Clam (\u3ci\u3eCorbicula fluminea\u3c/i\u3e) as a Potential Sentinel of Human Norovirus Contamination in Freshwater

The purpose of this study was to evaluate and validate the use of the clam Corbicula fluminea as a sentinel of human noroviruses (HuNoV) contamination in freshwater. The first specific aim was to develop a new method to extract HuNoV RNA from contaminated bivalves (e.g. oysters, clams) that would be much faster than existing methods. The procedure developed includes an initial total RNA extraction using TRI Reagent, followed by HuNoV RNA concentration and purification using biotinylated probe-capture technology. HuNoV RNA is finally detected by real-time RT-PCR. Using bivalve homogenates spiked with HuNoV, 100 PCR detection units of the virus was detectable. Compared to published methods that require an initial virus purification step, the new method is much faster to complete. Approximately 3 h are needed to purify NoV RNA using the new method compared to at least 8 h using conventional methods. Coupled with real-time RT-PCR, the new method can detect HuNoV in contaminated bivalves within 8 h. The detection limit of the method was 10 -100 PDU of HuNoV. In addition, the method was successfully applied for HuNoV detection in live artificiallycontaminated oysters, wild oysters, and also for murine norovius (MNV-1) and HuNoV detection in clams. The second specific aim was to evaluate the ability of C. fluminea to bioaccumulate and depurate HuNoV using MNV-1 as a surrogate of HuNoV. Clams were exposed to MNV-1 in 10 L artificial pond water for 6 h, 1, 2, and 3 d in an environmental chamber. Depuration experiments were carried out in 80 L artificial pond water for 0, 1, 4, 7, 10, and 15 d at 10°C and 20°C. MNV-1 was detectable after 6 h and 1 d exposure in clams exposed to virus concentrations of 106 PFUL-1 and 104 PFUL-1, respectively. The amount of bioaccumulated MNV-1 increased as the exposure period increased from 6 h to 3 d. The lowest virus concentration at which exposed clams were PCR-positive was 102 PFUL-1 after 2 d exposure at 20°C. Clams bioaccumulated MNV-1 more quickly at 20°C than at 10°C (p \u3c 0.05). The virus was persistently detected in contaminated clams during depuration at both 10°C and 20°C. Depuration occurred significantly more quickly at 20°C than at 10°C (p \u3c 0.05). The results indicate that the clam is likely to be useful as a sentinel for detecting NoV contamination in freshwater. The third specific aim was to determine whether C. fluminea is effective as a sentinel of HuNoV contamination in natural freshwater. Clams were collected from Lake Serene in Hattiesburg, Mississippi where HuNoV has never been detected from Oct 2010 to Jul 2011, and translocated to 9 sites at 4 freshwater creeks in Gulfport and Long Beach where the creek water flowed into Mississippi Sound. HuNoV RNA was isolated from clams (n = 588) using the biotinylated probe hybridization method mentioned above and detected by qRT-PCR. Correct identity of the virus was accomplished by sequencing some of the amplified RT-PCR products (HuNoV capsid N-terminal/shell domain). qRTPCR results showed that HuNoV GI and GII were detectable in the translocated C. fluminea mainly during the warmer months (Apr to Jul and Oct), but not during the colder months (Dec to Mar). Based on sequence comparisons, the HuNoV detected in translocated clams were classified into GI/17 and GII/4, respectively. Statistical analysis using binary logistic regression showed that water temperature and turbidity (p = 0.026 and p = 0.038, respectively), but not pH, salinity, or current velocity (p = 0.476, p = 0.425, and p = 0.174, respectively), were significant factors affecting HuNoV presence/absence in clams in freshwater creeks. In conclusion, it was found that the freshwater clam C. fluminea can be translocated and serve as an effective sentinel of HuNoV contamination in freshwater of low turbidity during warm months

Development of a Probe Hybridization Method to Facilitate Detection of Noroviruses in Oysters

Centers for Disease Control (CDC) reports that at least 50% of all foodborne outbreaks of gastroenteritis are due to noroviruses (NoV). Since NoV is mainly transmitted through the fecal-oral route and the infectious dose may be as low as 10 viral particles, the risk of infection after consumption of raw or improperly cooked seafood or after exposure to contaminated water is considered high. Although highly sensitive methods to detect NoV using RT-PCR are already available, isolation of either NoV RNA or virions from shellfish remains a cumbersome process. We developed a new hybridization method to extract NoV RNA from contaminated shellfish that is much faster compared to existing methods. Using the new method, NoV detection includes three basic steps: an initial extraction of total RNA using TRIZol, followed by isolation of NoV RNA using biotinylated DNA probe hybridization and then NoV detection by TaqMan RT-PCR. With oyster (Crassostrea virginica) homogenate spiked with 100 PCR detection units (PDU) of NoV, the virus can be detected with CT values at about 30. Compared to published methods that require an initial virus purification step, the new method is much faster, requiring approximately 3 hr compared to at least 8 hr using conventional methods. Coupled with TaqMan RT-PCR, the new method can be used to detect NoV in contaminated oysters and clams (Corbicula fluminea) within 8 hr. The detection limit was 100 PDU of NoV in spiked oyster tissue samples. The method has been successfully used to detect NoV in oysters artificially contaminated in the laboratory and in rare cases, oysters collected from the field

A Comparison of Methods to Concentrate Viruses from Environmental Waters Using MS2 as a Model

Viruses such as Rotavirus, Adenovirus and Norovirus are important etiological agents of gastroenteritis worldwide. With the high sensitivity and specificity of PCR, it is now possible to develop PCR-based methods to detect and quantify pathogenic viruses in environmental water samples. To develop reliable methods however, an effective procedure to concentrate viruses from large volumes of water is required. Because of the scale of concentration required, the procedure often requires two steps. The first to reduce tens of liters of water to less than half a liter and then a second to concentrate the sample to a final volume of less than 10 mL for RNA/DNA extraction. The objectives of the study were to compare the efficacy of hollow fiber ultrafiltration (HFUF) using F200B to that of an adsorption/elution method (AEM) using positively charged filters for concentrating viruses for the first step and to compare polyethylene glycol (PEG) precipitation to centrifugal ultrafiltration for the second step. A third objective was to determine the viral detection limit using real-time RT-PCR. Using beach water spiked with a singlestranded RNA bacteriophage (MS2) as a model, our results show a virus recovery rate of 84±6% and 18±8% for the HFUF method and AEM, respectively. For the second concentration step, we obtained a recovery rate of 49±5 % and 87±7% using PEG precipitation and centrifugal ultrafiltration, respectively. A potential limiting factor to more widespread using of HFUF is the higher cost and we found that cost can be reduced by using reusable filters. We were able to sanitize and reuse the same filter at least six times without affecting the virus recovery rate or the processing time

Antigenic Site-Specific Competitive Antibody Responses to the Fusion Protein of Respiratory Syncytial Virus Were Associated With Viral Clearance in Hematopoietic Cell Transplantation Adults

Background: Recent studies of human sera showed that the majority of the respiratory syncytial virus (RSV) neutralizing antibodies are directed against pre-fusion conformation of the fusion (F) protein of RSV and revealed the importance of pre-fusion antigenic site Ø specific antibodies. However, detailed analysis of multiple antigenic site-specific competitive antibody responses to RSV F protein and their contribution to virus clearance in humans are lacking.Methods: We prospectively enrolled a cohort of RSV infected hematopoietic cell transplantation (HCT) adults (n = 40). Serum samples were collected at enrollment (acute, n = 40) and 14 to 60 days post-enrollment (convalescent, n = 40). Antigenic site-specific F protein antibodies were measured against pre-fusion site Ø, post-fusion site I, and sites II and IV present in both the pre-fusion and post-fusion F protein conformations utilizing four different competitive antibody assays developed with biotinylated monoclonal antibodies (mAb) D25, 131-2A, palivizumab, and 101F, respectively. The lower limit of detection were 7.8 and 1.0 μg/mL for the competitive antibody assays that measured site Ø specific response, as well as sites I, II, and IV specific responses, respectively. Neutralizing antibody titers to RSV A and B subgroups was determined by microneutralization assays.Results: The overall findings in RSV infected HCT adults revealed: (1) a significant increase in antigenic site-specific competitive antibodies in convalescent sera except for site Ø competitive antibody (p < 0.01); (2) comparable concentrations in the acute and convalescent serum samples of antigenic site-specific competitive antibodies between RSV/A and RSV/B infected HCT adults (p > 0.05); (3) significantly increased concentrations of the antigenic site-specific competitive antibodies in HCT adults who had genomic RSV detected in the upper respiratory tract for <14 days compared to those for ≥14 days (p < 0.01); and (4) statistically significant correlation between the antigenic site-specific competitive antibody concentrations and neutralizing antibody titers against RSV/A and RSV/B (r ranged from 0.33 to 0.83 for acute sera, and 0.50–0.88 for convalescent sera; p < 0.05).Conclusions: In RSV infected HCT adults, antigenic site-specific antibody responses were induced against multiple antigenic sites found in both the pre-fusion and post-fusion F conformations, and were associated with a more rapid viral clearance and neutralizing antibody activity. However, the association is not necessarily the cause and the consequence

Antibody Response to the Furin Cleavable Twenty-Seven Amino Acid Peptide (p27) of the Fusion Protein in Respiratory Syncytial Virus (RSV) Infected Adult Hematopoietic Cell Transplant (HCT) Recipients

Background: Cleavage of the inactive precursor fusion protein (F0) of respiratory syncytial virus (RSV) at two furin-recognition sites is required for membrane fusion activity, and the cleavage releases the twenty-seven amino acid peptide (p27). However, a recent study shows that p27 was an immunodominant epitope in RSV infected children, indicating that p27 was recognized as an immunogen. In the present study, we investigated the immunogenicity of p27 in an immunocompromised population of adults by measuring serum and mucosal antibody responses to p27 in samples from adult hematopoietic cell transplant (HCT) recipients. Methods: We prospectively enrolled a cohort of RSV infected HCT recipients. Serum and nasal-wash samples were obtained within the first week of RSV infection (acute) and 3 to 5 weeks post-infection (convalescent). We quantified the serum and mucosal IgG and IgA anti-p27 antibodies by a RSV/A p27 peptide enzyme-linked immunosorbent assay (ELISA) and serum and mucosal p27 like antibodies (P27LA) by a p27 competitive antibody (P27CA) assay. Results: The lower limit of detection for the ELISA and P27CA assays was 0.2 and 50 ng/mL, respectively with no cross-reaction detected with a panel of monoclonal antibodies targeting pre-fusion and post-fusion antigenic sites. P27 antibodies were detected at nanogram concentration in sera and nasal washes in the majority of RSV infected HCT recipients. However, there was no significant difference in the geometric mean antibody concentrations between the acute and convalescent sera (except for serum P27LA), between HCT recipients who shed RSV <14 days and ≥14 days, as well as between RSV/A and RSV/B infected HCT recipients. In addition, approximately 30% of HCT recipients had a 4-fold or greater decrease in mucosal IgG and IgA anti-p27 antibodies during viral clearance. Conclusion: In conclusion, in RSV naturally infected adult HCT recipients, the antibodies against p27 were detectable in both serum and nasal wash samples with higher concentration in serum than that in nasal washes. However, nearly 30% of RSV infected HCT recipients had a significant decrease in their mucosal anti-p27 antibody, suggesting that IgG and IgA anti-p27 antibodies were binding to either free viruses or RSV infected cells containing p27, and that anti-p27 antibodies in the respiratory tract were part of the mucosal antibody response in controlling RSV infection

A Faster Method to Detect Norovirus in Oysters Using Probe Hybridization to Isolate Target RNA Before RT-PCR

Human Noroviruses (HuNoVs) are the most frequent cause of outbreaks of acute gastroenteritis following the ingestion of raw or improperly cooked oysters. Although highly sensitive methods to detect HuNoV in oysters using reverse transcriptase-polymerase chain reaction (RT-PCR) are available, rapid methods to process samples for RT-PCR are still needed. The conventional approach is to concentrate the virus first before RNA purification to maximize assay sensitivity, but the procedures used are cumbersome. We developed a new hybridization method that is much faster and more effective compared to existing technology. The procedure includes an initial extraction of total RNA from the digestive diverticula of oysters using TRI Reagent, followed by HuNoV RNA purification using a capture probe and then HuNoV detection by real-time RT-PCR. The detection limit is approximately 100 PCR detection units of HuNoV per sample. Compared to published methods that require an initial virus concentration step before RNA extraction, the new method is much faster to complete. Approximately 3 h are needed to purify HuNoV RNA using the new method compared to at least 8 h using conventional methods. Coupled with real-time RT-PCR, the new method can detect HuNoV in contaminated oysters within 8 h. The effectiveness of the method was demonstrated using live artificially contaminated oysters and wild oysters

Validation and Field Testing of Library-Independent Microbial Source Tracking Methods in the Gulf of Mexico

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10−5–10−6 dilution). LODs for HPyVs and M. smithii assays were similar to each other (10−3–10−4), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among \u3e300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts’ workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2

Humoral and Mucosal Antibody Response to RSV Structural Proteins in RSV-Infected Adult Hematopoietic Cell Transplant (HCT) Recipients

Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in infants, the elderly, and immunocompromised patients. RSV antibodies play a role in preventing reinfection and in clearance of RSV, but data regarding the levels of viral protein-specific antibodies elicited and their contribution to patient recovery from RSV-induced disease are limited. We prospectively enrolled a cohort of RSV-infected adult hematopoietic cell transplant (HCT) recipients (n = 40). Serum and nasal-wash samples were obtained at enrollment (acute samples) and convalescence (convalescent samples). We measured (1) humoral IgG and mucosal IgA binding antibody levels to multiple RSV proteins (F, G, N, P, and M2-1) by Western blot (WB); (2) neutralizing antibody (Nt Ab) titers by microneutralization assay; and (3) palivizumab-like antibody (PLA) concentrations by an ELISA-based competitive binding assay developed in the lab. Finally, we tested for correlations between protein-specific antibody levels and duration of viral shedding (normal: cleared in <14 days and delayed: cleared ≥14 days), as well as RSV/A and RSV/B subtypes. Convalescent sera from HCT recipients had significantly higher levels of anti-RSV antibodies to all 5 RSV structural proteins assayed (G, F, N, P, M2-1), higher Nt Abs to both RSV subtypes, and higher serum PLAs than at enrollment. Significantly higher levels of mucosal antibodies to 3 RSV structural proteins (G, N, and M2-1) were observed in the convalescent nasal wash versus acute nasal wash. Normal viral clearance group had significantly higher levels of serum IgG antibodies to F, N, and P viral proteins, higher Nt Ab to both RSV subtypes, and higher PLA, as well as higher levels of mucosal IgA antibodies to G and M2-1 viral proteins, and higher Nt Ab to both RSV subtypes compared to delayed viral clearance group. Normal RSV clearance was associated with higher IgG serum antibody levels to F and P viral proteins, and PLAs in convalescent serum (p < 0.05). Finally, overall antibody levels in RSV/A- and/B-infected HCT recipients were not significantly different. In summary, specific humoral and mucosal RSV antibodies are associated with viral clearance in HCT recipients naturally infected with RSV. In contrast to the humoral response, the F surface glycoprotein was not a major target of mucosal immunity. Our findings have implications for antigen selection in the development of RSV vaccines