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 pan-chimeric universal primer (JS15N14) containing 15 nucleotides with a random tetradecamer (14N) attached to the 3'-end was designed. The complimentary primer (JS15) was used for nucleic acid enrichment in a first round PCR. A second PCR was designed to create Illumina sequencer-compatible sequencing-ready libraries for NGS. The new LF-SISPA protocol was tested using six RNA and DNA viral genomes (10.8-229.4 kilobases, kb) from an ATCC virome nucleic acid mix (ATCC® MSA-1008™) followed by analysis using One Codex, an online identification tool. In addition, a human stool sample known to be positive for norovirus GII was sequenced, and de novo assembly was performed using the Genome Detective Virus Tool allowing for near complete genome identification in less than 24 h. The LF-SISPA method does not require prior knowledge of target sequences and does not require an expensive enzymatic library preparation kit, thereby providing a simple, fast, low-cost alternative for the identification of unknown viral pathogens.

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. Norovirus RNA could be detected in UNEXP-eluted nucleic acids by reverse transcription-quantitative PCR (RT-qPCR) from 54 stool samples after 2 weeks storage at room temperature on disks; a subset of seven samples were positive after 3 months storage. Genotyping was successful in 76% of 54 samples tested including six of seven samples stored on the UNEXP disks for up to one month. Comparison of UNEXP with the FTA elute card in a subset of 10 samples demonstrated similar detection and genotyping rates after two weeks of storage at room temperature. UNEXP disks could be useful for epidemiologic investigations of disease outbreaks in resource-limited areas by simplifying specimen transport to regional diagnostic laboratories or shipment to international centers without the need to ship samples on dry ice.

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. The host innate immune response to RNA virus infection primarily involves pathogen-sensing toll-like receptors (TLRs) TLR3 and TLR7 and retinoic acid-inducible gene I-like receptor RIG-I and melanoma differentiation associated gene 5 (MDA5). In this study, by using siRNA knockdown, we report that TV infection in LLC-MK2 cells results in an early [3 h post infection (h p.i.), P<0.05] RIG-I-dependent and type I interferon-mediated antiviral response, whereas an MDA5-mediated antiviral effect was observed at later (12 h p.i.; P<0.05) stages of TV replication. Induction of RIG-I and MDA5 was critical for inhibition of TV replication. Furthermore, pre-activation of the RIG-I/MDA5 pathway prevented TV replication (>900-fold decrease; P<0.05), suggesting that RIG-I and MDA5 ligands could be used to develop novel preventive and therapeutic measures against norovirus.

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. Negative stain electron microscopy was used to measure aggregation before and after addition of virus particles to surface water at different pH and specific conductance levels. The impact of aggregation on the efficacy of chlorine disinfection was also examined. Disinfection experiments with human adenovirus 2 (HAdV2) in source water were conducted using 0.2 mg/L free chlorine at 5 degrees C. Aggregation of HAdV2 in source water (≥3 aggregated particles) remained higher at higher specific conductance and pH levels. However, aggregation was highly variable, with the percentage of particles present in aggregates ranging from 43 to 71 %. Upon addition into source water, the aggregation percentage dropped dramatically. On average, chlorination CT values (chlorine concentration in mg/L x time in min) for 3-log10 inactivation of aggregated HAdV2 were up to three times higher than those for dispersed HAdV2, indicating that aggregation reduced the disinfection rate. This information can be used by water utilities and regulators to guide decision making regarding disinfection of viruses 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. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

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 the response to inactivation and disinfection methods. We compared the infectivity reduction of CSV and Aichi virus (AiV) by extreme pH, 56 degrees C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP) using the same matrix and identical test parameters for all viruses, and compared with reduction of human norovirus RNA. At pH 2 FCV was inactivated by 6 log10 whereas MNV, TuV and AiV were resistant. All CSV were completely inactivated at 56 degrees C within 20 min. MNV was inactivated 5 log10 by alcohols compared to 2 and 3 log10 for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log10 by 1,000 ppm chlorine compared to 1 log10 for the other CSV. All CSV except FCV dried on stainless steel surfaces were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at ≥300 megapascal (MPa), TuV at 600 MPa while AiV was completely resistant up to 800 MPa. By RT-qPCR, genogroup (G)I norovirus was more sensitive than GII norovirus to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, overall TuV and MNV were the most resistant CSV and therefore are best candidates to study public health outcomes of norovirus infections.

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. Samples were first tested for HRV RNA by real-time RT-PCR (rRT-PCR). A subset of rRT-PCR positive samples were cultured into MRC-5 and HeLa Ohio cells. Among 177 hand samples tested for HRV by real-time RT-PCR, 52 (29%) were positive. Among 15 RT-PCR positive hand samples that were cultured, two grew HRV. HRV was detected in each of the sampling months (January, February, June, July, November, and December). This study demonstrates in the natural setting that, at least after sneezing or nasal cleaning, hands were contaminated commonly with potentially infectious HRV. Future research could explore if HRV RNA is present consistently and is associated sufficiently with the incidence of respiratory illness in communities that it may provide a proxy measure of respiratory viral hand contamination.

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. The median duration of measured fever was 3.0 days and of virus detection in NP specimens was 4.2 days; however, few specimens were collected between days 5-9. The duration of virus detection (4.2 days) was similar to the duration of fever (3.5 days) (RR, 1.14; 95% CI, .66-1.95; P=.8), but was shorter than the duration of cough (11.0 days) (RR, .41; 95% CI, .24-.68; P.001). We detected viral RNA in two participants' stools. All cultures were negative. This investigation suggests that the duration of virus detection was likely similar to the seasonal influenza virus.

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 degrees C. The Efficiency Factor Hom (EFH) model was used to calculate CT values (mg-min/L) required to achieve 2-, 3-, and 4-log(10) reductions in viral titers. In all water types, monochloramine disinfection was most effective for MNV, with 3-log(10) CT values at 5 degrees C ranging from 27 to 110. Monochloramine disinfection was least effective for HAdV2 and E11, depending on water type, with 3-log(10) CT values at 5 degrees C ranging from 1200 to 3300 and 810 to 2300, respectively. Overall, disinfection proceeded faster at 15 degrees C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but there was no indication that overall disinfection efficacy was enhanced or inhibited in any one water type. CT values for HAdV2 in two types of source water exceeded federal CT value recommendations in the US. The results of this study demonstrate that water quality impacts the inactivation of viruses and should be considered when developing chloramination plans.

RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1 - 0.2ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration. Serial ten fold dilutions of two waterborne viruses were added to the water concentrates for detection for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8 PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

More information is needed on the disinfection efficacy of chlorine for viruses in source drinking 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 ground water source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate using 0.2 and 1 mg/L free chlorine at 5 and 15 degrees C. The efficiency factor Hom (EFH) model was used to calculate CT values (mg-min/L) required to achieve 2-, 3-, and 4-log10 reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log10 CT values at 5 degrees C ranging from ≤ 0.020-0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log10 CT values at 5 degrees C ranging from 2.3-7.9. Overall, disinfection proceeded faster at 15 degrees C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA's Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems Using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.

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. Participants in the investigation and their household contacts were interviewed, and serum samples and respiratory tract specimens were collected. Risk factors were evaluated by means of conditional logistic regression. RESULTS: Among 32 pneumonia case patients, 21 (65%) had confirmed or probable HAdV-14 infection. None of 32 matched control participants had evidence of HAdV-14 infection ([Formula: see text] for the difference). Factors independently associated with pneumonia included contact with a known HAdV-14-infected case patient (odds ratio [OR], 18.3 [95% confidence interval {CI}, 2.0]), current smoking (OR, 6.7 [95% CI, 0.9]), and having neither traveled off the island nor attended a large public gathering (OR, 14.7 [95% CI, 2.0]). Fourteen (67%) of 21 HAdV-14-positive case patients belonged to a single network of people who socialized and often smoked together and infrequently traveled off the island. HAdV-14 infection occurred in 43% of case-patient household contacts, compared with 5% of control-participant household contacts ([Formula: see text]) CONCLUSIONS: During a community outbreak in Alaska, HAdV-14 appeared to have spread mostly among close contacts and not widely in the community. Demographic characteristics and illness patterns among the case patients were similar to those observed in other recent outbreaks of HAdV-14 infection in the United States.

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 byproduct formation and biofilm growth. The inactivation of 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. Experiments were performed with 0.2 mg/L free chlorine or 1 mg/L monochloramine at pH 7 and pH 8 in buffered reagent grade water (RGW) at 5 degrees C. Ct values for 2-4-log10 (99-99.99%) reductions in viral titers were calculated using the Efficiency Factor Hom (EFH) model. The enteroviruses required the longest times for chlorine inactivation and MNV the least time. CVB5 required the longest exposure time, with Ct values of 7.4 and 10 mg-min/L (pH 7 and 8) for 4-log10 inactivation. Monochloramine disinfections was most effective for E1 (Ct values ranged from 8 to 18 mg-min/L for 2- and 3-log10 reductions, respectively). E11 and HAdV2 were the least susceptible to monochloramine disinfection, (Ct values of 1300 and 1600 mg-min/L for 3-log10 reductions, respectively). Monochloramine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disinfection. This data will be useful in drinking water risk assessment studies and disinfection system planning.