats. Immunoreactivity using polyclonal anti-ZIKV antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. Tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular Leydig cells. The virus likely localized to the brain via infection of Iba1+ macrophage/microglial cells. Jamaican fruit bats, therefore, may be a useful animal model for the study of ZIKV infection. This work also raises the possibility that bats may have a role in Zika virus ecology in endemic regions, and that ZIKV may pose a wildlife disease threat to bat populations.

Birth defects surveillance: experiences in Argentina and Colombia.

Birth defects (BDs) are structural or functional anomalies, sporadic or hereditary, of prenatal origin. Public health surveillance is defined as the ongoing systematic collection, analysis, and interpretation of outcome-specific data for use in the planning, implementation, and evaluation of public health practice. BD surveillance systems may have different characteristics according to design, coverage, type of surveillance, case ascertainment, case definition, BD description, maximum age of diagnosis, pregnancy outcomes, coding systems, and the location of the coding process (central or local). The aim of this article is to describe and compare methodology, applications, and results of birth defect surveillance systems in two South-American countries: Colombia and Argentina. In both countries, the surveillance systems developed activities in relation to the Zika virus emergency. For most BDs, a statistically significant higher prevalence is observed in Argentina-RENAC than in Colombian registries. This may be due to methodological reasons or real differences in prevalence. The strengths, weaknesses, and the future perspectives of the Argentine and Colombian systems are presented. When developing a surveillance system, the objectives, the available resources, and previous experiences in similar contexts must be taken into account. In that sense, the experience of Argentina and Colombia can be useful for others when developing a birth defect surveillance system.

Cell surface α2,3-linked sialic acid facilitates Zika virus internalization

ABSTRACT The emergence of neurotropic Zika virus (ZIKV) raised a public health emergency of global concern. ZIKV can cross the placental barrier and infect foetal brains, resulting in microcephaly, but the pathogenesis of ZIKV is poorly understood. With recent findings reporting AXL as a type I interferon antagonist rather than an entry receptor, the exact entry mechanism remains unresolved. Here we report that cell surface sialic acid plays an important role in ZIKV infection. Removal of cell surface sialic acid by neuraminidase significantly abolished ZIKV infection in Vero cells and human induced-pluripotent stem cells-derived neural progenitor cells. Furthermore, knockout of the sialic acid biosynthesis gene encoding UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase resulted in significantly less ZIKV infection of both African and Asian lineages. Huh7 cells deficient in α2,3-linked sialic acid through knockout of ST3 β-galactoside-α2,3-sialyltransferase 4 had significantly reduced ZIKV infection. Removal of membrane-bound, un-internalized virus with pronase treatment revealed the role of sialic acid in ZIKV internalization but not attachment. Sialyllactose inhibition studies showed that there is no direct interaction between sialic acid and ZIKV, implying that sialic acid could be mediating ZIKV-receptor complex internalization. Identification of α2,3-linked sialic acid as an important host factor for ZIKV internalization provides new insight into ZIKV infection and pathogenesis.

Genetic and biological characterisation of Zika virus isolates from different Brazilian regions.

BACKGROUND Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown.OBJECTIVES The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics.METHODS The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells.FINDINGS Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells.MAIN CONCLUSIONS Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.

Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells.

The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness.

Experimental transmission of Zika virus by Aedes japonicus japonicus from southwestern Germany

The invasive mosquito species Aedes japonicus japonicus (Ae. japonicus) is widely distributed in Central Europe and is a known vector of various arboviruses in the laboratory, including flaviviruses such as Japanese Encephalitis virus or West Nile virus. However, the vector competence of Ae. japonicus for the recently emerging Zika virus (ZIKV) has not been determined. Therefore, field-caught Ae. japonicus from Germany were orally infected with ZIKV and incubated at 21, 24, or 27 °C to evaluate the vector competence under climate conditions representative of the temperate regions (21 °C) in the species’ main distribution area in Europe and of Mediterranean regions (27 °C). Aedes japonicus was susceptible to ZIKV at all temperatures, showing infection rates between 10.0% (21 °C) and 66.7% (27 °C). However, virus transmission was detected exclusively at 27 °C with a transmission rate of 14.3% and a transmission efficiency of 9.5%. Taking into account the present distribution of Ae. japonicus in the temperate regions of Central Europe, the risk of ZIKV transmission by the studied Ae. japonicus population in Central Europe has to be considered as low. Nevertheless, due to the species’ vector competence for ZIKV and other mosquito-borne viruses, in combination with the possibility of further spread to Mediterranean regions, Ae. japonicus must be kept in mind as a potential vector of pathogens inside and outside of Europe.

Growth and adaptation of Zika virus in mammalian and mosquito cells.

The recent emergence of Zika virus (ZIKV) in the Americas coincident with increased caseloads of microcephalic infants and Guillain-Barre syndrome has prompted a flurry of research on ZIKV. Much of the research is difficult to compare or repeat because individual laboratories use different virus isolates, growth conditions, and quantitative assays. Here we obtained three readily available contemporary ZIKV isolates and the prototype Ugandan isolate. We generated stocks of each on Vero mammalian cells (ZIKVmam) and C6/36 mosquito cells (ZIKVmos), determined titers by different assays side-by-side, compared growth characteristics using one-step and multi-step growth curves on Vero and C6/36 cells, and examined plaque phenotype. ZIKV titers consistently peaked earlier on Vero cells than on C6/36 cells. Contemporary ZIKV isolates reached peak titer most quickly in a multi-step growth curve when the amplifying cell line was the same as the titering cell line (e.g., ZIKVmam titered on Vero cells). Growth of ZIKVmam on mosquito cells was particularly delayed. These data suggest that the ability to infect and/or replicate in insect cells is limited after growth in mammalian cells. In addition, ZIKVmos typically had smaller, more homogenous plaques than ZIKVmam in a standard plaque assay. We hypothesized that the plaque size difference represented early adaptation to growth in mammalian cells. We plaque purified representative-sized plaques from ZIKVmos and ZIKVmam. ZIKVmos isolates maintained the initial phenotype while plaques from ZIKVmam isolates became larger with passaging. Our results underscore the importance of the cells used to produce viral stocks and the potential for adaptation with minimal cell passages. In addition, these studies provide a foundation to compare current and emerging ZIKV isolates in vitro and in vivo.

Biomarkers and immunoprofiles associated with fetal abnormalities of ZIKV-positive pregnancies.

An intricate fetal-maternal immune crosstalk during pregnancy is essential for a healthy birth. Hence, the infection-induced alterations of maternal immunity often lead to adverse outcomes for mother and/or child. The emergence of Zika virus (ZIKV) infection in pregnant women has been associated with more than 3,000 cases of microcephaly and nervous system malformations. To explore the potential correlation of ZIKV-induced alteration of maternal immunity with fetal abnormalities, we performed extensive sera immunoprofiling of 74 pregnant women: 30 symptomatic ZIKV+ pregnant patients and 30 healthy pregnant controls in ZIKV-endemic Rio de Janeiro, along with 14 healthy pregnant controls in non-endemic Los Angeles. Extensive multiplexing analysis of 69 cytokines revealed that CXCL10, CCL2, and CCL8 chemokines were specifically associated with symptomatic ZIKV+ infection during pregnancy, and distinct immunoprofiles were detected at different trimesters in ZIKV-infected pregnant women. Intriguingly, the high CCL2 level and its inverse correlation with CD163, TNFRSF1A, and CCL22 levels was apparently associated with ZIKV-induced abnormal birth. Our findings provide insights into the alteration of ZIKV-elicited maternal immunity, serving as a potential clinical biomarker platform. NIH (CA200422, CA180779, DE023926, AI073099, AI116585, AI129496, AI140705, AI069120, AI056154, AI078389, AI28697, AI40718 and AI129534-01), Hastings Foundation, Fletcher Jones Foundation, Departamento de Ciência e Tecnologia (DECIT/25000.072811/2016-17) do Ministério da Saúde do Brasil, and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior CAPES/88887.116627/2016-01.

Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression

The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.