NS1 glycoprotein detection in serum and urine as an electrochemical screening immunosensor for dengue and Zika virus.

The incidence of infection by the dengue virus (DENV) has grown dramatically, reaching 128 countries in tropical and subtropical regions worldwide, with a pattern of hyper-endemicity. DENV is a mosquito-borne disease having four serotypes, one or two circulating in epidemic outbreaks. The diagnosis of DENV is challenging mainly due to the circulation of new viruses with remarkable similarities, such as Zika (ZIKV) that may cause fetal microcephaly. DENV affects 390 million people per year, but these numbers may be higher due to the underreported and misclassified cases. Recently, the NS1 nonstructural protein has been described in serum and urine of DENV and ZIKV patients, suggesting its use as a biomarker for screening since a negative NS1 sample confirms the absence of these infections. Herein, a label-free immunosensor comprising an assembled nanostructured thin film of carbon nanotube-ethylenediamine is described. The advantage of in situ electrosynthesis of polymer film is to allow major control of thickness and conductivity, in addition to designing the reactive groups for functionalization. A quartz crystal microbalance system was used to estimate the thickness of the polymeric film obtained. The anti-NS1 monoclonal antibodies were immobilized to carbon nanotubes by covalent linkage, permitting a high stability during measurements. Analytical responses to NS1 were obtained by differential pulse voltammetry (DPV), showing a linear range from 20 to 800 ng mL-1 and reproducibility of 3.0%, with a limit of detection (LOD) of 6.8 ng mL- 1. This immunosensor was capable of detecting ZIKV and DENV NS1 in spiked urine and real serum in a clinical range.Graphical abstract.

Detection of Zika virus in paired urine and amniotic fluid samples from symptomatic and asymptomatic women and their babies during a disease outbreak: association with neurological symptoms in newborns.

Paired maternal and newborn urine and amniotic fluid from 138 subjects collected during a Zika virus (ZIKV) outbreak was analyzed for ZIKV by gene amplification (RT-qPCR), and the findings were correlated with clinical symptoms and neurological anomalies in the babies. ZIKV was detected in 1 of 9 symptomatic women (11.1%) and in 19 of 129 asymptomatic women (14.7%). Neurological manifestations were present in 19 babies (13.7%), 10 of 20 (50%) positive and 9 of 119 (7.6%) negative (p < 0.001) for ZIKV. Twelve (8.6%) urines collected during gestation were ZIKV-positive; only 2 remained positive for ZIKV postpartum. Six (4.1%) newborn urines collected within 1 day of delivery were ZIKV-positive cases. In 3 of these cases, ZIKV was detected in mother's urine pre- and postpartum and in both mother's urine and babies' urine. Four of the amniotic fluid samples (2.9%) were ZIKV-positive. Among ZIKV-negative babies with neurological sequel, 87.5% were female; in contrast, 72.7% ZIKV-positive babies with neurological abnormalities were male (p = 0.019). We conclude that during a ZIKV outbreak, clinical symptoms and ZIKV detection in biological fluids are poor predictors of infection and adverse neurologic sequel in newborns.

Zika Virus in Rectal Swab Samples.

We detected Zika virus RNA in rectal swab samples from 10 patients by using real-time reverse transcription PCR, and we isolated the virus from 1 patient. The longest interval from symptom onset to detection was 14 days. These findings are applicable to diagnosis and infection prevention recommendations.

Zika virus found in brain tissue of a multiple sclerosis patient undergoing an acute disseminated encephalomyelitis-like episode.

BACKGROUND: A range of different neurological manifestations has been reported in fetuses and adults after Zika virus (ZIKV) infection. OBJECTIVE: We describe a detection of the ZIKV in the brain tissue from a multiple sclerosis (MS) patient with acute disseminated encephalomyelitis (ADEM)-like event in Rio de Janeiro, Brazil. METHODS: Biological samples collected during the hospitalization were tested by serology and molecular diagnostic for various infectious agents. Histopathological analysis was performed using the anti-flavivirus group 4G2 monoclonal antibody, anti-ZIKV non-structural 1 (NS1) monoclonal antibody, and anti-CD4, CD8, and CD11b antibodies. RESULTS: Anti-ZIKV IgM and IgG antibodies were positive in the serum and urine. A brain biopsy showed ZIKV protein in brain cells and T CD8 infiltration in brain tissue. CONCLUSION: Our data describe the coexistence of a recent central nervous system (CNS) ZIKV infection accompanied by a severe ADEM-like syndrome outcome in a patient with clinical history of MS. A de novo immune response concomitant with ZIKV infection might be involved in the mechanism of the ADEM-like syndrome and response to immunotherapy. The present report reinforces the importance of providing the differential diagnosis of acute episodes of MS exacerbation in an environment prone to ZIKV expression.

Immune Cell Dynamics in Rhesus Macaques Infected with a Brazilian Strain of Zika Virus.

Zika virus (ZIKV) is a mosquito-borne and sexually transmitted flavivirus that is associated with fetal CNS-damaging malformations during pregnancy in humans. This study documents the viral kinetics and immune responses in rhesus macaques infected with a clinical ZIKV Brazilian isolate. We evaluated the viral kinetics and immune responses induced after an i.v. infection with a Brazilian ZIKV clinical isolate (HS-2015-BA-01) in rhesus macaques for up to 142 d. ZIKV-specific Ab-secreting cells, germinal center reactions, and monocyte, dendritic cell, NK, and T cell frequencies were monitored. ZIKV loads were readily detected in plasma (until day 5 or 7), semen and urine (until days 7 and 14), and saliva (until day 42), but the viremia was rapidly controlled. No detectable clinical manifestations were observed. However, lymph node hyperplasia was clearly visible postviremia but was associated with low frequencies of ZIKV-specific Ab-secreting cells in lymph nodes and bone marrow, correlating with low Ab titers. CD14+/CD16- monocytes and myeloid CD11chi dendritic cells decreased in blood, whereas NK and T cell numbers were only marginally altered during the course of the study. ZIKV infection caused a significant lymphoid tissue activation but limited induction of ZIKV-specific B cells, suggesting that these parameters need to be considered for ZIKV vaccine design.

Importation of Zika Virus from Vietnam to Japan, November 2016.

We report a case of Zika virus infection that was imported to Japan by a traveler returning from Vietnam. We detected Zika virus RNA in the patient's saliva, urine, and whole blood. In the Zika virus strain isolated from the urine, we found clearly smaller plaques than in previous strains.

Prolonged Detection of Zika Virus in Vaginal Secretions and Whole Blood.

Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies.

Persistent Zika Virus Detection in Semen in a Traveler Returning to the United Kingdom from Brazil, 2016.

Zika virus is normally transmitted by mosquitos, but cases of sexual transmission have been reported. We describe a patient with symptomatic Zika virus infection in whom the virus was detected in semen for 92 days. Our findings support recommendations for 6 months of barrier contraceptive use after symptomatic Zika virus infection.

Zika virus infection in a traveller returning from the Maldives, June 2015.

We report a Zika virus (ZIKV) infection in a patient with fever and rash after returning to Finland from Maldives, June 2015. The patient had dengue virus (DENV) IgG and IgM antibodies but pan-flavivirus RT-PCR and subsequent sequencing showed presence of ZIKV RNA in urine. Recent association of ZIKV with microcephaly highlights the need for laboratory differentiation of ZIKV from DENV infection and the circulation of ZIKV in areas outside its currently known distribution range.

Detection of Zika virus in urine.

We describe the kinetics of Zika virus (ZIKV) detection in serum and urine samples of 6 patients. Urine samples were positive for ZIKV >10 days after onset of disease, which was a notably longer period than for serum samples. This finding supports the conclusion that urine samples are useful for diagnosis of ZIKV infections.

Zika virus isolation, propagation, and quantification using multiple methods.

Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106-107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10-100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.

Development of Universal and Lineage-Specific Primer Sets for Rapid Detection of the Zika Virus (ZIKV) in Blood and Urine Samples Using One-Step Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).

Zika is a mosquito-borne disease that has been posing a significant threat to public health in recent years. The Zika virus (ZIKV), the causative agent of this disease, is classified into 2 distinct genetic lineages, namely Asian and African. While molecular nucleic acid analysis methods have been shown to be useful for the diagnosis of ZIKV infection, the development of assays based on one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers several advantages, such as shorter incubation times, ease of handling, and rapid detection. In this study, a universal LAMP primer set was developed to target conserved sequences of known ZIKV lineages. Additionally, the Af7462 and As1788 primer sets were designed based on LAMP-based single-nucleotide polymorphism (SNPs) typing for the specific detection of the African and Asian lineages. The developed RT-LAMP assays could specifically detect the African and Asian lineages of ZIKV, with a detection limit ranging from 0.17 FFU/mL to 2.3×102 FFU/mL. As ZIKV viremia ranges between 102 to 106 PFU/mL or 103 to 106 copies/mL, the data indicate that the viremia range of clinical samples is within the detection range of our assay. Due to the high specificity and sensitivity, as well as the ease of use of our assay, it could potentially be used for early clinical diagnosis applications.

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.

Detection of Zika Virus Using Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP).

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) allows amplification and detection of RNA or DNA rapidly and relatively inexpensively. Here we describe how RT-LAMP can be utilized to detect Zika virus in human urine or serum samples or Aedes mosquito samples. This can be completed in under 30 min and without first isolating the RNA from the sample.

Susceptibility of Armigeres subalbatus Coquillett (Diptera: Culicidae) to Zika virus through oral and urine infection.

BACKGROUND: Zika virus (ZIKV) disease outbreaks have been occurring in South America since 2015, and has spread to North America. Because birth defects and cases of Guillain Barré have been associated with infection with ZIKV, this has drawn global attention. ZIKV is generally considered an Aedes-transmitted pathogen. The transmission of ZIKV through blood by Aedes mosquito bites has been recognized as the major transmission route. However, it is not clear whether there are other transmission routes that can cause viral infection in mosquitos. The aim of the present study is to describe the susceptibility of Armigeres subalbatus, which often develop in human waste lagoons, to ZIKV, through oral infection in adult mosquitoes and urine infection in larvae. METHODOLOGY/PRINCIPAL FINDINGS: Five-day-old female Ar. subalbatus ingested infectious blood meals containing ZIKV. After 4, 7, and 10 days of ingesting infectious blood meals, ZIKV could be detected in the midguts, salivary glands, ovaries, and collected saliva of mosquitoes. The ZIKV infection rate (IR) on day 10 reached 40% in salivary glands and 13% in saliva, indicating that these mosquitoes were able to transmit ZIKV. In addition, ZIKV infection was also discovered in mosquito ovaries, suggesting the possibility of vertical transmission of virus. Moreover, Ar. subalbatus transmitted ZIKV to infant mice bitten by infectious mosquitoes. In a second experiment, 1st-instar larvae of Ar. subalbatus were reared in water containing ZIKV and human urine. After pupation, pupae were placed in clean water and transferred to a mosquito cage for emergence. Although ZIKV RNA was detected in all of the larvae tested, ZIKV was not detected in the saliva of any adult Ar. subalbatus. Considering that there are more uncontrollable factors in nature than in the laboratory environment, the possibility that the virus is transmitted to adult mosquitoes via larvae is very small period. CONCLUSIONS/SIGNIFICANCE: Adult Ar. subalbatus could be infected with ZIKV and transmit ZIKV through mosquito bites. Therefore, in many rural areas in China and in undeveloped areas of other Asian countries, the management of human waste lagoons in the prevention and control of Zika disease should be considered. Corresponding adjustments and modifications should also be made in prevention and control strategies against ZIKV.

Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: A case-control study.

BACKGROUND: Beginning August 2017, we conducted a prospective case-control investigation in Monterrey, Mexico to assess the association between Zika virus (ZIKV) and Guillain-Barré syndrome (GBS). METHODS: For each of 50 GBS case-patients, we enrolled 2-3 afebrile controls (141 controls in total) matched by sex, age group, and presentation to same hospital within 7 days. RESULTS: PCR results for ZIKV in blood and/or urine were available on all subjects; serum ZIKV IgM antibody for 52% of case-patients and 80% of controls. Subjects were asked about antecedent illness in the two months prior to neurological onset (for case-patients) or interview (for controls). Laboratory evidence of ZIKV infection alone (PCR+ or IgM+) was not significantly different between case-patients and controls (OR: 1.26, 95% CI: 0.45-3.54) but antecedent symptomatic ZIKV infection [a typical ZIKV symptom (rash, joint pain, or conjunctivitis) plus laboratory evidence of ZIKV infection] was higher among case-patients (OR: 12.45, 95% CI: 1.45-106.64). GBS case-patients with laboratory evidence of ZIKV infection were significantly more likely to have had typical ZIKV symptoms than controls with laboratory evidence of ZIKV infection (OR: 17.5, 95% CI: 3.2-96.6). This association remained significant even when only GBS case-patients who were afebrile for 5 days before onset were included in the analysis, (OR 9.57 (95% CI: 1.07 to 85.35). CONCLUSIONS: During ZIKV epidemics, this study indicates that increases in GBS will occur primarily among those with antecedent symptomatic ZIKV.

Absence of Evidence of Zika Virus Infection in Cord Blood and Urine from Newborns with Congenital Abnormalities, Indonesia.

Zika virus (ZIKV) has recently been confirmed as endemic in Indonesia, but no congenital anomalies (CA) related to ZIKV infection have been reported. We performed molecular and serological testing for ZIKV and other flaviviruses on cord serum and urine samples collected in October 2016 to April 2017 during a prospective, cross-sectional study of neonates in Jakarta, Indonesia. Of a total of 429 neonates, 53 had CA, including 14 with microcephaly. These 53, and 113 neonate controls without evidence of CA, were tested by ZIKV-specific real-time reverse transcription polymerase chain reaction (RT-PCR), pan-flavivirus RT-PCR, anti-ZIKV and anti-DENV IgM ELISA, and plaque reduction neutralization test. There was no evidence of ZIKV infection among neonates in either the CA or non-CA cohorts, except in three cases with low titers of anti-ZIKV neutralizing antibodies. Further routine evaluation throughout Indonesia of pregnant women and their newborns for exposure to ZIKV should be a high priority for determining risk.

Aedes mosquitoes acquire and transmit Zika virus by breeding in contaminated aquatic environments.

Zika virus (ZIKV) is a mosquito-borne flavivirus that predominantly circulates between humans and Aedes mosquitoes. Clinical studies have shown that Zika viruria in patients persists for an extended period, and results in infectious virions being excreted. Here, we demonstrate that Aedes mosquitoes are permissive to ZIKV infection when breeding in urine or sewage containing low concentrations of ZIKV. Mosquito larvae and pupae, including from field Aedes aegypti can acquire ZIKV from contaminated aquatic systems, resulting in ZIKV infection of adult females. Adult mosquitoes can transmit infectious virions to susceptible type I/II interferon receptor-deficient (ifnagr-/-) C57BL/6 (AG6) mice. Furthermore, ZIKV viruria from infected AG6 mice can causes mosquito infection during the aquatic life stages. Our studies suggest that infectious urine could be a natural ZIKV source, which is potentially transmissible to mosquitoes when breeding in an aquatic environment.