1. Continuing Studies of Viruses in Hampton Roads and Shellfish Howard Kator, Kimberly Reece, Corinne Audemard, Wendi Ribiero, Martha Rhodes With support from: Hampton Roads District Commission NOAA Sea Grant Virginia Institute of Marine Science

2. SETTING THE STAGE: PREVIOUS WORK REPORTED AT THE LAST ISS MEETING IN VIRGINIA BEACH

3. VIMS/VDH/VMRC and HRSD Combined Hampton Roads Clam Study

4. Background In 2005 the Division of Shellfish Sanitation (DSS), approached VIMS with regard to evaluating the sanitary quality of the Hampton Roads clam (Mercenaria mercenaria) resource. Specifically, although much of the area is closed to direct harvesting, questions arose concerning the safety of clams harvested from these waters for relay to approved waters for marketing. VDH concerns centered on the presence of effluents from four waste water treatment plants (WWTP) discharging to the waters of Hampton Roads. WWTP effluents have been shown to be potential sources of pathogenic human enteroviruses (e. g., van den Berg et al. 2005) and the effectiveness of the relaying process for elimination of viruses such as norovirus (NoV) from clams is unknown.

5. Background A Fall 2007 VIMS/VDH study design involved placement of on- bottom cages containing clams at two locations in Hampton Roads followed by analysis of microbial burdens that included NoV, FRNA coliphage and fecal coliforms/Escherichia coli. Two locations were chosen on the basis of proximity to the HRSD Nansemond WWTP outfall. Exposure studies were conducted (Fall 2007) when viral persistence or occurrence would be favored, i. e., in the fall when water temperatures were decreasing.

6. Figure 1. Location of clam exposure sites for Fall 2007 samples. NANSEMOND WWTP OUTFALL CLAMS OUTSIDE WWTP BUFFER ZONE CLAMS WITHIN WWTP BUFFER ZONE

7. *Fecal coliforms and E. coli densities determined using the APHA 5-tube MPN with EC-MUG as the medium. FRNA coliphage measured following a proposed FDA method but using Salmonella typhimurium WG49 as the assay host. NoV occurrence indicated as ratio of analytical replicates that were positive by real-time PCR. Water samples collected at the time of shellfish retrieval: "Cond 1 " - <1.8 fecal coliforms and E. coli per 100 ml; <1 FRNA phage per 100 ml; 22.8 psu, 12.1  C Nansemond WWTP outfall - 2.0 fecal coliforms and E. coli per 100 ml; <1 FRNA phage per 100 ml; 22.6 psu, 11.8  C. Table 1. VIMS microbiological results for clams exposed for ca. 2 weeks at the Nansemond WWTP outfall and Condemnation Line 1 ("Cond 1") in Hampton Roads. Samples retrieved and analyzed Nov. 28, 2007.* Clams were sourced from a commercial dealer (Cherrystone Aquaculture) and approved for human consumption. For some samples that were positive for NoV the PCR amplification products were sequenced to determine whether genogroup I, II or both were detected. Detected norovirus on both "sides" of line FRNA coliphage higher near outfall

8. *Fecal coliforms and E. coli densities determined using the APHA 5-tube MPN with EC-MUG as the medium. FRNA coliphage measured following a proposed FDA method but using Salmonella typhimurium WG49 as the assay host. NoV occurrence indicated as ratio of analytical replicates that were positive by nested PCR. Water samples collected at the time of shellfish retrieval: Cond 1 – 2.0 fecal coliforms and E. coli per 100 ml; <1 FRNA phage per 100 ml; 23.1 psu, 10.1°C Nansemond WWTP outfall - <1.8 fecal coliforms and E. coli per 100 ml; <1 FRNA phage per 100 ml; 22.5 psu, 9.9°C Table 2. VIMS microbiological results for clams exposed for ca. 3 weeks at the Nansemond WWTP outfall and Condemnation Line 1 (Cond 1) in Hampton Roads. Samples retrieved and analyzed Dec. 4, 2007.* Clams were sourced from a commercial dealer (Cherrystone Aquaculture) and approved for human consumption. Detected norovirus on both "sides" of line FRNA coliphage higher near outfall

9. Figure 2. Clam deployment sites and WWTP effluent locations- Spring 2008 experiments

10. GI GII Effluents-GIGII

11. DNA sequences from sequencing the PCR fragment amplified with GI specific primers and probe. Note the relatively highly conserved sequences with a few nucleotide differences suggesting that there is some genetic strain variation both within and between sites.

12. 2009 COMPARATIVE RELAY STUDY Contamination under natural conditions Relay into approved waters Crassostrea virginica Mercenaria mercenaria Detect and relate norovirus to FRNA coliphage, measure virus elimination kinetics

13. 2009- A "strange" year for obtaining shellfish contaminated with norovirus?

14. Objective: norovirus and FRNA coliphage uptake at densities high enough to follow elimination kinetics over 14 day time course

15. Cont'd

18. *Detection of norovirus GI/GII in 2 qPCR analytical replicates of post-chlorinated effluent

21. Plan one more set of experiments for the Fall of 2010 Either in situ contamination or tank contamination with relay in approved waters? Evaluate virus recovery using APHA 10-12 oyster homogenate versus homogenizing digestive diverticula from 1, 3 or 5 oyster samples to improve detection

22. VIRUS DETECTION METHODS

23. Shellfish sample processing for norovirus (based on Jothikumar et al. 2005; Gentry et al. 2009) Aliquot 1.5 g (can freeze at this point) qPCR for the detection of GI and for GII 37°C for 1 hr with shaking 65°C for 15 min Centrifuge 3000 x g for 5 min, collect supernatant Homogenization of clam tissues RNA extraction using the MagMAX kit Add Buffer + Proteinase K Reverse transcription to obtain cDNA Virus release from the shellfish tissues RNA extraction Virus detection DNA sequencing to identify the strain(s) detected

24. Adenovirus Qualities supporting its evaluation as a candidate indicator: Appears to be stable in aqueous environments Resistant to UV radiation, chlorination Doesn’t appear to have substantial seasonal variation (as does norovirus) Unlike norovirus it can be cultured to address the question of viral infectivity when recovered from the environment

25. Shellfish sample processing for adenovirus (based on Woods (2006), Puig et al. (1994), and Heim et al. (2003)) Adjust conductivity to below 2,000 μS/cm qPCR and nested PCR for all strains of human adenovirus Resuspend pellet in glycine/NaCl, adjust pH to 7.5, centrifuge to pellet Resuspend pellet in threonine/NaCl, centrifuge to pellet, collect supernatant Homogenization of clam tissues (can freeze at this point) PEG 8000 precipitation, centrifuge to pellet, resuspend in 1X TE Adjust pH to 4.8, centrifuge to pellet DNA extraction using DNeasy tissue kit Virus release from the shellfish tissues DNA extraction Virus detection DNA sequencing to identify the strain(s) detected

26. Effluent sample processing for norovirus and adenovirus (based on Katayama et al. 2002) Filter through negatively charged membrane filter (0.45µm) Reverse transcription Elute the virus with NaOH Neutralize filtrate with H 2 SO 4 and 100X TE Effluent sample + 25mM MgCl 2 Concentrate using Vivaspin 6 ultrafilter Rinse out cations with H 2 SO 4 RNA extraction using MagMAX qPCR for the detection of GI and for GII Adsorption of the virus to a membrane Elution of the virus Virus concentration Virus detection DNA extraction using DNeasy tissue Kit qPCRNested PCR Automated DNA sequencing

27. Combined FDA/HRSD/VDH Dye Study of Menchville HRSD Waste Water Treatment Plant-James River Virginia

28. VIMS participation: (1) Provide a second, independent analysis of oyster (Crassostrea virginica) and effluent samples for indicator and virus presence for HRSD (2) Allow for comparison of results using different viral detection methods.

29. Parameters to be measured in oysters: Fecal coliforms Escherichia coli FRNA male-specific coliphage Norovirus (strains GI and GII) Adenovirus (human-specific primer set) Parameters to be measured in WWTP samples: Norovirus (strains GI and GII) Adenovirus (human-specific primer set)

30. Collection date Sample type # of qPCR replicate positives for GI # of qPCR replicate positives for GII # of qPCR replicates positive for adenovirus # of nested- PCR replicates positive for adenovirus 4/21/10Influent-High flow (#5)0/22/2 4/21/10Influent-Low flow (#6)0/22/2 4/21/10Postchlorination-High flow (#7)0/22/20/22/2 4/21/10Postchlorination-Low flow (#8)0/22/2 4/23/10Postchlorination-Low flow (#16)0/22/20/22/2 4/24/10Postchlorination-Low flow (#20)0/22/20/2 4/26/10Postchlorination-Low flow (#28)0/22/20/2 Samples positive for target virus 0/77/73/75/7 Norovirus and human adenovirus in HRSD James River WWTP samples taken during FDA dye release and exposure of sentinel shellfish

31. Norovirus and human adenovirus in sentinel oysters deployed in the James River during the FDA dye release: first exposure study (4/13/2010 - 4/27/2010) MPN /100g oyster PFU/100g oyster Norovirus (qPCR) Adenovirus StationFCECFRNAGIGII FDA-1<18 <398Not detected FDA-2<18 <45Not detected FDA-3<20 20<48Not detected FDA-4<18 <49Not detected Detected FDA-5<19 <55Not detected Detected HRSD -1<18 <44Not detected HRSD-2<18 <44Not detected Detected HRSD-2 (duplicate) <18 <44Not detected

32. Norovirus and human adenovirus in sentinel oysters deployed in the James River during the FDA dye release: second exposure study (4/27/2010 - 5/11/2010) MPN /100g shellfish PFU/100g shellfishNorovirus (qPCR) Adenovirus StationFCECFRNAGIGII FDA-123 315Not detected FDA-2<18 <44Not detected FDA-3<2020<44Not detected FDA-4<18 <45DetectedNot detected FDA-5<18 <46DetectedNot detected HRSD-1<18 <47DetectedNot detected HRSD-2<18 <45Not detected HRSD-2 (duplicate) <18 <46Not detected

33. FDA deployed oyster cages +

34. Outfall NoV FRNA AdV 1 2 3 4 5

35. Location of "control" sites in Burwell Bay

36. Proposed Future Work NOAA- Evaluation of conventional and an innovative immunomagnetic method for detecting and monitoring pathogenic human norovirus in bivalve shellfish "….. improving the sensitivity of norovirus detection….." Evaluate adenovirus as a viral indicator