深圳市输入性寨卡病毒分离株的进化分析及生物学特性研究

目的 对从深圳宝安国际机场口岸入境的1例自柬埔寨归国发热人员进行寨卡病毒实验室检测,研究寨卡病毒分离株的遗传进化和生物学特征,为寨卡病毒病的预防控制提供参考依据。方法 采集患者血液、唾液和尿液样本,采用荧光RT-PCR方法进行寨卡病毒核酸检测;选用多种组织培养细胞分离病毒,分别观察病毒株的致病变效应、空斑形成及病毒滴度等,并对两株病毒基因序列进行分子遗传进化研究,分析输入性病例来源。结果 荧光RT-PCR结果显示,该病例血清、唾液以及尿液样本寨卡病毒核酸阳性并持续一段时间。首次成功从唾液和尿液标本中分离到寨卡病毒株,将其分别命名为ZIKV/S/SZ1901和ZIKV/U/SZ1901。两分离株均可引起Vero细胞病变,不引起BHK-21细胞病变;均可在Vero细胞中形成空斑,滴度分别为3.4×10⁵ pfu/mL、1.4×10³ pfu/mL。RT-PCR扩增出预期大小约10 272 bp片段,测序结果表明该片段与ZIKV/Hu/Thai/KngSG/17-D501株相应序列的同源性为99.6%。系统进化树显示该病毒属亚洲谱系,与输入至我国的其它寨卡病毒株位于不同的进化分支。寨卡病毒编码区氨基酸位点分析提示,SZ1901毒株在结构基因的氨基酸位点(D683E、V763M、T777M)和非结构基因NS1基因的(A188V)变异与近几年流行株完全相同。结论 根据患者流行病学史、临床表现和实验室检测结果,确诊该病例为深圳市从柬埔寨输入性寨卡病毒感染病例,且寨卡病毒分离株具备与2016年以来流行的寨卡病毒大部分相同的分子基础。     

Update on the Transmission of Zika Virus Through Breast Milk and Breastfeeding: A Systematic Review of the Evidence

We systematically searched regional and international databases and screened 1658 non-duplicate records describing women with suspected or confirmed ZIKV infection, intending to breastfeed or give breast milk to an infant to examine the potential of mother-to-child transmission of Zika virus (ZIKV) through breast milk or breastfeeding-related practices. Fourteen studies met our inclusion criteria and inform this analysis. These studies reported on 97 mother–children pairs who provided breast milk for ZIKV assessment. Seventeen breast milk samples from different women were found positive for ZIKV via RT-PCR, and ZIKV replication was found in cell cultures from five out of seven breast milk samples from different women. Only three out of six infants who had ZIKV infection were breastfed, no evidence of clinical complications was found to be associated with ZIKV RNA in breast milk. This review updates our previous report by including 12 new articles, in which we found no evidence of ZIKV mother-to-child transmission through breast milk intake or breastfeeding. As the certainty of the present evidence is low, additional studies are still warranted to determine if ZIKV can be transmitted through breastfeeding.     

广东首例苏里南输入性寨卡病毒感染病例的实验室检测

目的对从广州白云国际机场口岸入境的一例自苏里南归国发热人员进行寨卡病毒实验室检测。方法采集发热人员的血液和唾液样本,同时使用两种寨卡病毒实时荧光RT-PCR试剂进行寨卡病毒核酸检测。RT-PCR扩增寨卡病毒部分基因片段,并进行序列测定和同源性比较。基于扩增片段构建系统进化树,分析输入性病例的来源。结果实时荧光RT-PCR结果显示,该病例血清样本寨卡病毒核酸弱阳性、唾液样本寨卡病毒核酸阳性。RT-PCR扩增出预期大小约1.5Kb片段,测序结果表明该片段与巴西寨卡病毒株(GenBank号KX197250)相应序列的同源性为99%。系统进化树显示该病毒属亚洲谱系。结论根据患者流行病学史、临床表现和病例标本核酸检测情况,确诊该病例为广东首例从苏里南地区输入性寨卡病例。     

Serological Evidence of Widespread Zika Transmission across the Philippines

Zika virus (ZIKV) exposure across flavivirus-endemic countries, including the Philippines, remains largely unknown despite sporadic case reporting and environmental suitability for transmission. Using laboratory surveillance data from 2016, 997 serum samples were randomly selected from suspected dengue (DENV) case reports across the Philippines and assayed for serological markers of short-term (IgM) and long-term (IgG) ZIKV exposure. Using mixture models, we re-evaluated ZIKV IgM/G seroprevalence thresholds and used catalytic models to quantify the force of infection (attack rate, AR) from age-accumulated ZIKV exposure. While we observed extensive ZIKV/DENV IgG cross-reactivity, not all individuals with active DENV presented with elevated ZIKV IgG, and a proportion of dengue-negative cases (DENV IgG-) were ZIKV IgG-positive (14.3%, 9/63). We identified evidence of long-term, yet not short-term, ZIKV exposure across Philippine regions (ZIKV IgG+: 31.5%, 314/997) which was geographically uncorrelated with DENV exposure. In contrast to the DENV AR (12.7% (95%CI: 9.1–17.4%)), the ZIKV AR was lower (5.7% (95%CI: 3–11%)) across the country. Our results provide evidence of widespread ZIKV exposure across the Philippines and suggest the need for studies to identify ZIKV infection risk factors over time to better prepare for potential future outbreaks.     

Zika Virus Antibody Titers Three Years after Confirmed Infection

Background: In 2015–2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. Methods: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. Results: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. Conclusions: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.     

Seroprevalence of Zika Virus in Amphawa District,Thailand, after the 2016 Pandemic

In 2016, Zika virus (ZIKV) infection was declared a public health emergency of international concern because of the neurological consequences in babies born to infected people. Because of the mild and nonspecific symptoms, serological tests are essential in epidemiological studies. However, cross-reactive antibodies between other Flaviviridae members may complicate the interpretation of results of these tests. This study investigated the seroprevalence of ZIKV infection in Samut Songkhram in central Thailand which was affected by the Zika outbreak of 2016. Three hundred and fifty volunteers aged 5–50 years in Amphawa District, Samut Songkhram, were enrolled between April 2017 and April 2018. ZIKV nonstructural protein 1 (NS1) immunoglobulin G enzyme-linked immunosorbent assay (ELISA) was used to screen serum samples collected on the first day of enrollment and after 6 and 12 months. The seroprevalence and seroconversion of ZIKV were assessed. Cases of ZIKV seroconversion were verified as evidence of ZIKV infection by NS1 blockade-of-binding ELISA and plaque reduction neutralization test (PRNT50). ZIKV seroprevalence in Amphawa was 15.1–17.8% with no significant change over the year. The total seroconversion rate throughout the year was 7/100 person-years. The ratio of asymptomatic to symptomatic infections was 4.5:1. The cases in our study confirmed the occurrence of occult ZIKV infections in the community. These undetected infections might promote the spread of ZIKV in vulnerable groups of the community.     

Zika病毒:虫媒病毒传染病的新挑战

Zika病毒(Zika virus,ZIKV)是一种新发的虫媒病毒,伊蚊是其主要的传播媒介。Zika病毒感染后主要引起发热,皮疹,关节痛等轻症症状。2015年起,Zika病毒在拉丁美洲及南美洲多个国家流行,并且Zika病毒感染患者不仅出现发热,皮疹和关节痛等症状,还发现Zika病毒病的流行与婴儿小头畸形,格林巴利综合征,病毒性脑膜炎病例的发生相关。不仅如此,Zika病毒感染还可以通过性途径传播。因此,从病毒感染症状的复杂性来看,目前流行的Zika病毒已经与1947年刚发现时的Zika病毒完全不同,而是成为了一种“新病毒”。 本文系统地梳理总结了Zika病毒感染相关的最新研究进展,并提出Zika病毒及其感染是对虫媒病毒传染病的新挑战的概念。     

Flavivirus cross‐reactivity in serological tests and Guillain‐Barré syndrome in a hematopoietic stem cell transplant patient: A case report

Serological diagnosis of flavivirus infection is a challenge, particularly in the context of a disease associated with immune response enhancement in a transplant patient, where aspects such as previous flavivirus infections may be involved with the outcome. We report a case of a pediatric patient who developed Guillain‐Barré syndrome (GBS) after matched‐unrelated hematopoietic stem cell transplantation (HSCT). The patient lives in a Brazilian region that is experiencing an epidemic of Zika virus (ZIKV) and dengue virus (DENV). Because an increasing number of cases of GBS, likely triggered by ZIKV infection, are being reported in Brazil, samples from the patient were tested for both ZIKV and DENV infection. Serological assays strongly suggested a recent ZIKV infection, although infection by DENV or co‐infection with both viruses cannot be ruled out. The presence of anti‐DENV immunoglobulin‐G in donor serum led to the hypothesis that antibodies from the donor could have enhanced the severity of the ZIKV infection. This hypothesis is in agreement with the recent findings that DENV sero‐cross‐reactivity drives antibody‐dependent enhancement of ZIKV infection. These findings highlight the need for discussion of the indication to perform previous flavivirus tests in HSCT donors, especially in areas where ZIKV and other flaviviruses co‐circulate.     

Pregnant Women Infected with Zika Virus Show Higher Viral Load and Immunoregulatory Cytokines Profile with CXCL10 Increase

Background: Zika virus (ZIKV) infection during pregnancy usually shows only mild symptoms and is frequently subclinical. However, it can be vertically transmitted to the fetus, causing microcephaly and other congenital defects. During pregnancy, the immune environment modifications can alter the response to viruses in general and ZIKV in particular. Objective: To describe the role of pregnancy in the systemic pro- and anti-inflammatory response during symptomatic ZIKV infection. Materials and Methods: A multiplex assay was used to measure 25 cytokines, chemokines, and receptors in 110 serum samples from pregnant and nonpregnant women with and without ZIKV infection with and without symptoms. Samples were collected through an epidemiological surveillance system. Results: Samples from pregnant women with ZIKV infection showed a higher viral load but had similar profiles of inflammatory markers as compared with nonpregnant infected women, except for CXCL10 that was higher in infected pregnant women. Notably, the presence of ZIKV in pregnancy favored a regulatory profile by significantly increasing anti-inflammatory cytokines such as interleukin (IL)-10, receptors IL-1RA, and IL-2R, but only those pro-inflammatory cytokines such as IL-6, interferon (IFN)-α, IFN-γ and IL-17 that are essential for the antiviral response. Interestingly, there were no differences between symptomatic and weakly symptomatic ZIKV-infected groups. Conclusion: Our results revealed a systemic anti-inflammatory cytokine and chemokine profile that could participate in the control of the virus. The anti-inflammatory response in pregnant women infected with ZIKA was characterized by high CXCL10, a cytokine that has been correlated with congenital malformations.     

Healthcare workers' knowledge towards Zika virus infection in Indonesia:A survey in Aceh

Objective:To assess the knowledge on Zika virus(ZIKV) infection among healthcare providers(doctors) in Aceh province,Indonesia.Methods:A self-administered internet based survey was conducted from 3 May to 3 June 2016 among the members of doctor organizations in Aceh province.A set of validated,pre-tested questionnaire was used to measure knowledge regarding ZIKV infection and to collect a range of explanatory variables.A two-steps logistic regression analysis was employed to assess the association of participants' demographic,workplace characteristics and other explanatory variables with the knowledge.Results:A total of 442 participants included in the final analysis and 35.9% of them(159) had a good knowledge on ZIKV infection.Multivariate model revealed that type of occupation,type of workplace,availability of access to medical journals and experience made Zika disease as differential diagnose were associated with knowledge on ZIKV infection.In addition,three significant source of information regarding ZIKV were online media(60%),medical article or medical news(16.2%) and television(13.2%).Conclusion:The knowledge of the doctors in Aceh regarding ZIKV infection is relatively low.Doctors who have a good knowledge on ZIKV infection are more confident to established ZIKV disease as differential diagnosis in their clinical setting.Therefore,such program to increase healthcare providers' knowledge regarding ZIKV infection is needed to screen potential carriers of ZIKV infection.     

High Incidence of Zika or Chikungunya Infection among Pregnant Women Hospitalized Due to Obstetrical Complications in Northeastern Brazil—Implications for Laboratory Screening in Arbovirus Endemic Area

The diagnostic of arbovirus-related obstetric complications in high-risk pregnancy and childbirth care is challenging, especially in endemic areas. We conducted a prospective study to track active or recent Zika (ZIKV), dengue (DENV), or chikungunya (CHIKV) virus infection among hospitalized pregnant women (PW) with obstetric complications in a hospital at the epicenter of Zika outbreak and ZIKV-related microcephaly in Brazil. Clinical data and blood samples were collected at enrollment and 10 days after the admission of study participants, between October 2018 and May 2019. Further clinical data were extracted from medical records. Samples were screened by molecular and serological tests. Out of 780 participants, 93.1% (95% CI: 91.1–94.7%) presented previous DENV exposure (IgG). ZIKV, CHIKV, and/or DENV laboratory markers of recent or active infection were detected in 130 PW, yielding a prevalence of 16.6% (95% CI: 14.2–19.5%); 9.4% (95% CI: 7.4–11.7%), 7.4% (95% CI: 5.7–9.7%), and 0.38% (95% CI: 0.1–1.2%) of CHIKV, ZIKV, and DENV infections, respectively. Most ZIKV infections were detected by molecular assays (89.6%), while CHIKV infections were detected by serology (95.9%). Our findings highlight the need for arbovirus infections screening in PW with obstetrical complications, potentially associated to these infections in endemic areas regardless of the signs or symptoms suggestive of arboviral disease.     

Can Zika Virus Infection in High Risk Pregnant Women Be Differentiated on the Basis of Symptoms?

Zika virus (ZIKV) infection in pregnancy is associated with congenital neurological abnormalities. Our understanding of the full clinical spectrum of ZIKV infection is incomplete. Using data from this prospective cohort study consisting of 650 women attending a high-risk pregnancy clinic during the Zika virus outbreak in Brazil, we investigated the extent to which specific symptoms can be utilized to differentiate ZIKV-infected pregnant women from those with other pregnancy-related problems. All were tested for ZIKV in urine by RT–qPCR. Demographic and clinical data including physical symptoms during follow-up were recorded and analyzed with respect to Zika virus exposure status. Forty-eight (7.4%) women were positive for ZIKV by RT–qPCR. The majority (70.8%) were asymptomatic, and only four ZIKV-positive women (8.3%) reported symptoms during pregnancy that met the WHO case definition. Zika-positive and -negative women reported similar frequencies of ZIKV-like symptoms (as per the WHO definition): fever (16.7% vs. 13.6%), arthralgia/arthritis (10.4% vs. 11.3%), rash (4.2% vs. 5.3%), and conjunctivitis (2.1% vs. 3.2%). Most pregnant women positive for ZIKV in urine are asymptomatic and do not deliver a baby with microcephaly. Physical symptoms alone did not differentiate between high-risk pregnant women positive or negative for ZIKV.     

Inhibition of Brazilian ZIKV strain replication in primary human placental chorionic cells and cervical cells treated with nitazoxanide

Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.     

寨卡病毒动物感染模型研究进展

寨卡病毒是一种经由蚊媒传播的重要的黄病毒属病毒,已导致全球数百万人感染,引发严峻的公共卫生危机。2015年以来,尤其是中南美洲地区暴发的寨卡疫情出现了新生儿小头畸形、大脑发育异常与格林巴列综合征等不同以往的严重症状,引发高度关注。合适的动物感染模型对于研究寨卡病毒的传播方式、致病机理及研发防控寨卡病毒感染的新药、疫苗及诊断试剂等都有十分重要的意义。本文就近年来建立的寨卡病毒动物感染模型进展进行综述。     

A Glimmer of Hope: Recent Updates and Future Challenges in Zika Vaccine Development

The emergence and rapid spread of Zika virus (ZIKV) on a global scale as well as the establishment of a causal link between Zika infection and congenital syndrome and neurological disorders triggered unprecedented efforts towards the development of a safe and effective Zika vaccine. Multiple vaccine platforms, including purified inactivated virus, nucleic acid vaccines, live-attenuated vaccines, and viral-vectored vaccines, have advanced to human clinical trials. In this review, we discuss the recent advances in the field of Zika vaccine development and the challenges for future clinical efficacy trials. We provide a brief overview on Zika vaccine platforms in the pipeline before summarizing the vaccine candidates in clinical trials, with a focus on recent, promising results from vaccine candidates that completed phase I trials. Despite low levels of transmission during recent years, ZIKV has become endemic in the Americas and the potential of large Zika outbreaks remains real. It is important for vaccine developers to continue developing their Zika vaccines, so that a potential vaccine is ready for deployment and clinical efficacy trials when the next ZIKV outbreak occurs.     

Knowledge of Zika Virus Transmission and Its Prevention among High-Risk Pregnant Women in Brazil

Recent outbreaks of Zika virus (ZIKV) infection highlight the urgent need to evaluate the efficacy of current public health measures to educate susceptible groups about how to prevent infection, modes of viral transmission, and consequences of infection. We performed a cross-sectional study in the city of Jundiaí, São-Paulo, from March 2016 to August 2017. In 315 high-risk pregnant women we evaluated the rate of ZIKV infection, knowledge of pathways of ZIKV transmission, and the use of protective measures. Data were analyzed and correlated with sociodemographic variables. The rate of ZIKV infection was 10.8%. ZIKV transmission by mosquitoes was the best-known means of virus acquisition, while transmission of ZIKV by sexual intercourse as well as mother–fetus transmission was known by less than half of the women. The use of insect repellent, reported by 53% of participants, was correlated with higher education and personal directives from health professionals. Condom use was reported by 19.5% of subjects. Improved strategies to increase awareness of ZIKV infection and its consequences, designed to appeal to specific, targeted populations, are clearly necessary to more accurately prevent the spread of this infection and diminish adverse consequences in the pregnant population.     

Lack of Association between Adverse Pregnancy Outcomes and Zika Antibodies among Pregnant Women in Thailand between 1997 and 2015

Data about Zika virus infection and adverse pregnancy outcomes in Southeast Asia are scarce. We conducted an unmatched case-control study of Zika virus (ZIKV) serology in pregnant women enrolled in human immunodeficiency virus (HIV) or hepatitis B virus (HBV) perinatal prevention trials between 1997 and 2015 in Thailand. Case and control groups included women with and without adverse pregnancy outcomes. Plasma samples collected during the last trimester of pregnancy were tested for ZIKV IgG/IgM and Dengue IgG/IgM (Euroimmun, AG, Germany). Case newborn plasma samples were tested for ZIKV IgM and ZIKV RNA (Viasure, Spain). The case group included women with stillbirth (n = 22) or whose infants had microcephaly (n = 4), a head circumference below the first percentile (n = 14), neurological disorders (n = 36), or had died within 10 days after birth (n = 11). No women in the case group were positive for ZIKV IgM, and none of their live-born neonates were positive for ZIKV IgM or ZIKV RNA. The overall ZIKV IgG prevalence was 29%, 24% in the case and 34% in the control groups (Fisher’s exact test; p = 0.13), while the dengue IgG seroprevalence was 90%. Neither neonatal ZIKV infections nor ZIKV-related adverse pregnancy outcomes were observed in these women with HIV and/or HBV during the 18-year study period.     

Incorporation of CD55 into the Zika Viral Envelope Contributes to Its Stability against Human Complement

The rapid spread of the virus in Latin America and the association of the infection with microcephaly in newborns or Guillain–Barré Syndrome in adults prompted the WHO to declare the Zika virus (ZIKV) epidemic to be an international public health emergency in 2016. As the virus was first discovered in monkeys and is spread not only by mosquitos but also from human to human, we investigated the stability to the human complement of ZIKV derived from mosquito (ZIKVInsect), monkey (ZIKVVero), or human cells (ZIKVA549 and ZIKVFibro), respectively. At a low serum concentration (10%), which refers to complement concentrations found on mucosal surfaces, the virus was relatively stable at 37 °C. At higher complement levels (up to 50% serum concentration), ZIKV titers differed significantly depending on the cell line used for the propagation of the virus. While the viral titer of ZIKVInsect decreased about two orders in magnitude, when incubated with human serum, the virus derived from human cells was more resistant to complement-mediated lysis (CML). By virus-capture assay and Western blots, the complement regulator protein CD55 was identified to be incorporated into the viral envelope. Blocking of CD55 by neutralizing Abs significantly increased the sensitivity to human complement. Taken together, these data indicate that the incorporation of CD55 from human cells contributes to the stability of ZIKV against complement-mediated virolysis.