Efficiencies and kinetics of infection in different cell types/lines by African and Asian strains of Zika virus

After recent outbreaks, Zika virus (ZIKV) was linked to severe neurological diseases including Guillain-Barré syndrome in adults and microcephaly in newborns. The severities of pathological manifestations have been associated with different ZIKV strains. To better understand the tropism of ZIKV, we infected 10 human and four nonhuman cell lines (types) with two African (IbH30656 and MR766) and two Asian (PRVABC59 and H/FP/2013) ZIKV strains. Cell susceptibility to ZIKV infection was determined by examining viral titers, synthesis of viral proteins, and replication of positive and negative strands of viral genome. Among nonhuman cell lines, only Vero cells were efficiently infected by ZIKV. Among human cell lines, all were permissive to ZIKV infection. However, 293T and HeLa cells showed differential susceptibility towards African strains. In 293T cells, the NS1 protein was expressed at the high level by African strains but was almost not expressed by Asian strains though there was no obvious difference in viral genome replication, suggesting that the differential susceptibility might be controlled at the stage of viral protein translation. This study provides comprehensive results of the permissiveness of different cell types to both African and Asian ZIKV strains, which might help clarify their different pathogenesis.     

Congenital Zika syndrome: Pitfalls in the placental barrier

Much progress with respect to congenital Zika virus (ZIKV) pathogenesis has been achieved after the 2015 outbreak in Brazil. It is now accepted that ZIKV is vertically transmitted, infects cells of the developing central nervous system and the placenta, yet it is unclear to what extent placental affection contributes to the development of congenital ZIKV. The association between fulminant villitis and severe fetal involvement emerges as a possibility. ZIKV is unique among the Flaviviruses in its ability to be sexually transmitted, possibly responsible for its teratogenicity. Furthermore, there is controversy over the participation of antibody dependent enhancement (ADE) in patients with non‐neutralizing anti‐Flavivirus antibodies, a phenomenon previously recognized in serious DENV infections. Our aim was to analyze information regarding the contribution of the placental barrier as an actual player in neonatal ZIKV. Therefore, we underwent a systematic review with keywords “Zika virus” and “ZIKV”. Articles were screened for relevance concerning the topics of microcephaly, transplacental transmission, sexual transmission, and ADE. We identified variables that affect the severity of congenital Zika syndrome: age of gestation at maternal infection, the extent of placental disruption (villitis), sexual transmission, initial viral replication at the uterine wall, anti‐DENV antibodies, and the possibility of antibody‐mediated transcytosis of ZIKV through the placenta. These questions may not seem relevant when Zika becomes endemic, and we are no longer witness to the extreme clinical sequelae seen when the virus moves through an immunologically naïve population; however, characterizing the pathogenesis of congenital Zika syndrome will continue to further our understanding.     

Zika virus transmission via breast milk in suckling mice

ObjectivesInfectious Zika viral particles were detected in human milk; however, whether they can be transmitted via breastfeeding remains unknown, so our objective was to clarify this.MethodsHere, in a natural breastfeeding model, wild-type (C57Bl/6; WT) or interferon α/β (IFNα/β) receptor-deficient (A129; KO) murine dams on day 1 post-delivery were infected with Zika virus (ZIKV) intraperitoneally, and the neonates were suckled. In a novel artificial feeding model, WT suckling mice at 1 day old were fed with ZIKV alone or ZIKV and human breast milk mixtures. Thereafter, the virus distribution, clinical progression and neuropathology in the WT or KO neonates were characterized to evaluate the risk of ZIKV transmission through breast milk.ResultsIn natural breastfeeding, viral RNAs (8/8) and infectious viral particles (7/8) were extensively present in the mammary glands of KO dams. All tested KO neonates (5/5), and none of WT neonates (0/9), were infected with ZIKV. In artificial feeding, 100% of the WT neonates (two groups, 12/12 and 16/16) were infected and developed some signs of neurodegeneration. ZIKV tended to seed and accumulate in the lungs and were subsequently disseminated to other tissues in both 16 naturally suckled and 19 artificially fed infected neonates. As human breast milk was mixed with ZIKV and fed to WT neonates, 45% individuals (9/20) were infected; in the infected neonates, the viral spread to the brain was delayed, and the clinical outcomes were alleviated.ConclusionsThese results demonstrated that suckling mice can be infected with ZIKV through suckling, and breast milk has potential antiviral activity, inhibiting ZIKV infection.     

Zika virus infection of Hofbauer cells

Recent studies have linked antenatal infection with Zika virus (ZIKV) with major adverse fetal and neonatal outcomes, including microcephaly. There is a growing consensus for the existence of a congenital Zika syndrome (CZS). Previous studies have indicated that non‐placental macrophages play a key role in the replication of dengue virus (DENV), a closely related flavivirus. As the placenta provides the conduit for vertical transmission of certain viruses, and placental Hofbauer cells (HBCs) are fetal—placental macrophages located adjacent to fetal capillaries, it is not surprising that several recent studies have examined infection of HBCs by ZIKV. In this review, we describe congenital abnormalities associated with ZIKV infection, the role of HBCs in the placental response to infection, and evidence for the susceptibility of HBCs to ZIKV infection. We conclude that HBCs may contribute to the spread of ZIKV in placenta and promote vertical transmission of ZIKV, ultimately compromising fetal and neonatal development and function. Current evidence strongly suggests that further studies are warranted to dissect the specific molecular mechanism through which ZIKV infects HBCs and its potential impact on the development of CZS.     

Zika virus: Future reproductive concerns

The pandemic spread of Zika virus (ZIKV), a member of the flavivirus genus of the Flaviviridae family, has become a major public health concern. Reproductive specialists are particularly concerned over the spread of ZIKV as it is now known to have both sexual and transplacental routes of transmission resulting in fetal congenital abnormalities. Other members of the Flaviviridae family, hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) (which primarily affects cattle), are well known to reproductive specialists as both sexually transmitted illnesses that are capable of vertical transmission. Congenital infection with BVDV also has a predilection for neuro‐teratogenicity as has been seen with ZIKV. HCV and BVDV are also known to be capable of persistent infection in offspring. Could this be the case with ZIKV? Examining what we know about HCV and BVDV, in addition to what we have already learned about ZIKV, may answer some of the questions that remain about ZIKV. Herein, we review the current literature as it pertains to ZIKV vertical transmission and neuro‐teratogenicity and compare it to what is known about HCV and BVDV.     

Zika virus: Molecular responses and tissue tropism in the mammalian host

Zika virus (ZIKV) outbreaks have raised alarm because of reports of congenital Zika virus syndrome in infants. The virus is also known to cause the debilitating Guillain–Barré syndrome in adults. As a result, extensive research has been carried out on the virus over the past few years. To study the molecular responses of viral infectivity in mammals, in vitro two‐dimensional and three‐dimensional cellular models have been employed. The in vivo models of mouse, pig, chicken, and nonhuman primates are primarily used to investigate the teratogenicity of the virus, to study effects of the virus on specific tissues, and to study the systemic effects of a proposed antiviral agent. The virus exhibits wide tissue tropism in the mammalian host. The major host tissues of viral persistence and propagation are neural tissue, ocular tissue, testicular tissue and placental tissue. An understanding of the function of viral components, viral replication cycle, and the molecular responses elicited in the host tissues is imperative for designing antiviral treatment strategies and for development of vaccines. This review provides an update on ZIKV research models and mammalian host responses with respect to ZIKV tissue infection.     

Modulation of cellular machineries by Zika virus-encoded proteins

The strain of Zika virus (ZIKV) that circulated during the 2015 epidemic in Brazil has been associated with more than 2000 cases of microcephaly from September 2015 through November 2016. The viral genome determines the biology and pathogenesis of a virus and the virus employs its own gene products to evade host immune surveillance, manipulate cellular machineries, and establish efficient replication. Therefore, understanding the functions of virus-encoded protein not only aids the knowledge of ZIKV biology but also guides the development of anti-ZIKV drugs. In this review, we focus on 10 proteins encoded by ZIKV and summarize their functions in ZIKV replication and pathogenesis according to studies published in the past 6 years.     

Synergistic antiviral activity of Sofosbuvir and type‐I interferons (α and β) against Zika virus

Zika virus (ZIKV) is transmitted by mosquitoes and causes Dengue‐like illness, neurological symptoms such as Guillain‐Barré Syndrome and microcephaly in children born to infected pregnant mothers. Recently, the World Health Organization (WHO) declared ZIKV infection as a Global Health Emergency. However, there are no known prophylactic or therapeutic measures against this virus. As a proof of concept toward combination therapeutic strategy against ZIKV, combinations of host‐targeted (Interferon‐α and Interferon‐β) and direct acting (Sofosbuvir) antivirals were evaluated in a hepatic cell line (Huh7) using a Cytoprotection (CP) assay. The combination of these antivirals resulted in synergistic inhibition of ZIKV infection in the in vitro CP assay. Additional testing in a ZIKV yield assay demonstrated that combination treatment of these antivirals conferred >2‐log reduction in the release of viral RNA. Measurement of ZIKV proteins in the cells infected with multiple ZIKV strains isolated from different geographical regions (Americas, Asia, and Africa) using an immunofluorescence assay confirmed the effective antiviral activity of this combination against ZIKV. These results demonstrate the in vitro proof of concept (POC) for using a combination approach utilizing the strengths of both virus and host‐targeted antivirals. These results suggest the effectiveness of the combination strategy in combating ZIKV, in the in vitro systems. Further evaluation of such combination therapies in vivo might provide an impetus for the development of effective ZIKV therapeutic strategies.     

The antimalarial drug amodiaquine possesses anti‐ZIKA virus activities

Zika virus (ZIKV) outbreak has emerged as a global health threat, particularly in tropical areas, over the past few years. No antiviral therapy or vaccine is available at present. For these reasons, repurposing clinically approved drugs against ZIKV infection may provide rapid and cost‐effective global health benefits. Here, we explored this strategy and screened eight FDA‐approved drugs for antiviral activity against ZIKV using a cell‐based assay. Our results show that the antimalarial drug amodiaquine has anti‐ZIKV activity with EC₅₀ at low micromolar concentrations in cell culture. We further characterized amodiaquine antiviral activity against ZIKV and found that it targets early events of the viral replication cycle. Altogether, our results suggest that amodiaquine may be efficacious for the treatment of ZIKV infection.

     

Molecular detection of Zika virus in blood and RNA load determination during the French Polynesian outbreak

Zika virus (ZIKV) viremia is reported as low and transient; however, these estimates rely on limited data. We report RNA loads in sera collected from symptomatic patients during the 2013‐2014 French Polynesian ZIKV outbreak. We performed molecular detection of ZIKV RNA in sera from 747 patients presenting with suspected acute phase ZIKV infection. Among patients with confirmed infection, we analyzed the duration of viremia, assessed viral RNA loads and recorded the main clinical symptoms. A total of 210/747 (28.1%) sera tested positive using a ZIKV‐specific RT‐PCR. Viral RNA loads in symptomatic patients that ranged from 5 to 3.7 × 10⁶ copies/mL (mean 9.9 × 10⁴ copies/mL) were not related to a particular clinical presentation, and were significantly lower than those previously obtained from asymptomatic ZIKV infected blood donors. The rate of detection of ZIKV RNA in sera from suspected cases of acute phase ZIKV infection was low. ZIKV RNA loads were lower in symptomatic patients compared to asymptomatic blood donors and were lower than RNA loads usually reported in dengue infections. As there is no abrupt onset of symptoms in ZIKV infections, we suggest that infected patients sought for medical attention when viremia was already decreasing or had resolved.

     

Using immunocompromised mice to identify mechanisms of Zika virus transmission and pathogenesis

Zika virus (ZIKV) is responsible for a recent global epidemic that has been associated with congenital brain malformations in fetuses and with Guillain–Barré syndrome in adults. Within the last 2 years, a major effort has been made to develop murine models to study the mechanism of viral transmission, pathogenesis and the host immune response. Here, we discuss the findings from these models regarding the role that the innate and adaptive immune responses have in controlling ZIKV infection and pathogenesis. Additionally, we examine how innate and adaptive immune responses influence sexual and vertical transmission of ZIKV infection as well as how these responses can influence the ability of ZIKV to cross the placenta and to induce damage in the developing brain.     

Molecular and serological techniques to detect co-circulation of DENV,ZIKV and CHIKV in suspected dengue-like syndrome patients

BackgroundArboviruses are important emerging viruses worldwide. The signs and symptoms of Zika virus (ZIKV) infection are similar to those presented by infections with dengue virus (DENV) and chikungunya virus (CHIKV). Furthermore, diagnosis of ZIKV infection is particularly challenging in dengue endemic regions and with co-circulation of DENV, CHIKV, and ZIKV, making diagnosis based solely on clinical and epidemiological data unreliable. As these three viral infections share similar clinical manifestations, differential diagnosis is crucial.ObjectivesIn this study, diagnoses of ZIKV, CHIKV and DENV infections were investigated in 30 patients with suspected dengue fever residing in the area of co-circulation of these three arboviruses.Study designThe study included whole blood and/or serum samples obtained from 30 patients with suspected dengue fever. All patients were tested for DENV infection as well as for CHIKV and ZIKV infections. Assays for detecting anti-DENV IgM and DENV RNA by semi-nested RT-PCR and ZIKV and CHIKV RNA by real-time RT-PCR were performed.ResultsDENV RNA was not detectable in any of the clinical samples, whereas ZIKV RNA was detectable in 17 samples (56.7%). Co-infection by ZIKV and CHIKV was documented in one case. Of the 17 ZIKV-positive individuals, 8 showed reactivity for anti-DENV IgM, which suggested recent DENV infection, cross-reactivity or co-infection.ConclusionOur findings confirm that accurate laboratory testing is of paramount importance for differential diagnosis in areas of simultaneous transmission of different arboviruses with similar clinical presentations.     

Zika and pregnancy: A comprehensive review

Zika virus (ZIKV) infection is a well‐nurtured topic for healthcare personnel nowadays. Central nervous system involvement including microcephaly and ocular involvements has already been reported in neonates of affected pregnant ladies. In this article, we have discussed these effects on the newborns of ZIKV‐infected mothers. The proposed pathogenesis, modes of transmission of this infection from mothers to the fetuses, diagnosis of the cases and precaution for the pregnant ladies have also been discussed. We have gathered the recently available data on the risk of ZIKV for expectant mothers from PubMed, https://www.gov.uk/guidance/zika-virus as well as from centers for disease control and prevention websites.     

First detection of autochthonous Zika virus transmission in a HIV-infected patient in Rio de Janeiro,Brazil

Since May 2015, Brazil’s Ministry of Health has reported autochthonous transmission of Zika virus (ZIKV) in some states of the country. Simultaneous circulation of Dengue, Chikungunya and ZIKV in the country hinder both the diagnosis and the therapeutic approach of patients seeking care with acute febrile illnesses especially in patients with comorbidities. The association between HIV infection and endemic diseases has been described especially in tropical regions with varying levels of complications, although there has been no report of ZIKV in HIV-infected patients. We report the first autochthonous case of laboratory confirmed ZIKV infection in a HIV-infected patient in Rio de Janeiro, Brazil. He evolved with only mild symptoms and recovered well without major laboratory abnormalities. Phylogenetic analysis of the ZIKV detected in the patient sera clustered within the Asian clade. To the best of our knowledge, this is the first time that Zika virus co-infection is reported in a HIV-infected patient.     

Kinetics of antigen-specific IgM/IgG/IgA antibody responses during Zika virus natural infection in two patients

Understanding of kinetics of antibody responses is crucial for developing rapid serological tests and studying the mechanisms of Zika virus (ZIKV) infection. Most of the serological diagnostic assays previously published are based on either IgM or IgG titer, little is known on the level of IgA antibody in saliva and urine. In this study, we investigated the kinetics of IgM/IgG/IgA antibody responses in serum, saliva, and urine obtained from two ZIKV infected individuals from as early as the second day of onset of symptoms to as long as 2 years postinfection. Other than detecting robust early IgM response, long lasting IgG response, we discovered strong early IgA response specific for ZIKV in saliva in both patients. This unique observation provides a novel strategy and scientific basis for the development of noninvasive rapid tests for ZIKV infection.     

The potential contribution of impaired brain glucose metabolism to congenital Zika syndrome

The Zika virus (ZIKV) became a major worldwide public concern in 2015 due to the congenital syndrome which presents the highest risk during the first trimester of pregnancy and includes microcephaly and eye malformations. Several cellular, genetic and molecular studies have shown alterations in metabolic pathways, endoplasmic reticulum (ER) stress, immunity and dysregulation of RNA and energy metabolism both in vivo and in vitro. Here we summarise the main metabolic complications, with a particular focus on the possibility that brain energy metabolism is altered following ZIKV infection, contributing to developmental abnormalities. Brain energetic failure has been implicated in neurological conditions such as autism disorder and epilepsy, as well as in metabolic diseases with severe neurodevelopmental complications such as Glut‐1 deficiency syndrome. Therefore, these energetic alterations are of wide‐ranging interest as they might be directly implicated in congenital ZIKV syndrome. Data showing increased glycolysis during ZIKV infection, presumably required for viral replication, might support the idea that the virus can cause energetic stress in the developing brain cells. Consequences may include neuroinflammation, cell cycle dysregulation and cell death. Ketone bodies are non‐glycolytic brain fuels that are produced during neonatal life, starvation or fasting, ingestion of high‐fat low‐carbohydrate diets, and following supplementation with ketone esters. We propose that dietary ketones might alter the course of the disease and could even provide some degree of prevention of ZIKV‐associated abnormalities and potentially related neurological conditions characterised by brain glucose impairment.