Vector Competence of American Mosquitoes for Three Strains of Zika Virus

In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas.     

Characterization of Lethal Zika Virus Infection in AG129 Mice

BackgroundMosquito-borne Zika virus (ZIKV) typically causes a mild and self-limiting illness known as Zika fever, which often is accompanied by maculopapular rash, headache, and myalgia. During the current outbreak in South America, ZIKV infection during pregnancy has been hypothesized to cause microcephaly and other diseases. The detection of ZIKV in fetal brain tissue supports this hypothesis. Because human infections with ZIKV historically have remained sporadic and, until recently, have been limited to small-scale epidemics, neither the disease caused by ZIKV nor the molecular determinants of virulence and/or pathogenicity have been well characterized. Here, we describe a small animal model for wild-type ZIKV of the Asian lineage.Conclusions/SignificanceFoot pad injection of AG129 mice with ZIKV represents a biologically relevant model for studying ZIKV infection and disease development following wild-type virus inoculation without the requirement for adaptation of the virus or intracerebral delivery of the virus. This newly developed Zika disease model can be exploited to identify determinants of ZIKV virulence and reveal molecular mechanisms that control the virus-host interaction, providing a framework for rational design of acute phase therapeutics and for vaccine efficacy testing.     

Molecular evolution of Zika virus as it crossed the Pacific to the Americas

Zika virus was previously considered to cause only a benign infection in humans. Studies of recent outbreaks of Zika virus in the Pacific, South America, Mexico and the Caribbean have associated the virus with severe neuropathology. Viral evolution may be one factor contributing to an apparent change in Zika disease as it spread from Southeast Asia across the Pacific to the Americas. To address this possibility, we have employed computational tools to compare the phylogeny, geography, immunology and RNA structure of Zika virus isolates from Africa, Asia, the Pacific and the Americas. In doing so, we compare and contrast methods and results for tree search and rooting of Zika virus phylogenies. In some phylogenetic analyses we find support for the hypothesis that there is a deep common ancestor between African and Asian clades (the “Asia/Africa” hypothesis). In other phylogenetic analyses, we find that Asian lineages are descendent from African lineages (the “out of Africa” hypothesis). In addition, we identify and evaluate key mutations in viral envelope protein coding and untranslated terminal RNA regions. We find stepwise mutations that have altered both immunological motif sets and regulatory sequence elements. Both of these sets of changes distinguish viruses found in Africa from those in the emergent Asia–Pacific–Americas lineage. These findings support the working hypothesis that mutations acquired by Zika virus in the Pacific and Americas contribute to changes in pathology. These results can inform experiments required to elucidate the role of viral genetic evolution in changes in neuropathology, including microcephaly and other neurological and skeletomuscular issues in infants, and Guillain‐Barré syndrome in adults.     

Vectors,Hosts, and Control Measures for Zika Virus in the Americas

We examine Zika virus (ZIKV) from an ecological perspective and with a focus on the Americas. We assess (1) the role of wildlife in ZIKV disease ecology, (2) how mosquito behavior and biology influence disease dynamics, and (3) how nontarget species and ecosystems may be impacted by vector control programs. Our review suggests that free-ranging, non-human primates may be involved in ZIKV transmission in the Old World; however, other wildlife species likely play a limited role in maintaining or transmitting ZIKV. In the Americas, a zoonotic cycle has not yet been definitively established. Understanding behaviors and habitat tolerances of Aedes aegypti and Aedes albopictus, two ZIKV competent vectors in the Americas, will allow more accurate modeling of disease spread and facilitate targeted and effective control efforts. Vector control efforts may have direct and indirect impacts to wildlife, particularly invertebrate feeding species; however, strategies could be implemented to limit detrimental ecological effects.     

Zika Virus,a New Threat for Europe?

BackgroundSince its emergence in 2007 in Micronesia and Polynesia, the arthropod-borne flavivirus Zika virus (ZIKV) has spread in the Americas and the Caribbean, following first detection in Brazil in May 2015. The risk of ZIKV emergence in Europe increases as imported cases are repeatedly reported. Together with chikungunya virus (CHIKV) and dengue virus (DENV), ZIKV is transmitted by Aedes mosquitoes. Any countries where these mosquitoes are present could be potential sites for future ZIKV outbreak. We assessed the vector competence of European Aedes mosquitoes (Aedes aegypti and Aedes albopictus) for the currently circulating Asian genotype of ZIKV.Conclusions/SignificanceIn combination with the restricted distribution of European Ae. albopictus, our results on vector competence corroborate the low risk for ZIKV to expand into most parts of Europe with the possible exception of the warmest regions bordering the Mediterranean coastline.     

Inhibition of innate immune response ameliorates Zika virus-induced neurogenesis deficit in human neural stem cells

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.     

Zika virus and Zika fever

An emerging mosquito-borne arbovirus named Zika virus (ZIKV), of the family Flaviviridae and genus Flavivirus, is becoming a global health threat. ZIKV infection was long neglected due to its sporadic nature and mild symptoms. However, recently, with its rapid spread from Asia to the Americas, affecting more than 30 countries, accumulating evidences have demonstrated a close association between infant microcephaly and Zika infection in pregnant women. Here, we reviewed the virological, epidemiological, and clinical essentials of ZIKV infection.

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Zika infection and the development of neurological defects

Starting with the outbreak in Brazil, Zika virus (ZIKV) infection has been correlated with severe syndromes such as congenital Zika syndrome and Guillain‐Barré syndrome. Here, we review the status of Zika virus pathogenesis in the central nervous system (CNS). One of the main concerns about ZIKV exposure during pregnancy is abnormal brain development, which results in microcephaly in newborns. Recent advances in in vitro research show that ZIKV can infect and obliterate cells from the CNS, such as progenitors, neurons, and glial cells. Neural progenitor cells seem to be the main target of the virus, with infection leading to less cell migration, neurogenesis impairment, cell death and, consequently, microcephaly in newborns. The downsizing of the brain can be directly associated with defective development of the cortical layer. In addition, in vivo investigations in mice reveal that ZIKV can cross the placenta and migrate to fetuses, but with a significant neurotropism, which results in brain damage for the pups. Another finding shows that hydrocephaly is an additional consequence of ZIKV infection, being detected during embryonic and fetal development in mouse, as well as after birth in humans. In spite of the advances in ZIKV research in the last year, the mechanisms underlying ZIKV infection in the CNS require further investigation particularly as there are currently no treatments or vaccines against ZIKV infection.     

Diagnostic differentiation of Zika and dengue virus exposure by analyzing T cell receptor sequences from peripheral blood of infected HLA-A2 transgenic mice

Zika virus (ZIKV) is a significant global health threat due to its potential for rapid emergence and association with severe congenital malformations during infection in pregnancy. Despite the urgent need, accurate diagnosis of ZIKV infection is still a major hurdle that must be overcome. Contributing to the inaccuracy of most serologically-based diagnostic assays for ZIKV, is the substantial geographic and antigenic overlap with other flaviviruses, including the four serotypes of dengue virus (DENV). Within this study, we have utilized a novel T cell receptor (TCR) sequencing platform to distinguish between ZIKV and DENV infections. Using high-throughput TCR sequencing of lymphocytes isolated from DENV and ZIKV infected mice, we were able to develop an algorithm which could identify virus-associated TCR sequences uniquely associated with either a prior ZIKV or DENV infection in mice. Using this algorithm, we were then able to separate mice that had been exposed to ZIKV or DENV infection with 97% accuracy. Overall this study serves as a proof-of-principle that T cell receptor sequencing can be used as a diagnostic tool capable of distinguishing between closely related viruses. Our results demonstrate the potential for this innovative platform to be used to accurately diagnose Zika virus infection and potentially the next emerging pathogen(s).     

Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere

Zika virus (ZIKV) had remained a relatively obscure flavivirus until a recent series of outbreaks accompanied by unexpectedly severe clinical complications brought this virus into the spotlight as causing an infection of global public health concern. In this review, we discuss the history and epidemiology of ZIKV infection, recent outbreaks in Oceania and the emergence of ZIKV in the Western Hemisphere, newly ascribed complications of ZIKV infection, including Guillain-Barré syndrome and microcephaly, potential interactions between ZIKV and dengue virus, and the prospects for the development of antiviral agents and vaccines.     

Xanthenedione (and intermediates involved in their synthesis) inhibit Zika virus migration to the central nervous system in murine neonatal models

Zika Virus (ZIKV), an arbovirus that belongs to the Flaviviridae family, has become a global concern since its outbreak in the Americas in 2015. With symptoms similar to other Flavivirus as Dengue and Yellow Fever viruses, infections by ZIKV have also been related to several neurological complications such as microcephaly in newborns and Guillain-Barre syndrome. Considering the high prevalence of ZIKV infection in certain areas, the risks that the virus poses to fetal brain development, and the fact that there is no vaccine or specific prophylaxis available, an effective treatment capable of preventing the infection is of potential interest. Therefore, in the present investigation, the antiviral activity on ZIKV of a group of xanthenodiones and intermediate ketones involved in their synthesis was evaluated for the first time. It was found that the compound 2-(2,6-dichlorobenzylidene)cyclohexane-1,3-dione 27 was able to completely inhibit the viral infection of Vero cells as well as to significantly reduce viral load in the brains of newborn Swiss mice. These effects are related to a direct interaction of the compound with the viral particle, blocking the viral adsorption.     

Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies

Here we propose a strategy allowing implementing efficient and practicable large-scale seroepidemiological studies for Zika Virus (ZIKV). It combines screening by a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (CPE-based VNT). In post-epidemic samples from Martinique Island blood donors (a population with a dengue seroprevalence above 90%), this strategy allowed reaching specificity and sensitivity values over 98%. The CPE-based VNT consists of recording CPE directly under the optical microscope, which is easy to identify with ZIKV strain H/PF/2013 at day 5 pi. Overall, considered that CPE-based VNT is cost effective and widely automatable, the NS1 protein-based Zika IgG ELISA+CPE-based VNT combination strategy represents a convenient tool to expedite ZIKV seroprevalence studies.     

Working with Zika and Usutu Viruses In Vitro

Usutu (USUV) and Zika (ZIKV) viruses are emerging arboviruses of significant medical and veterinary importance. These viruses have not been studied as well as other medically important arboviruses such as West Nile (WNV), dengue (DENV), or chikungunya (CHIKV) viruses. As such, information regarding the behavior of ZIKV and USUV viruses in the laboratory is dated. Usutu virus re-emerged in Austria in 2001 and has since spread throughout the European and Asian continents causing significant mortality among birds. Zika virus has recently appeared in the Western Hemisphere and has exhibited high rates of birth defects and sexual transmission. Information about the characteristics of USUV and ZIKV viruses are needed to better understand the transmission, dispersal, and adaptation of these viruses in new environments. Since their initial characterization in the middle of last century, technologies and reagents have been developed that could enhance our abilities to study these pathogens. Currently, standard laboratory methods for these viruses are limited to 2–3 cell lines and many assays take several days to generate meaningful data. The goal of this study was to characterize these viruses in cells from multiple diverse species. Cell lines from 17 species were permissive to both ZIKV and USUV. These viruses were able to replicate to significant titers in most of the cell lines tested. Moreover, cytopathic effects were observed in 8 of the cell lines tested. These data indicate that a variety of cell lines can be used to study ZIKV and USUV infection and may provide an updated foundation for the study of host-pathogen interactions, model development, and the development of therapeutics.     

Optimization of Zika virus envelope protein production for ELISA and correlation of antibody titers with virus neutralization in Mexican patients from an arbovirus endemic region

Background

Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications.

Methods

We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera.

Results

Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro.

Conclusions

Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.

     

Clinical manifestations and health outcomes associated with Zika virus infections in adults: A systematic review

BackgroundZika virus (ZIKV) has generated global interest in the last five years mostly due to its resurgence in the Americas between 2015 and 2016. It was previously thought to be a self-limiting infection causing febrile illness in less than one quarter of those infected. However, a rise in birth defects amongst children born to infected pregnant women, as well as increases in neurological manifestations in adults has been demonstrated. We systemically reviewed the literature to understand clinical manifestations and health outcomes in adults globally.MethodsThis review was registered prospectively with PROPSERO (CRD 42018096558). We systematically searched for studies in six databases from inception to the end of September 2020. There were no language restrictions. Critical appraisal was completed using the Joanna Briggs Institute Critical Appraisal Tools.FindingsWe identified 73 studies globally that reported clinical outcomes in ZIKV-infected adults, of which 55 studies were from the Americas. For further analysis, we considered studies that met 70% of critical appraisal criteria and described subjects with confirmed ZIKV. The most common symptoms included: exanthema (5,456/6,129; 89%), arthralgia (3,809/6,093; 63%), fever (3,787/6,124; 62%), conjunctivitis (2,738/3,283; 45%), myalgia (2,498/5,192; 48%), headache (2,165/4,722; 46%), and diarrhea (337/2,622; 13%). 36/14,335 (0.3%) of infected cases developed neurologic sequelae, of which 75% were Guillain-Barré Syndrome (GBS). Several subjects reported recovery from peak of neurological complications, though some endured chronic disability. Mortality was rare (0.1%) and hospitalization (11%) was often associated with co-morbidities or GBS.ConclusionsThe ZIKV literature in adults was predominantly from the Americas. The most common systemic symptoms were exanthema, fever, arthralgia, and conjunctivitis; GBS was the most prevalent neurological complication. Future ZIKV studies are warranted with standardization of testing and case definitions, consistent co-infection testing, reporting of laboratory abnormalities, separation of adult and pediatric outcomes, and assessing for causation between ZIKV and neurological sequelae.     

Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China

Zika virus(ZIKV) has been isolated from mosquitoes such as Aedes, Mansonia uniformis, and Culex perfuscus; However,the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported. In June and July2018, 22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China. Among them, six strains of ZIKV were isolated from mosquitoes: four from An. sinensis and two from Cx. tritaeniorhynchus. Molecular genetic analysis showed that the ZIKV isolated from An. sinensis and Cx. tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV. In addition, the ZIKV strains isolated from An. sinensis and Cx. tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015. This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province, China; This is also the first time that ZIKV has been isolated from An. sinensis and Cx. tritaeniorhynchus. Given that An. sinensis and Cx. tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia, the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.     

寨卡疫苗研究进展

寨卡病毒系由伊蚊传播的黄病毒,超过20亿人在其流行区域生活。近几年,在中南美洲乃至世界范围内爆发的寨卡疫情已对全球公共卫生事业构成严重威胁。已有研究证实寨卡病毒感染为格林巴利综合征的病因之一;孕妇感染寨卡病毒后,可造成新生儿小头症。早日研制出安全有效的寨卡疫苗之重要性不言而喻。全球率先报道的寨卡疫苗为一款DNA疫苗,其在设计、制备和生产方面均较其他类型疫苗更加简易,且可避免可复制型疫苗因毒力回复而造成对孕妇和胎儿的健康威胁,在寨卡疫苗的研究中具有显著优势。其他传统类型疫苗和基于抗体的新型疫苗等均有研究机构开展研究,并已取得阶段性进展,本文将扼要综述寨卡疫苗的研究现状与进展。     

Spatial and temporal invasion dynamics of the 2014–2017 Zika and chikungunya epidemics in Colombia

Zika virus (ZIKV) and chikungunya virus (CHIKV) were recently introduced into the Americas resulting in significant disease burdens. Understanding their spatial and temporal dynamics at the subnational level is key to informing surveillance and preparedness for future epidemics. We analyzed anonymized line list data on approximately 105,000 Zika virus disease and 412,000 chikungunya fever suspected and laboratory-confirmed cases during the 2014–2017 epidemics. We first determined the week of invasion in each city. Out of 1,122, 288 cities met criteria for epidemic invasion by ZIKV and 338 cities by CHIKV. We analyzed risk factors for invasion using linear and logistic regression models. We also estimated that the geographic origin of both epidemics was located in Barranquilla, north Colombia. We assessed the spatial and temporal invasion dynamics of both viruses to analyze transmission between cities using a suite of (i) gravity models, (ii) Stouffer’s rank models, and (iii) radiation models with two types of distance metrics, geographic distance and travel time between cities. Invasion risk was best captured by a gravity model when accounting for geographic distance and intermediate levels of density dependence; Stouffer’s rank model with geographic distance performed similarly well. Although a few long-distance invasion events occurred at the beginning of the epidemics, an estimated distance power of 1.7 (95% CrI: 1.5–2.0) from the gravity models suggests that spatial spread was primarily driven by short-distance transmission. Similarities between the epidemics were highlighted by jointly fitted models, which were preferred over individual models when the transmission intensity was allowed to vary across arboviruses. However, ZIKV spread considerably faster than CHIKV.