Growth of Zika virus in human reconstituted respiratory,intestinal, vaginal and neural tissues

ObjectivesZika virus (ZIKV) is mostly mosquito borne but it can also be transmitted via the sexual route and persists in semen for a prolonged time. Moreover, viral RNA has been detected in breast milk, saliva, lacrimal fluids and urine, suggesting other possible transmission routes. The aim of our research is to better define ZIKV tropism.MethodsWe investigated the tropism of Asian and African strains of ZIKV using human-derived neural, vaginal, intestinal and respiratory tissues.ResultsAsian and African strains of ZIKV were able to grow in all tissues tested, although with different efficiency (7.3 log RNA copies released apically in vaginal tissues versus 9.8 log RNA copies released in intestinal tissues), without the need for major adaptation.ConclusionsOur results underline that ZIKV tropism may be broader than expected in humans and stress the need to better explore all possible virus-shedding sites and transmission routes.     

Detection of Zika virus in saliva

BackgroundDuring the largest Zika virus (ZIKV) outbreak ever reported that occurred from October 2013 to March 2014 in French Polynesia, we observed that several patients presenting the symptoms of acute phase Zika fever were tested negative in blood by ZIKV real-time PCR (RT-PCR).ObjectivesAs we have previously detected ZIKV RNA in the saliva of a young child, we investigated the use of saliva as an alternative sample for routine ZIKV RNA detection.Study designOver a 6 month period, 1,067 samples collected from 855 patients presenting symptoms of Zika fever (saliva only, blood only or both samples) were tested using a specific ZIKV RT-PCR. A medical questionnaire was available for most of the patients.ResultsZIKV was more frequently detected in saliva compared to blood. For the 182 patients with both samples collected, tests were positive for 35 (19.2%) in saliva while negative in blood and tests were positive for 16 (8.8%) in blood while negative in saliva; the difference in mean days after symptoms onset and the percentage of the main symptoms of Zika fever for patients only positive in saliva or in blood was not significant.ConclusionThe use of saliva sample increased the rate of molecular detection of ZIKV at the acute phase of the disease but did not enlarge the window of detection of ZIKV RNA. Saliva was of particular interest when blood was difficult to collect (children and neonates especially).     

Transmission of maternal antibody prenatally and from milk into serum of neonatal rabbits.

Rabbit dams fed 0.1% BSA for various periods before and during lactation produced anti-BSA of low avidity and IgG isotype in serum and milk. Milk anti-BSA and IgG concentrations were one-third to one-half of those in the serum. At birth, kits had IgG and anti-BSA serum concentrations approximately equal to their dams. Both fell rapidly for the first 10-20 days, levelling off at about 1 mg IgG/ml. Kits born to unimmunized dams and suckled by dams with anti-BSA in the milk showed increasing anti-BSA in serum for the first 12-16 days, falling by 20 days. Foster-suckling on immunized dams beginning at various times after birth showed antibody uptake from birth through 12 days of age. Thus immunoglobulins are among factors absorbed from milk that have potential for regulating the immune responses of rabbit neonates.     

Viability of infectious viral particles of HIV and BMCs in breast milk.

BACKGROUND: Infectious factors in breast milk such as viral particles and living infected cells are of prime importance in the transmission of HIV by breastfeeding. OBJECTIVES: To perform effective approaches for reducing HIV transmission via breastfeeding, we investigated the biological importance of infectious viral particles and infected BMCs in breast milk. STUDY DESIGN: Alteration of viral infectivity was monitored using a modified experimental infection assay that exploited the cytotoxicity of breast milk, and BMC viability was evaluated by flow-cytometric analysis. RESULTS: Infectious viral particles were found to decrease time-dependently after contact with breast milk, whereas BMCs showed prolonged survival in breast milk. CONCLUSIONS: The biological importance of infected BMCs in breast milk for the transmission of HIV via breastfeeding was considered.     

Zika virus infection and implications for kidney disease

High-level and persistent viruria observed in patients infected by Zika virus (ZIKV) has been well documented. However, renal pathology in acutely infected, immunocompetent patients remains subclinical. Moreover, the long-term impact of ZIKV infection, replication, and persistence in the renal compartment of adults and infants as well as immunosuppressed patients and solid organ transplant (SOT) recipients is unknown. Mechanisms involving host and viral factors that limit or control ZIKV pathogenesis in the renal compartment are important yet unexplored. The observation that long-term viral shedding occurs in the renal compartment in the absence of clinical disease requires further investigation. In this review, I explore Zika virus-induced renal pathology in animal models, the dynamics of virus shedding in urine, virus replication in glomerular cells, ZIKV infection in human renal transplantation, and the potential impact of long-term persistent ZIKV infection in the renal compartment.     

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.     

Suckling: Developmental indicator of genetic obesity in mice

The Bar Harbor obese-hyperglycemic mouse (ob/ob) characteristically develops its obese phenotype during the postweaning period, when access to food is unrestricted and its behavioral phenotype of hyperphagia appears. In the present study suckling behavior (viz., nipple attachment latencies and total suckling time) of lean and preobese mouse pups, derived from matings of heterozygous lean parents, was measured in either fed or fasted conditions from 6 to 21 days postpartum on anesthetized dams. Twenty-hour milk deprivation selectively channeled pup activity into suckling for both preobese and lean mice, although, in general, preobese mice nipple-attached sooner and suckled longer than leans. In particular, fed preobese pups suckled longer than fed lean pups from 18 days on, providing a preweaning behavioral indicant of a genetic tendency to obesity when environmental constraints on overeating are minimized.     

Effective immunity to dental caries: passive transfer to rats to antibodies to Streptococcus mutans elicits protection.

Rat dams, given intravenous injections of heat-killed Streptococcus mutans 6715, mutant C211 demonstrated significant agglutinin activity to the homologous S. mutans in colostrum, milk, and serum. This antibody activity was associated with the immunoglobulin G (IgG) class. High titers of anti-S. mutans antibody associated with the IgG class were also exhibited in the sera and saliva of the offspring that suckled these dams. After challenge with the homologous, live S. mutans, these offspring developed significantly fewer caries on all molar surfaces than did nonimmunized infected controls. A secretory immune response (manifested by the presence of specific IgA antibody to S. mutans in colostrum and milk) was elicited (i) in rat dams locally injected, in the region of the mammary gland, with heat-killed S. mutans antigen, and (ii) in other rat dams that were provided formalin-killed S. mutans in their drinking water. Offspring suckling these dams were challenged with virulent S. mutans before weaning and developed significantly fewer caries than did their infected controls. These findings clearly suggest that passively derived IgG or IgA antibodies to S. mutans are protective against dental caries.     

Fatal encephalitis associated with Zika virus infection in an adult

BackgroundZika virus (ZIKV) was first identified in the Americas in 2015, when an outbreak of an exanthematous illness occurred in Brazil. Subsequentely, there was an increase of microcephaly cases, suggesting an association between ZIKV and this neurological complication. Currently, ZIKV has been recognised as causing a wide range of neurological complications including Guillain Barré syndrome, and myelitis.ObjectivesIn this report, we describe the first fatal case of encephalitis in a 47 years old non pregnant woman, infected during the Brazilian zika epidemic of 2016.Study designThe diagnosis of encephalitis was determined by the presence of a disturbed level of consciousness and focal neurological signs during an exanthemous viral infection.ResultsCSF analysis supported the diagnosis of viral encephalitis, revealing lymphocytic pleocytosis, a high protein concentration, and the presence of IgM zika antibodies. RT-PCR analysis for ZIKV was positive in the urine. A brain computed tomography showed massive brain swelling. Our case differs from previous reports, because her neurological picture developed rapidly and in a very aggressive manner leading to brain death after eleven days of admission.ConclusionIn endemic areas, ZIKV should be considered as an aetiological agent in cases of encephalitis, and clinicians should be aware of its potential severity.     

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.     

Retroviral gene insertion in breast milk mediated lymphomagenesis

We have demonstrated breast milk transmitted MoMuLV-ts1 retrovirus infection and subsequent lymphoma development in offspring of uninfected mothers suckled by infected surrogate mothers. Additionally, we have shown that the lymphoma development occurs as a result of viral gene integration into host genome. A total of 146 pups from Balb/C mice were divided into 5 groups; one control and 4 experimental. All offspring suckled from surrogate infected or control mothers, except one group of infected pups left with their biological mothers. Thirteen of 91 infected pups developed lymphoma. Inverse-PCR, DNA cloning, and quantitative real-time PCR (qRT-PCR) were used to study the virus integration sites (VIS) and alterations in gene expression. VIS were randomly distributed throughout the genome. The majority of insertion sites were found in chromosomes 10, 12 and 13. A total of 209 proviral genomic insertion sites were located with 52 intragenic and 157 intergenic sites. We have identified 29 target genes. Four genes including Tacc3, Aurka, Gfi1 and Ahi1 showed the maximum upregulation of mRNA expression. These four genes can be considered as candidate genes based on their association with cancer. Upregulation of these genes may be involved in this type of lymphoma development. This model provides an important opportunity to gain insight into the relationship of viral gene insertion into host genome and development of lymphoma via natural transmission route such as breast milk.     

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.     

Effects of unilateral suckling on nursing behavior and c-fos activity in the caudal periaqueductal gray in rats

In rats, suckling elicits kyphosis-the bilaterally symmetrical, upright, humpbacked nursing posture-and maximal expression of the immediate early gene c-fos in a region of the caudal periaqueductal gray (cPAG) that mediates the sensorimotor integration of kyphosis. We determined the effects of prepartum unilateral nipple removal on nursing behavior and c-fos expression during a 60-min mother-litter interaction on Day 7 postpartum. Compared with dams suckled by 6 pups bilaterally, dams suckled unilaterally displayed essentially normal maternal behaviors, including kyphosis. Unilaterally suckled dams, however, showed an increase in the abnormal prone nursing posture, a decrease in proportion of kyphotic nursing of total time over pups, and a 20% higher contralateral/ipsilateral ratio of cPAG neurons expressing c-fos. These results are consistent with an incompletely lateralized neural pathway conveying suckling stimulation to the cPAG and provide a mechanism whereby kyphosis is elicited by unilateral suckling when pups initiate nursing from their supine dam.     

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.

     

Investigation of body temperature as a possible feeding control in the suckling rat

Under natural conditions, rat pups are deprived of milk only when the dam leaves the nest. At this time the body temperatures of the pups will fall. In this study we have investigated the possibility that lowered body temperature of the pups stimulates behavioral changes in either the pups or the dam, leading to increased milk intake. To study behavioral changes of the pups, the milk intake of warm-fasted, cool-fasted, and fed littermates was compared to 10, 14 and 16 days of age. There was no effect of body temperature on milk intake at any age. To study behavioral changes in the dam, milk release by dams suckling entire cool litters was compared with that by dams suckling entire warm litters. Milk release was not significantly affected by the litter temperature. We conclude that lowered body temperature of suckling rat pups does not cause increased milk intake.     

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.