Functional evaluation of human papillomavirus type 31 long control region variants.

Persistent infections by high-risk human papillomavirus (HR-HPV) are a necessary condition, but not sufficient for cervical cancer development. Genetic variants of HR-HPV appear to be related to the risk of persistent infections. The study performed a functional evaluation of variants of the HPV-31 promoter region (LCR). For this, cloning and subcloning of variants HPV-31/UFPE-21 HPV-31/UFPE-89, HPV-31/UFPE-66, E2 gene and prototype HPV-31 were performed. Transfection with different concentrations of E2 was done and the concentration of 25 ng was determined to be ideal for LCR activation. HPV-31/UFPE-21 and HPV-31/UFPE-89 have a greater ability to alter Nluc reporter gene expression levels and HPV-31/UFPE-66 showed decreased levels of gene expression of Nluc reporter gene compared to control. Statistical analysis showed a significant difference between the polymorphic LCR regions and the control (p < 0.0001). A more refined profile of variants of HPV-31 and its importance for the prognosis of cervical lesions begins to be drawn.

Searching Anti-Zika Virus Activity in 1H-1,2,3-Triazole Based Compounds.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flaviviridae family and is responsible for an exanthematous disease and severe neurological manifestations, such as microcephaly and Guillain-Barré syndrome. ZIKV has a single strand positive-sense RNA genome that is translated into structural and non-structural (NS) proteins. Although it has become endemic in most parts of the tropical world, Zika still does not have a specific treatment. Thus, in this work we evaluate the cytotoxicity and antiviral activities of 14 hybrid compounds formed by 1H-1,2,3-triazole, naphthoquinone and phthalimide groups. Most compounds showed low cytotoxicity to epithelial cells, specially the 3b compound. After screening with all compounds, 4b was the most active against ZIKV in the post-infection test, obtaining a 50% inhibition concentration (IC50) of 146.0 µM and SI of 2.3. There were no significant results for the pre-treatment test. According to the molecular docking compound, 4b was suggested with significant binding affinity for the NS5 RdRp protein target, which was further corroborated by molecular dynamic simulation studies.

Evolutionary study of potentially zoonotic hepatitis E virus genotype 3 from swine in Northeast Brazil.

Hepatitis E virus (HEV), an emerging virus associated with acute hepatic disease, leads to thousands of deaths worldwide. HEV has already been reported in Brazil; however, there is a lack of epidemiological and molecular information on the genetic variability, taxonomy, and evolution of HEV. It is thus unclear whether hepatitis E is a neglected disease in Brazil or it has low relevance for public health in this country. Here, for the first time, we report the presence of HEV in Northeast Brazil. A total of 119 swine faecal samples were screened for the presence of HEV RNA using real-time polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR; among these, two samples were identified as positive. Molecular evolution analyses based on capsid sequences revealed that the samples had close proximities to HEV sequences belonging to genotype 3 and were genetically related to subtype 3f isolated in humans. Parsimony ancestral states analysis indicated gene flow events from HEV cross-species infection, suggesting an important role of pig hosts in viral spillover. HEV's ability for zoonotic transmission by inter-species host switching as well as its possible adaptation to new animal species remain important issues for human health.

Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

BACKGROUND: Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates. METHODS: We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated. RESULTS: Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001). CONCLUSIONS: Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases.

In Vitro and In Vivo Models for Studying SARS-CoV-2, the Etiological Agent Responsible for COVID-19 Pandemic.

The emergence and rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted the scientific community to rapidly develop in vitro and in vivo models that could be applied in COVID-19 research. In vitro models include two-dimensional (2D) cultures of immortalized cell lines or primary cells and three-dimensional (3D) cultures derived from lung, alveoli, bronchi, and other organs. Although cell-based systems are economic and allow strict control of experimental variables, they do not always resemble physiological conditions. Thus, several in vivo models are being developed, including different strains of mice, hamsters, ferrets, dogs, cats, and non-human primates. In this review, we summarize the main models of SARS-CoV-2 infection developed so far and discuss their advantages, drawbacks and main uses.

Bisbenzylisoquinoline Alkaloids of Cissampelos Sympodialis With in Vitro Antiviral Activity Against Zika Virus.

In search of new antiviral compounds against Zika virus we conducted a bioassay-guided fractionation of bisbenzyilisoquinoline alkaloids isolated from Cissampelos sympodialis (Menispermaceae), a medicinal plant species endemic to Brazil. Six subfractions were obtained from a tertiary alkaloidal fraction of the rhizomes (TAFrz) using preparative high-performance liquid chromatography. All the subfractions were tested against Zika virus-infected Vero cells as the cellular model to evaluate cytotoxicity and antiviral effective concentrations. The results showed that three of the six TAFrz subfractions tested were active. The most active ones were the subfraction 6 (that consisted of the alkaloids methylwarifteine and warifteine present as a mixture at a ratio of 8.8:1.2 respectively) and the subfraction 5, that was later identified as warifteine, the major tertiary alkaloid of this species. Warifteine was able to significantly reduce virus titer in Zika virus-infected Vero cells with an IC50 of 2.2 µg/ml and this effect was selective (selectivity index, SI = 68.3). Subfraction 6 had an IC50 = 3.5 µg/ml and was more cytotoxic than pure warifteine, with SI = 6.14. Fraction 5 and fraction 6 were more potent in decreasing the viral titer of Zika virus-infected Vero cells than 6-methylmercaptopurine riboside (IC50 = 24.5 µg/ml and SI = 11.9), a mercaptopurine riboside with ZIKV antiviral activity used as a positive control. Our data demonstrate that alkaloids of the bisbenzylisoquinoline type may be explored as new antiviral agents or as an useful pharmacophore for investigating ZIKV antiviral activity.

Bisbenzylisoquinoline alkaloids of Cissampelos sympodialis with antiviral activity against dengue virus.

A number of bisbenzyilisoquinoline alkaloids have been previously isolated from Cissampelos sympodialis (Menispermaceae). The tertiary alkaloid fraction of the rhizomes (TAFrz) was prepared and the major alkaloid warifteine was isolated. Five TAFrz subfractions in addition to warifteine were tested against Dengue virus (DENV). We then used an epithelial (Vero) cell line to evaluate the cytotoxicity and effective concentrations of the samples against DENV. All TAFrz subfractions were active, but subfraction 6 (a mixture of the alkaloids methylwarifteine and warifteine) in particular showed a promising antiviral effect against DENV-2 with an IC50 of 2.00 µg/mL and a selectivity index (SI) of 10.74. Warifteine was the second most active sample and had an IC50 of 8.13 µg/mL and SI = 10.94. The antiviral activity of the samples compared favorably with that of 6-methylmercaptopurine riboside (IC50 = 7.31 µg/mL and SI = 11.8). These results suggest that bisbenzylisoquinoline alkaloids may prove interesting leading antiviral compounds.

Guillain-Barré syndrome during the Zika virus outbreak in Northeast Brazil: An observational cohort study.

OBJECTIVE: To determine the clinical phenotype of Guillain-Barré syndrome (GBS) after Zika virus (ZIKV) infection, the anti-glycolipid antibody signature, and the role of other circulating arthropod-borne viruses, we describe a cohort of GBS patients identified during ZIKV and chikungunya virus (CHIKV) outbreaks in Northeast Brazil. METHODS: We prospectively recruited GBS patients from a regional neurology center in Northeast Brazil between December 2014 and February 2017. Serum and CSF were tested for ZIKV, CHIKV, and dengue virus (DENV), by RT-PCR and antibodies, and serum was tested for GBS-associated antibodies to glycolipids. RESULTS: Seventy-one patients were identified. Forty-eight (68%) had laboratory evidence of a recent arbovirus infection; 25 (52%) ZIKV, 8 (17%) CHIKV, 1 (2%) DENV, and 14 (29%) ZIKV and CHIKV. Most patients with a recent arbovirus infection had motor and sensory symptoms (72%), a demyelinating electrophysiological subtype (67%) and a facial palsy (58%). Patients with a recent infection with ZIKV and CHIKV had a longer hospital admission and more frequent mechanical ventilation compared to the other patients. No specific anti-glycolipid antibody signature was identified in association with arbovirus infection, although significant antibody titres to GM1, GalC, LM1, and GalNAc-GD1a were found infrequently. CONCLUSION: A large proportion of cases had laboratory evidence of a recent infection with ZIKV or CHIKV, and recent infection with both viruses was found in almost one third of patients. Most patients with a recent arbovirus infection had a sensorimotor, demyelinating GBS. We did not find a specific anti-glycolipid antibody signature in association with arbovirus-related GBS.

Structural and functional impacts of E5 genetic variants of human papillomavirus type 31.

Persistent infections caused by high-risk human papillomavirus (HR-HPV) are important, for the development of cervical lesions, but environmental and genetic factors are also related in the process of carcinogenesis. Among the genetic factors, the genetic variants of HR-HPV appear to be related to the risk of persistent infections. Therefore, the present study investigates variants of HPV31 E5 oncogene in cervical scraping samples from Brazilian women to assess their functional and structural effects, in order to identify possible repercussions of these variants on the infectious and carcinogenic process. Our results detected nucleotide changes previously described in the HPV31 E5 oncogene, which may play a critical role in the development of cancer due to its ability to promote cell proliferation and signal transmission. In our study, the interaction percentage of the 31E5 sequence generated by the Immune Epitope Server database and the Analysis Resource (IEDB) allowed us to include possible immunogenic epitopes with the MHC-I and MHC-II molecules, which may represent a possible relationship between protein suppression of the immune system. In the structural analysis of the HPV31 E5 oncoprotein, the N5D, I48 V, P56A, F80I and V64I polymorphisms can be found inserted within transmembrane regions. The P56A mutation has been predicted to be highly stabilizing and, therefore, can cause a change in protein function. Regarding the interaction of the E5 protein from HPV31 with the signaling of NF-kB pathway, we observed that in all variants of the E5 gene from HPV-31, the activity of the NF-kB pathway was increased compared to the prototype. Our study contributes to a more refined design of studies with the E5 gene from HPV31 and provides important data for a better understanding of how variants can be distinguished under their clinical consequences.

Isatin Derivatives and Their Antiviral Properties Against Arboviruses: A Review.

Arboviruses have been spreading rapidly throughout the Western Hemisphere in recent decades. Among the arboviruses with high morbidity and mortality are the members of the Alphavirus and Flavivirus genera. Within the first genus, Chikungunya Virus (CHIKV) is considered one of the most challenging human arboviral infection worldwide, against which there is no specific antivirals. Flaviviruses are some of the main viruses responsible for encephalitis, haemorrhagic disease and developmental defects. Dengue virus (DENV), Japanese Encephalitis Virus (JEV), West Nile Virus (WNV) and Zika Virus (ZIKV) are examples of flaviviruses without clinically approved antiviral agents. Thus, the search for new antivirals becomes highly important. One of the strategies that can be employed to obtain new drugs is the identification and utilization of privileged structures. Isatin is an example of a privileged molecular framework, displaying a broad spectrum of biological activities, including antiviral action. Obtaining and studying the antiviral properties of isatin derivatives have helped to identify important agents with potential activity against different arboviruses. This article reviews some of these isatin derivatives, their structures and antiviral properties reported against this important group of viruses.

Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.

Zika virus tropism and interactions in myelinating neural cell cultures: CNS cells and myelin are preferentially affected.

The recent global outbreak of Zika virus (ZIKV) infection has been linked to severe neurological disorders affecting the peripheral and central nervous systems (PNS and CNS, respectively). The pathobiology underlying these diverse clinical phenotypes are the subject of intense research; however, even the principal neural cell types vulnerable to productive Zika infection remain poorly characterised. Here we used CNS and PNS myelinating cultures from wild type and Ifnar1 knockout mice to examine neuronal and glial tropism and short-term consequences of direct infection with a Brazilian variant of ZIKV. Cell cultures were infected pre- or post-myelination for various intervals, then stained with cell-type and ZIKV-specific antibodies. In bypassing systemic immunity using ex vivo culture, and the type I interferon response in Ifnar1 deficient cells, we were able to evaluate the intrinsic infectivity of neural cells. Through systematic quantification of ZIKV infected cells in myelinating cultures, we found that ZIKV infection is enhanced in the absence of the type I interferon responses and that CNS cells are considerably more susceptible to infection than PNS cells. In particular, we demonstrate that CNS axons and myelinating oligodendrocytes are especially vulnerable to injury. These results have implications for understanding the pathobiology of neurological symptoms associated with ZIKV infection. Furthermore, we provide a quantifiable ex vivo infection model that can be used for fundamental and therapeutic studies on viral neuroinvasion and its consequences.

Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

BACKGROUND: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. CONCLUSIONS/SIGNIFICANCE: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.