Continuing Studies of Viruses in Hampton Roads and Shellfish

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

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SETTING THE STAGE:

PREVIOUS WORK REPORTED AT THE LAST ISS MEETING IN VIRGINIA BEACH



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



BackgroundBackground

• 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.



BackgroundBackground

• 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.



Figure 1. Location of clam exposure sites for Fall 2007 Figure 1. Location of clam exposure sites for Fall 2007 samples.samples.



NANSEMOND WWTP OUTFALL

CLAMS OUTSIDE WWTP BUFFER ZONE

CLAMS WITHIN WWTP BUFFER ZONE



Density 100 g-1 shellfish NoV Genogroup Site

Replicate Fecal coliforms E. coli FRNA phage

“Cond 1” A 20 20 17 0/2 B 90 0/2 A 20 20 330 0/2 B 226 0/2 A 18 18 106 0/2 B 92 1/2 GI A <18 20 18 2/2 GI and GII B <18 2/2 GI

Means

<19

20

<159

Nansemond

A

20

<18

140 2/2

B 198 0/2 A 45 45 334 0/2 B 398 1/2 A 100 18 488 1/2 B 388 0/2 A <18 <18 570 0/2 B 424 0/2

Means

<46

<25

368

*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, 12.1C Nansemond WWTP outfall - 2.0 fecal coliforms and E. coli per 100 ml; <1 FRNA phage per 100 ml; 22.6 psu, 11.8C.

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



Density 100g-1 shellfish NoV Genogroup Site

Replicate Fecal coliforms E. coli FRNA phage

“Cond 1” A <18 <18 <18 2/2

B 18 2/2 A 20 20 36 2/2 B 54 2/2 A <18 <18 36 1/2 B 54 2/2 A 20 20 268 2/2 B 214 2/2

Means

<19

<19

<87

Nansemond

A

7 8

78

54 2/2 GI

B 2/2 GII A 45 45 72 2/2 B 72 2/2 A 78 78 320 2/2 GI and GII B 410 2/2 A 230 230 178 2/2 B 90 2/2

Mean

108

108

159

*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



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



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

GIGIGI

GI

GIGII

Effluents- GI GII

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.

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



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

Date Shellfish Source

FRNA PFU/100g shellfish


G1 G2 G1 G2

(1) Vendor Baseline Oysters- Wicomico R. <44 2/2 0/2 174 1/2 0/2

3/24/09 Clam- James R. <44 0/2 0/2 44 2/2 1/2

Deployed within Nansemond WWTP outfall zone

Exposed <42 2/2 0/2 320 2/2 0/2

for 14d 2/2 0/2 2/2 0/2

(4/9/2009) <40 2/2 0/2 160 2/2 0/2

2/2 0/2 2/2 0/2

(2) Vendor Baseline Oysters- Ware R. <46 2/2 0/2 1246 2/2 0/2

4/9/09 Clams- York R. <46 2/2 0/2 498 1/2 0/2

Deployment conditions: PSU= 23, Temp= 9.3 C

Deployment conditions: PSU=18, Temp= 13.3 C

Conclusion: shellfish were apparently contaminated with norovorus at time of purchase; could not determine whether contamination occurred at WWTP outfall or was initially present. Oysters FRNA coliphage negative; clams FRNA positive

Crassostrea virginica Mercenaria mercenaria

Norovirus Norovirus

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

Date Shellfish Source FRNA FRNA G1 G2 G1 G2

(5) Vendor Baseline Oysters-Mobjack Bay <46 0/2 0/2 36 0/2 0/24/28/09 Clams-Mobjack Bay <46 0/2 0/2 36 0/2 0/2

512 0/2 1/2 110 0/2 0/2340 0/2 2/2 146 0/2 0/2

Oysters but not CLAMS show norovirus uptake; both show FRNA uptake

90 0/2 0/2 540 0/2 0/2180 0/2 0/2 250 0/2 0/2

Only FRNA coliphage detected in exposed shellfish

Exposed 4/28/09 at VIP HRSD WWTP (Elizabeth R) for 10 weeks (7/7/09) 1304 0/2 0/2 <40 0/2 0/2

1036 0/2 0/2 <42 0/2 0/2

Only FRNA coliphage detected in exposed oysters

Exposed 4/28/09 at VIP HRSD WWTP (Elizabeth R) for 7d (5/5/09)

Exposed 4/28/09 at VIP HRSD WWTP (Elizabeth R) for 22d (5/20/09)

Crassostrea virginica Mercenaria mercenariaNorovirus Norovirus

Cont'd

Date Shellfish Source



G1 G2 G1 G2(8) Vendor Baseline, 5/5/2009 Oysters-Mobjack <48 1/2 0/2 270 0/2 0/2

Clams-Mobjack <48 1/2 0/2 200 0/2 0/2

5760 0/2 0/2 <38 0/2 0/25086 0/2 0/2 <38 0/2 0/2

Deployment conditions: Warwick River, 5/5/09 - PSU= 12. Temp=19.9 C, 5/20/09- PSU= 10, Temp= 17.9 C

FRNA coliphage detected in exposed oysters at sufficient density, norovirus not detected in clams or oysters


Norovirus Norovirus

Exposed 5/5/09 in

Warwick R (James) for 15 d (5/20/09) within WWTP buffer zone

Norovirus FRNA coliphage detected at time of purchase

Cont'd

Date Shellfish SourceFRNA

PFU/100g FRNA

PFU/100g G1 G2 G1 G2

Oysters-Mobjack <46 0/2 0/2 <16 0/2 0/2Clams-Mobjack <46 0/2 0/2 34 0/2 0/2

<46 0/2 0/2 <18 0/2 0/2<46 0/2 0/2 <18 0/2 0/2

Norovirus not detected at time of purchase

Oysters-York <46 0/2 0/2 <16 0/2 0/2Clams-York <46 0/2 0/2 <16 0/2 0/2

<46 0/2 0/2 <16 0/2 0/2<46 0/2 0/2 <16 0/2 0/2

Norovirus not detected at time of purchase

Transfered 10/22/09 to Warwick R (James) WWTP buffer zone

Exposed in Warwick for 11 d, (11/02/09)

130 0/2 0/2 16 0/2 0/288 0/2 0/2 <16 0/2 0/2

316 0/2 0/2 16 0/2 0/2496 0/2 0/2 34 0/2 0/2

(9) Vendor baseline, 10/13/2009

(10) Vendor baseline, 10/22/09

Deployment conditions: Warwick River, 10/22/09, 20 PSU, 15.9 C

FRNA coliphage detected only in oysters at sufficient densities, norovirus not detected in clams/oysters

Norovirus


Norovirus

Cont'd

Date NP VIP James River Williamsburg

4/3/08 .5/1* 0/14/7/08 1/1

4/15/08 1/1 .5/14/21/08 1/14/28/08 0/1 0/.55/5/08 .5/1

5/13/08 0/1

11/5/09 0/011/10/09 0/.511/18/09 0/0

1/19/10 .5/.5 0/.5 0/.52/16/10 0/.5 0/1 0/13/23/10 0/0 0/0 0/0

HRSD Treatment Plant

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

Relay in York River following in situ contamination of Crassostrea virginica and Mercenaria mercenaria adjacent to WWTP outflall in James River, Virginia (4-19-10)

Crassostrea vir ginica Mercenaria mercenaria

Date FC EC FRNA

coliphage Norovirus FC EC FRNA

coliphage Norovirus

MPN /100g PFU

/100g* G1 G2 MPN /100g PFU

/100g* G1 G2

Vendor 3/31/10- virus baseline <46

not detected

not detected 279

not detected

not detected

0 day relay to York R.

(4-19-10, following exposure in James R.) <333 <317 7728

not detected

not detected <19 <19 1033

not detected

not detected

(James- 12 PSU, 16.5 C; York River, 17 PSU, 15 C)

3d

(4/22/10) <32 <25 612 not

detected not

detected <68 <37 805 not

detected not

detected (18 PSU, 14.6 C)

7d

(4/26/10) <18 <18 <47 <19 <19 72 (18 PSU, 17.1 C)

15d

(5/3/10) <19 <19 <45 <25 <19 <63 (18 PSU, 19.1 C)

*FRNA coliphage detection limits: oysters- <44 pfu 100-1 g; clams- <17 pfu

Relay in York River following in situ contamination of Crassostrea virginica and Mercenaria mercenaria adjacent to WWTP outflall in James River, Virginia (5-17-10)


Date FC EC FRNA

coliphage Norovirus FC EC FRNA

coliphage Norovirus

MPN /100g PFU/ 100g* G1 G2 MPN /100g

PFU/ 100g* G1 G2

Vendor 4/13/10- virus

baseline <46 not

detected not

detected <17 not

detected not

detected

0 day relay to York R. (5-17-10) (following 2 week exposure in James R.) <20 <19 1301

not detected detected <24 <23 615

not detected detected

(James- 17 PSU, 15.8 C; York River, 18 PSU, 19.7 C)

3d

(5/20/10) <25 <25 <168 not

detected not

detected <19 <19 331 not

detected not

detected (18 PSU, 19.6 C)

7d

(5/24/10) <29 <25 <45 <19 <19 <36 (20 PSU, 21 C)

15d

(6/1/10) <40 <40 <44 <25 <25 <17 (20 PSU, 23.9 C)

*FRNA coliphage detection limits: oysters- <44 pfu 100-1 g; clams- <17 pfu

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



VIRUS DETECTION METHODS

Shellfish sample processing for norovirusShellfish sample processing for norovirus(based on Jothikumar et al. 2005; Gentry et al. 2009)(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 shaking65°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

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

Shellfish sample processing for adenovirusShellfish sample processing for adenovirus(based on Woods (2006), Puig et al. (1994), and Heim et al. (2003))(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 detectionDNA sequencing to identify the strain(s) detected

Effluent sample processing for norovirus and adenovirusEffluent sample processing for norovirus and adenovirus(based on Katayama et al. 2002)(based on Katayama et al. 2002)

Filter through negatively charged membrane filter (0.45µm)

Reverse transcription

Elute the virus with NaOH

Neutralize filtrate with H2SO4 and 100X TE

Effluent sample + 25mM MgCl2

Concentrate using Vivaspin 6 ultrafilter

Rinse out cations with H2SO4

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

qPCR Nested PCR

Automated DNA sequencing

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

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.

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)

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

4/21/10 Influent-High flow (#5) 0/2 2/2 2/2 2/2

4/21/10 Influent-Low flow (#6) 0/2 2/2 2/2 2/2

4/21/10 Postchlorination-High flow (#7) 0/2 2/2 0/2 2/2

4/21/10 Postchlorination-Low flow (#8) 0/2 2/2 2/2 2/2




Samples positive for target virus

0/7 7/7 3/7 5/7

Norovirus and human adenovirus in HRSD James River WWTP samples taken during FDA dye release and exposure of sentinel shellfish

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 oysterPFU/100g

oysterNorovirus

(qPCR)Norovirus

(qPCR) Adenovirus

Station FC EC FRNA GI GII

FDA-1 <18 <18 <398 Not detected Not detected Not detected


FDA-3 <20 20 <48 Not detected Not detected Not detected

FDA-4 <18 <18 <49 Not detected Not detected Detected

FDA-5 <19 <19 <55 Not detected Not detected Detected

HRSD -1 <18 <18 <44 Not detected Not detected Not detected

HRSD-2 <18 <18 <44 Not detected Not detected Detected

HRSD-2 (duplicate)

<18 <18 <44 Not detected Not detected Not detected

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 shellfish Norovirus (qPCR)

Norovirus (qPCR)

Adenovirus

Station FC EC FRNA GI GII

FDA-1 23 23 315 Not detected Not detected Not detected


FDA-3 <20 20 <44 Not detected Not detected Not detected

FDA-4 <18 <18 <45 Detected Not detected Not detected

FDA-5 <18 <18 <46 Detected Not detected Not detected

HRSD-1 <18 <18 <47 Detected Not detected Not detected

HRSD-2 <18 <18 <45 Not detected Not detected Not detected

HRSD-2 (duplicate)

<18 <18 <46 Not detected Not detected Not detected





FDA deployed oyster cages +



Outfall

NoV

NoV

NoV FRNA

AdV

AdV

AdV

1

2

3

4

5





Location of "control" sites in Burwell Bay

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

Continuing Studies of Viruses in Hampton Roads and Shellfish

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