Cross-Protection of Dengue Virus Infection against Congenital Zika Syndrome, Northeastern Brazil.

The Zika virus outbreak in Latin America resulted in congenital malformations, called congenital Zika syndrome (CZS). For unknown reasons, CZS incidence was highest in northeastern Brazil; one potential explanation is that dengue virus (DENV)-mediated immune enhancement may promote CZS development. In contrast, our analyses of historical DENV genomic data refuted the hypothesis that unique genome signatures for northeastern Brazil explain the uneven dispersion of CZS cases. To confirm our findings, we performed serotype-specific DENV neutralization tests in a case-control framework in northeastern Brazil among 29 Zika virus-seropositive mothers of neonates with CZS and 108 Zika virus-seropositive control mothers. Neutralization titers did not differ significantly between groups. In contrast, DENV seroprevalence and median number of neutralized serotypes were significantly lower among the mothers of neonates with CZS. Supported by model analyses, our results suggest that multitypic DENV infection may protect from, rather than enhance, development of CZS.

Waterline Disinfectants Reduce Dental Bioaerosols: A Multitracer Validation.

Oral microbes are dispersed during dental treatment and reduction methods have been proposed, but dental unit waterline (DUWL) disinfectants have received little attention; specifically, the effect on viruses has not been studied. This study aims to 1) investigate the effect of DUWL disinfectants on viral dispersion in dental bioaerosols and 2) establish a dual-tracer system using live bacteriophage and fluorescein supported by optical particle measurement. Bacteriophage MS2 was used as a viral tracer and fluorescein as a fluorescent tracer. Validation experiments were conducted to exclude interference of one tracer with the other or of DUWL disinfectants on detection methods. Simulated "saliva" containing the tracers was infused into the mouth of a dental mannequin during 10-min dental procedures with an air turbine handpiece (n = 3 replicates). Aerosols and droplets were sampled in an enclosed dental operatory using air samplers and settlement onto sterile filter papers. Bacteriophage was quantified using plaque assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Fluorescein was quantified fluorometrically. The effect of DUWL disinfectants on total aerosol concentration was assessed in separate experiments using an optical particle counter. DUWL disinfectants reduced bacteriophage viability, and interference between tracers was not observed. In simulated clinical procedures, the disinfectant ICX reduced bacteriophage detection substantially (P < 0.001; 2-way analysis of variance). MS2 RNA was detected in all experimental samples but not negative controls. Samples positive on RT-qPCR but not plaque assays may indicate that virions at distant sites are nonviable. Fluorescein tracer showed good agreement with the bacteriophage tracer. DUWL disinfectants designed for continuous presence in irrigants reduce the dispersion of viable virus in dental bioaerosols during simulated procedures. Their use may therefore be important for routine infection control and as a mitigation factor during infectious disease outbreaks. Future studies should explore this using a range of viruses and other microbes.

Machine-learning-based automated quantification machine for virus plaque assay counting.

The plaque assay is a standard quantification system in virology for verifying infectious particles. One of the complex steps of plaque assay is the counting of the number of viral plaques in multiwell plates to study and evaluate viruses. Manual counting plaques are time-consuming and subjective. There is a need to reduce the workload in plaque counting and for a machine to read virus plaque assay; thus, herein, we developed a machine-learning (ML)-based automated quantification machine for viral plaque counting. The machine consists of two major systems: hardware for image acquisition and ML-based software for image viral plaque counting. The hardware is relatively simple to set up, affordable, portable, and automatically acquires a single image or multiple images from a multiwell plate for users. For a 96-well plate, the machine could capture and display all images in less than 1 min. The software is implemented by K-mean clustering using ML and unsupervised learning algorithms to help users and reduce the number of setup parameters for counting and is evaluated using 96-well plates of dengue virus. Bland-Altman analysis indicates that more than 95% of the measurement error is in the upper and lower boundaries [±2 standard deviation]. Also, gage repeatability and reproducibility analysis showed that the machine is capable of applications. Moreover, the average correct measurements by the machine are 85.8%. The ML-based automated quantification machine effectively quantifies the number of viral plaques.

Mouse Models for Cytomegalovirus Infections in Newborns and Adults.

This article describes procedures for infecting adult mice with murine cytomegalovirus (MCMV) and for infecting newborn mice to model congenital CMV infection. Methods are included for propagating MCMV in cell cultures and preparing a more virulent form of MCMV from the salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, methods are described for preparing the murine embryonic fibroblasts used for propagating MCMV, and for the plaque assay. © 2022 Wiley Periodicals LLC.

Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13.

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

Cytomegalovirus Infection: Mouse Model.

This unit describes procedures for infecting newborn and adult mice with murine cytomegalovirus (MCMV). Methods are included for propagating MCMV in cell cultures and for preparing a more virulent form of MCMV from salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, a method is described for preparing the murine embryonic fibroblasts used for propagating MCMV and for the plaque assay. © 2018 by John Wiley & Sons, Inc.

Characterization and Transferring of Human Rotavirus Double-Layered Particles in MA104 Cells.

BACKGROUND: Rotavirus (RV) is a major cause of gastroenteritis in infants and children and is one of the most severe public health problems. Rotaviruses outer layer contains two proteins including VP4 and VP7. These proteins are necessary for host-cell binding and penetration. TLP (triple layer virus particle) of RV is a complete infectious virion that binds to the target cells and internalized at the cytoplasm. The DLP (double layer virus particle) is a non-infectious particle that is formed through exclusion of the outer layer proteins including VP4 and VP7. These DLPs are the transcriptionally active forms of rotavirus. OBJECTIVES: The aim of this study was to transfer DLP of RV into cytoplasm of MA104 cells by Lipofectamine and to analyze their replication. MATERIALS AND METHODS: Initially, rotavirus was purified by CsCl discontinuous gradient and DLP was separated from TLP based on density differences. For confirmation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins were conducted Then the purified DLP of RV was transferred into MA104 cells using Lipofectamine. RESULTS: We attempt to avoid the attachment and entry of the rotavirus by using Lipofectamine to mediate the delivery of viral particles directly into the cytoplasm. DLP was endocytosed into the cytoplasm following treatment by Lipofectamine and then replicated in cytoplasm. CONCLUSIONS: Therefore the non-infectious DLPs were became infectious if introduced into the cytoplasm of permissive and cancerous cells, without passing attachment and entry process.

Growth curve of murine xenotropic leukemia virus-related virus grown in Chinese hamster ovary cells.

BACKGROUND: Murine xenotropic leukemia virus-related virus (XMulV) is used as a model virus in the evaluation of viral inactivation in Chinese hamster ovary (CHO) cell-derived pharmaceutical proteins. Mus dunni cells and mink lung cells are used to produce XMulV particles. In consideration of the characteristics of XMulV, we tried to propagate the viruses on CHO cells, a nonmurine cell line. METHODS: The viruses were harvested from CHO cells from Day 2 to Day 7 postinfection, and reverse transcription-quantitative polymerase chain reaction was performed to quantify the viruses on different days. A cell-based infectivity assay was used to evaluate the XMulV titers. RESULTS: The content of the XMulV virions began to increase on Day 5 and grew exponentially from Day 6 to Day 7 postinfection. The growth curve was a typical single-step growth curve. Titers of the viral stock harvested on Day 7 were assayed on PG-4 cells, and the titers were 8.78 ± 0.25 log10 PFU/mL. CONCLUSION: Based on these data, we conclude that CHO cells could be a host cell line for XMulV particles. XMulV produced on Day 7 in CHO cells could be used at a laboratory scale for the evaluation of XMulV clearance in pharmaceutical proteins derived from CHO cells.