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      Differential virulence between Asian and African lineages of Zika virus

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          Abstract

          Zika virus (ZIKV) is a small enveloped positive-sense single-stranded RNA virus belonging to the genus Flavivirus of the Flaviviridae family that has reemerged in recent years as a human pathogen with epidemic potential. To date, the ongoing epidemic in the Americas has led to tens of thousands of confirmed cases in Brazil alone (Pan American Health Organization Zika-Epidemiological Report, 2 March 2017). More concerning are the reports of severe neurological disorders such as Guillain-Barré syndrome and congenital Zika syndrome (microcephaly and other neurodevelopmental defects), which led the World Health Organization to declare the ZIKV epidemic as a Public Health Emergency of International Concern in 2016. Phylogenetic analyses show that ZIKV can be classified into 2 main lineages: the African lineage and the Asian lineage, the latter of which is responsible for the recent epidemics. Because recent ZIKV infections were associated with the development of congenital and neurological disorders, a key question was raised as to whether Asian-lineage ZIKV strains were phenotypically different from the African lineage strains. It is well described that mutations acquired during Flavivirus evolution can alter their virulence and/or tropism [1]. ZIKV is primarily transmitted by the mosquito species Aedes aegypti and Aedes albopictus. Several studies show that vector transmission can differ between ZIKV strains, as the overall ZIKV infection prevalence and transmission rates of African strains may be higher in A. aegypti than Asian strains [2], suggesting that viral adaptation may have occurred—similar to a single mutation in the chikungunya virus envelope protein that affected vector specificity and epidemic potential. During the recent outbreak, it became clear that ZIKV is sometimes able to cause a prolonged infection. Numerous studies showed that Asian-lineage strains isolated in South America replicate at low levels in tissues months after the initial infection. For example, viral genome can be found in stillborn babies who were infected early during gestation [3], weeks after initial infection in sperm, and in rhesus monkeys, ZIKV can be found in the cerebral spinal fluid (CSF) 42 days after infection, weeks after the virus was cleared from the blood [4]. However, to date, no data are available on the ability of African-lineage strains to cause prolonged infections, but early studies in 2016 suggested that an African ZIKV strain was highly pathogenic and led to cell death; when the first reports on a potential link between microcephaly and ZIKV emerged, laboratories started to study the effect of ZIKV on human neural precursor cells (hNPCs)/human neural stem cells (hNSCs) with MR766, the most available strain at the time (Fig 1 and Table 1). These first studies showed strong tropism and deleterious effects on NSC homeostasis and growth (e.g., [5]). However, some inconsistencies between this rather strong virulence and the long-term developmental effects associated with ZIKV infections led researchers to question the biological validity of this strain, which has been passaged many times in animals and in cells and acquired mutations and deletions at multiple positions during passaging (Table 1) [6–8]. The relevance of this strain was also questioned in a study that showed reduced toxicity and long-term persistence in human neural progenitors of an Asian-lineage ZIKV strain from Puerto Rico, which seemed more consistent with clinical manifestations [9]. Consequently, we compared the neurovirulence ex vivo of 2 low-passaged African- and Asian-lineage ZIKV strains [6]. The African ArB41644 strain was isolated in Central African Republic in 1989, whereas the Asian H/PF/2013 strain was isolated in 2013 during the French Polynesian epidemic (Table 1). We showed that the African ZIKV strain displayed a higher infectivity and replication rate in hNSCs than the Asian ZIKV strain. ArB41644 triggered more defects in proliferation and more apoptosis, which correlated with a stronger induction of the antiviral response [6]. In a subsequent study, we compared 2 Asian-lineage ZIKV strains (H/PF/2013 and ZIKVNL00013, isolated during the current epidemic in 2015) with 2 African-lineage ZIKV strains (MR766 and Uganda 927) in human neural progenitor cells and confirmed the phenotypic differences between ZIKV strains from the Asian and African lineages [10]. Both African strains infected more cells, replicated to higher titers, and induced early cell death more frequently than the Asian-lineage ZIKV strains [10]. The milder virulence of the Asian strains in these studies could be consistent with a persistent infection and effect of the Asian ZIKV lineage in developing neural cells. In addition, in dendritic cells, infection by African (MR766 and Dakar-1984) and Asian (PR-2015) strains led also to a difference in pathogenicity, as African strains triggered more cellular death [8]. Notably, this study also showed that despite differences in viral replication rates between the extensively passaged MR766 strain and the more recently isolated Dakar-1984 strain, the inductions of cell death were similar, suggesting that the ability to cause cell death seen in various cellular models with MR766 could still in certain circumstances be representative for African ZIKV lineage strains [8]. 10.1371/journal.pntd.0005821.g001 Fig 1 Phylogenetic analyses of ZIKV strains discussed. 10.1371/journal.pntd.0005821.t001 Table 1 Passage history of ZIKV strains. Lineage Strain name GenBank Country of origin Year Reference Passage histories African MR766 NC_012532; HQ234498.1; AY632535 Uganda 1947 [8] Unknown + 1x Vero [11] Not indicated [12] Not indicated [13] Not indicated [10] Not indicated [14] Not indicated [15] Not indicated [16] Not indicated [17] Not indicated [18] 146x SM, 1x C6/36, 1x Vero [19] 146x SM, 3x Vero [20] Not indicated [21] Not indicated Uganda 976 Uganda 1962 [10] 6x Vero P6-740 HQ234499.1 Malaysia 1966 [8] Unknown + 1x Vero [18] 6x SM, 1x BHK, 1x C6/36, 3x Vero IbH30656 HQ234500 Nigeria 1968 [16] Not indicated Dakar41519 HQ234501 Senegal 1984 [14] Not indicated [18] 1x AP61, 1x C6/36, 3x Vero Dakar 41671 Senegal 1984 [14] Not indicated Dakar 41677 Senegal 1984 [14] Not indicated DakAr41524 KX198134; KY348860; KX601166 Senegal 1984 [8] Unknown + 1x Vero DakAr41525 KU955591 Senegal 1985 [22] 5x Vero, 1x C6/36, 2x Vero [2] 1x AP61, 1x C6/36 ArB41644 Central African Republic 1989 [10] 5x Vero [6] 5x Vero HD78788 KF383039 Senegal 1991 [13] Not indicated MP1751 Uganda 1962 [23] 3x SM,4x unknown methods, 1x Vero Asian FSS13025 KU955593; JN860885.1 Cambodia 2010 [15] Not indicated [22] 1x AP-1, 1x C6/36, 5x Vero [2] 1x Vero, 1x C6/36 [17] Not indicated [18] 5x Vero [21] Not indicated H/PF/2013 KX369547; KJ776791 French Polynesia 2013 [12] Not indicated [10] 4x Vero [14] Not indicated [24] 6x C6/36 [6] 5x Vero [20] Limited passages in Vero BeH815744 KU365780 Brazil 2015 [24] 3x C6/36 BeH819015 KU365778.1 Brazil 2015 [20] Not indicated FB-GWUH-2016 KU870645 Guatemala 2015 [12] Not indicated FLR KU820897; KX087102 Colombia 2015 [16] Not indicated Mex1-7 Mexico 2015 [22] 3x Vero [2] 4x Vero PRVABC59 KU501215; KU501215.1 Puerto Rico 2015 [8] Unknown + 1x Vero [23] 4x Vero [15] Not indicated [16] Not indicated [18] 3x Vero [19] ZIKVBR Brazil 2015 [11] Not indicated ZKV2015 KU497555 Brazil 2015 [4] Not indicated ZIKVNL00013 KU937936 Surinam 2016 [10] 4x Vero GZ01 KU820898 Venezuela 2016 [15] Not indicated SZ01 KU866423 Samoa 2016 [16] Not indicated Abbreviations: BHK, baby hamster kidney, SM suckling mice Since infection with ZIKV strains from the Asian lineage is associated with microcephaly in humans, it is likely that ZIKV crosses the placental barrier early in gestation, when the brain is starting to develop. Some studies showed that cells of the placenta are susceptible to ZIKV infection, in line with the congenital infections and neurological symptoms associated. Because infections early in gestation seem to be more linked to microcephaly [25], it was hypothesized that the placenta vulnerability could differ depending on the gestational stage. While the placental syncytiotrophoblast, which is found in mature placenta, appears mostly resistant to ZIKV, a study that compared an African strain (MR766) to an Asian strain (FSS13025, Cambodia) showed that embryonic stem cell (ESC)-derived trophoblasts, which recapitulate primitive placenta cells during implantation, are highly susceptible to ZIKV infection [17]. Interestingly, infection with the African strain again resulted in higher viral replication and cell lysis. These data might indicate that infection with African-lineage ZIKV strains—if they enter the fetus—could result in an early termination of pregnancy, while infection with an Asian-lineage ZIKV strain would be less destructive and more chronic, thereby allowing the pregnancy to continue, leading to the development of congenital malformations [17]. While these ex vivo data suggested that African ZIKV lineage strains are more virulent, it was important to confirm the difference in (neuro)virulence of these 2 lineages in vivo. Interferon-α/β receptor (Ifnar)-/- mice injected subcutaneously with African ZIKV strains (MR766 and 3 strains from Senegal) or with the Asian-lineage H/PF/2013 strain all presented with weight loss and succumbed to the infection after 6 (for Senegal strains) to 10 (H/PF/2013) days. Interestingly, infection with the African-lineage strain MR766 resulted in 20% survival at 2 weeks [14]. Another study compared the symptoms induced by African (Uganda and Senegal) and Asian (Cambodia, Malaysia, and Puerto Rico) ZIKV strains in signal transducer and activator of transcription (Stat2)-/- and Ifnar -/- mice infected subcutaneously [18]. Importantly, infections with African strains resulted in more weight loss, mortality, and severe prolonged neurological symptoms compared to Asian strains. Moreover, induction of type 1 and 2 interferon (IFN) were higher following infection with African strains associated with enhanced levels of inflammatory cytokines such as interleukin 6 (IL6) or tumor necrosis factor (TNF). These observations were confirmed in another study showing that an African strain (MP1751) is pathogenic in A129 mice, contrary to an Asian strain (PRVABC59) that did not cause signs of illness [26]. These studies confirm the ex vivo results in neural and nonneural cells regarding the higher virulence of African strains and could suggest that different ZIKV strains may also display phenotypic differences in human subjects (Fig 1) (Table 2). Many of the findings from immunocompromised mice have been recapitulated in nonhuman primates, such as rhesus and cynomolgus macaques. The development of these different animal models led to new knowledge concerning the pathogenesis of ZIKV. However, these systems have limitations, in particular, studying viral pathogenesis using animals lacking a key component of antiviral immunity, which makes it still difficult to derive definitive conclusions regarding ZIKV virulence in humans. Therefore, many unanswered questions remain, in particular, regarding the mechanisms of host restriction, immune evasion, and inflammatory response as well as the long-term neurodevelopmental implications of congenital infection in humans. 10.1371/journal.pntd.0005821.t002 Table 2 Comparative analyses of ZIKV African and Asian lineages. Model Lineage: Strain Findings Reference Ex vivo Endothelial cells African: MR766, IbH30656; Asian: PRVABC59, FLR Asian-lineage strains replicate faster and isolates induced significant cell death compared to African-lineage strains. [16] Astrocytes African: HD78788, ArB41644; Asian: H/PF/2013 African-lineage strains led to a higher infection rate and viral production infection. Asian-lineage strain led to the expression of these innate immune response genes early, while their induction by the African strain was delayed. [6][13] Neuronal stem cells African: DakAr41525, MR766, ArB41644, Uganda 976; Asian: H/FP/2013, Mex1-7, FSS13025, FB-GWUH-2016, ZIKVNL00013 African-lineage strains led to a higher infection rate and virus production as well as stronger cell death and antiviral response compared to Asian-lineage strains. Protein 53 (p53) plays an important role in apoptosis induced by Asian-lineage strains but not by African-lineage strains. [22][12][6].[21][10] Cerebral organoids African: MR766; Asian: ZIKVBR Asian-lineage strain led to a higher viral production as well as stronger cell death compared to the African strain. [11] Embryonic stem cell–derived trophoblast African: MR766; Asian: FSS13025 African-lineage strain led to a higher viral production as well as stronger cell death compared to Asian-lineage strains. [17] Monocyte-derived dendritic cells African: DakAr41524, P6-740, MR766; Asian: PRVABC59 African-lineage strains led to a higher infection rate and viral production as well as stronger cell death and antiviral response compared to Asian-lineage strains. [8] Neuronal cell lines (SK-N-SH, U87 MG) African: MR766, Uganda 976; Asian: H/FP/2013, ZIKVNL00013 African-lineage strains led to a higher infection rate and virus production. [10] In Vivo Mosquito: Aedes aegypti African: DAKAR41525, MR766; Asian: MEX1–7, PRVABC59, GZ01, FSS13025, H/PF/2013, BeH815744 Infection prevalence and transmission rate of African-lineage strains is higher compared to Asian-lineage strains. Infection prevalence and transmission rate of Asian-lineage strains from the Americas are higher compared to Asian-lineage strains from Asia. [15][24][2][19] Mouse: A129 African: MR766, P6-740, DAKAR41519,41667,41671,MP1751; Asian: H/PF/2013, PRVABC59, FSS13025, BeH819015, PRVABC59 African lineage is pathogenic whereas Asian lineage does not cause sign of illness. [23] Ifnar1 -/- Irf3 -/- Irf5 -/- Irf7 -/- African-lineage strains induced higher mortality and severe neurological symptoms in a short period as compared to Asian-lineage strains. [14] Stat2 -/- Ifnar1 -/- Among Asian-lineage strains, the Cambodia strain presented more severe symptoms including front limb paralysis and lethality, followed by the Malaysia and Puerto Rico strains. The Brazilian strain is least severe. The Uganda strain showed orders of magnitude higher viral RNA level compared to all other strains including Senegal, especially in brain tissue. [18] Ifngr1 -/- Higher levels of interferon type I and type II and inflammatory cytokine induction by African-lineage strains as compared to the Asian-lineage strains. [20] Nonhuman primate Asian: ZKV2015, PRVABC59 No differences observed [4] However, one major challenge in interpreting and comparing the data from these studies is the lack of harmonization of the virus strains used. In this review alone, in which we focused on studies that compared multiple ZIKV strains, at least 12 African and 13 Asian strains were found (Fig 1, Tables 1 and 2). While most studies include the African lineage MR766 strain, this strain cannot be considered as a standard, as it has been extensively passaged and at least 3 MR766 strains exist with genetic differences, including a 4–6 codon deletion in the envelope (E) protein [7]. For other strains, multiple sequences exist, or in some cases, no sequence data are available (Table 1). Therefore, when comparing ZIKV lineages, the strains should be sequenced and experiments more standardized to allow proper comparison between studies. Ideally, more recent African ZIKV strains should become available (as the most recent isolate so far is from 1991) (Table 1). In conclusion, the data reviewed here indicate that there are intrinsic differences in the pathogenicity/virulence of African- and Asian-lineage ZIKV strains. Whether these differences are responsible for differences in clinical presentations should be confirmed, but one can speculate that the phenotype of Asian ZIKV strains (lower infection rate, less virus production, and poor induction of early cell death) might contribute, at least in part, to the ability to cause persistent infections within the CNS of fetuses, while African-lineage ZIKV strains could result in more acute infection. However, even though ex vivo and in vivo data point towards a stronger virulence for African strains, it is still premature to conclude that if an African epidemic declares itself (due to an African ZIKV strain), neurological symptoms with similar or worse gravity than those observed in South America will be found. If confirmed, recent reports of African ZIKV strain infection and potential microcephaly in Guinea Bissau may be proof of what could be expected in this continent [27]. Determining the virulence factors for ZIKV using reverse genetic systems that have now been developed for both African and Asian strains will be crucial to identify the molecular and cellular mechanisms behind differences in ZIKV pathogenicity and also those of other emerging arboviruses.

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          Increase in Reported Prevalence of Microcephaly in Infants Born to Women Living in Areas with Confirmed Zika Virus Transmission During the First Trimester of Pregnancy - Brazil, 2015.

          Widespread transmission of Zika virus by Aedes mosquitoes has been recognized in Brazil since late 2014, and in October 2015, an increase in the number of reported cases of microcephaly was reported to the Brazil Ministry of Health.* By January 2016, a total of 3,530 suspected microcephaly cases had been reported, many of which occurred in infants born to women who lived in or had visited areas where Zika virus transmission was occurring. Microcephaly surveillance was enhanced in late 2015 by implementing a more sensitive case definition. Based on the peak number of reported cases of microcephaly, and assuming an average estimated pregnancy duration of 38 weeks in Brazil (1), the first trimester of pregnancy coincided with reports of cases of febrile rash illness compatible with Zika virus disease in pregnant women in Bahia, Paraíba, and Pernambuco states, supporting an association between Zika virus infection during early pregnancy and the occurrence of microcephaly. Pregnant women in areas where Zika virus transmission is occurring should take steps to avoid mosquito bites. Additional studies are needed to further elucidate the relationship between Zika virus infection in pregnancy and microcephaly.
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            Zika Virus Infection and Stillbirths: A Case of Hydrops Fetalis, Hydranencephaly and Fetal Demise

            Background The rapid spread of Zika virus in the Americas and current outbreak of microcephaly in Brazil has raised attention to the possible deleterious effects that the virus may have on fetuses. Methodology/Principal Findings We report a case of a 20-year-old pregnant woman who was referred to our service after a large Zika virus outbreak in the city of Salvador, Brazil with an ultrasound examination that showed intrauterine growth retardation of the fetus at the 18th gestational week. Ultrasound examinations in the 2nd and 3rd trimesters demonstrated severe microcephaly, hydranencephaly, intracranial calcifications and destructive lesions of posterior fossa, in addition to hydrothorax, ascites and subcutaneous edema. An induced labor was performed at the 32nd gestational week due to fetal demise and delivered a female fetus. ZIKV-specific real-time polymerase chain reaction amplification products were obtained from extracts of cerebral cortex, medulla oblongata and cerebrospinal and amniotic fluid, while extracts of heart, lung, liver, vitreous body of the eye and placenta did not yield detectable products. Conclusions/Significance This case report provides evidence that in addition to microcephaly, there may be a link between Zika virus infection and hydrops fetalis and fetal demise. Given the recent spread of the virus, systematic investigation of spontaneous abortions and stillbirths may be warranted to evaluate the risk that ZIKV infection imparts on these outcomes.
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              A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses

              Zika virus (ZIKV) is a mosquito borne flavivirus, which was a neglected tropical pathogen until it emerged and spread across the Pacific Area and the Americas, causing large human outbreaks associated with fetal abnormalities and neurological disease in adults. The factors that contributed to the emergence, spread and change in pathogenesis of ZIKV are not understood. We previously reported that ZIKV evades cellular antiviral responses by targeting STAT2 for degradation in human cells. In this study, we demonstrate that Stat2 -/- mice are highly susceptible to ZIKV infection, recapitulate virus spread to the central nervous system (CNS), gonads and other visceral organs, and display neurological symptoms. Further, we exploit this model to compare ZIKV pathogenesis caused by a panel of ZIKV strains of a range of spatiotemporal history of isolation and representing African and Asian lineages. We observed that African ZIKV strains induce short episodes of severe neurological symptoms followed by lethality. In comparison, Asian strains manifest prolonged signs of neuronal malfunctions, occasionally causing death of the Stat2 -/- mice. African ZIKV strains induced higher levels of inflammatory cytokines and markers associated with cellular infiltration in the infected brain in mice, which may explain exacerbated pathogenesis in comparison to those of the Asian lineage. Interestingly, viral RNA levels in different organs did not correlate with the pathogenicity of the different strains. Taken together, we have established a new murine model that supports ZIKV infection and demonstrate its utility in highlighting intrinsic differences in the inflammatory response induced by different ZIKV strains leading to severity of disease. This study paves the way for the future interrogation of strain-specific changes in the ZIKV genome and their contribution to viral pathogenesis.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Funding acquisitionRole: Writing – original draft
                Role: Writing – original draft
                Role: Funding acquisitionRole: Writing – review & editing
                Role: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: Writing – original draft
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                21 September 2017
                September 2017
                : 11
                : 9
                : e0005821
                Affiliations
                [1 ] Pathogenesis and Control of Chronic Infections, INSERM/Université de Montpellier/ Etablissement Français du Sang, Inserm, Montpellier, France
                [2 ] Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands
                [3 ] CHU de Montpellier, Department of Bacteriology-Virology, Montpellier, France
                Baylor College of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-4492-2093
                Article
                PNTD-D-17-01090
                10.1371/journal.pntd.0005821
                5608167
                28934211
                6a04a2b4-f8f2-47f4-a047-e6fd41654eed
                © 2017 Simonin et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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                Figures: 1, Tables: 2, Pages: 8
                Funding
                This work was funded by REACting ( https://www.inserm.fr/zoom/reacting-une-approche-multidisciplinaire-pourrelever-le-defi-des-crises-epidemiques). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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