Development and evaluation of a ligation-free sequence-independent, single-primer amplification (LF-SISPA) assay for whole genome characterization of viruses.

Molecular identification and characterization of novel or re-emerging infectious pathogens is critical for disease surveillance and outbreak investigations. Next generation sequencing (NGS) using Sequence-Independent, Single-Primer Amplification (SISPA) is being used extensively in sequencing of viral genomes but it requires an expensive library preparation step. We developed a simple, low-cost method that enriches nucleic acids followed by a ligation-free (LF) 2-step Polymerase Chain Reaction (PCR) procedure for library preparation.

A new solid matrix for preservation of viral nucleic acid from clinical specimens at ambient temperature.

Stabilizing paper matrix methods for retaining nucleic acid from inactivated clinical specimens offer a solution for molecular diagnostics when specimens may be stored or shipped at ambient temperature. We developed cellulose disks (UNEXP) saturated with a total nucleic acid extraction buffer (UNEX) modified from a previously developed lysis buffer for multiple enteric pathogens. Infectivity of hepatitis A virus, adenovirus and poliovirus was destroyed after 2-3 h incubation at room temperature on the UNEXP disks.

Critical role of RIG-I and MDA5 in early and late stages of Tulane virus infection.

Human noroviruses are a major cause of acute gastroenteritis worldwide, but the lack of a robust cell culture system or small animal model have hampered a better understanding of innate immunity against these viruses. Tulane virus (TV) is the prototype virus of a tentative new genus, Recovirus, in the family Caliciviridae. Its epidemiology and biological properties most closely resemble human norovirus.

Aggregation of Adenovirus 2 in Source Water and Impacts on Disinfection by Chlorine.

It is generally accepted that viral particles in source water are likely to be found as aggregates attached to other particles. For this reason, it is important to investigate the disinfection efficacy of chlorine on aggregated viruses. A method to produce adenovirus particle aggregation was developed for this study.

Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance.

Comprehensive comparison of cultivable norovirus surrogates in response to different inactivation and disinfection treatments.

Human norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions.

Hand contamination with human rhinovirus in Bangladesh.

As one step in developing a measure of hand contamination with respiratory viruses, this study assessed if human rhinovirus (HRV) was detectable on hands in a low income non-temperate community where respiratory disease is a leading cause of child death. Research assistants observed residents in a low income community in Dhaka, Bangladesh. When they observed a resident sneeze or pick their nose, they collected a hand rinse and anterior nare sample from the resident.

Virus detection and duration of illness among patients with 2009 pandemic influenza A (H1N1) virus infection in Texas.

Knowledge from early outbreaks is limited regarding the virus detection and illness duration of the 2009 pandemic influenza A (H1N1) infections. During the period from April to May 2009 in Texas, we collected serial nasopharyngeal (NP) and stool specimens from 35 participants, testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) and culture. The participants were aged 2 months to 71 years; 25 (71%) were under 18.

Source water quality effects on monochloramine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus.

There is a need for more information regarding monochloramine disinfection efficacy for viruses in water. In this study, monochloramine disinfection efficacy was investigated for coxsackievirus B5 (CVB5), echovirus 11 (E11), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated ground water and two partially treated surface waters. Duplicate disinfection experiments were completed at pH 7 and 8 in source water at concentrations of 1 and 3 mg/L monochloramine at 5 and 15 °C.

Effects of source water quality on chlorine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus.

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.

Outbreak of pneumonia associated with emergent human adenovirus serotype 14--Southeast Alaska, 2008.

Background: In September 2008, an outbreak of pneumonia associated with an emerging human adenovirus (human adenovirus serotype 14 [HAdV-14]) occurred on a rural Southeast Alaska island. Nine patients required hospitalization, and 1 patient died.

Methods: To investigate the outbreak, pneumonia case patients were matched to control participants on the basis of age, sex, and community of residence.

Inactivation of adenoviruses, enteroviruses, and murine norovirus in water by free chlorine and monochloramine.

Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41 (HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and E11), and murine norovirus (MNV) are compared in this study.

Comparison of hollow-fiber ultrafiltration to the USEPA VIRADEL technique and USEPA method 1623.

Hollow-fiber ultrafiltration (UF) is a technique that is increasingly viewed as an effective alternative for simultaneously recovering diverse microbes (e.g., viruses, bacteria, parasites) from large volumes of drinking water.

Development of plaque assays for adenoviruses 40 and 41.

Enteric adenoviruses, important agents of infantile gastroenteritis, are difficult to culture with low titers and limited CPE. Consequently, few plaque assays have been reported and none are used routinely by investigators who may need reproducible quantitative assays for these viruses. CPE in A549 cells (an epithelial lung carcinoma cell line) was induced by isolates of human adenovirus (HAdV) serotypes 40 or 41 that were obtained by prior limited passage in primary cynmolgous monkey kidney (pCMK), human embryonic kidney (HEK), and Graham 293 cells.

Ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from 100-L tap water samples.

This study focused on ultrafiltration as a technique for simultaneously concentrating and recovering viruses, bacteria and parasites in 100-L drinking water samples. A chemical dispersant, sodium polyphosphate, and Tween 80 were used to increase microbial recovery efficiencies. Secondary concentration was performed to reduce sample volumes to 3-5 mL for analysis using tissue culture, microscopy, and real-time PCR and RT-PCR.

Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples.

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States.

Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants.

The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2, Escherichia coli, Enterococcus faecalis, Cryptosporidium parvum, 4.

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia).

UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR.

Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation.

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41.

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.

Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR.

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41.