- Email: [email protected]
Monitoring the marine environment for Small Round Structured Viruses (SRSVS): A new approach to combating the transmission of these viruses by molluscan shellfish
e>
Pergamon
PIT: S0273-1223(98)00799-9
Wat Sci. Tull. Vol. 38. No. 12. pp. SI-S6. 1998. @1998IAWQ Published by ElsevierScienceLId Printed in Greal Britain. All rights reserved 0273-1223/98 S19'00 + 0'00
MONITORING THE MARINE ENVIRONMENT FOR SMALL ROUND STRUCTURED VIRUSES (SRSVS): A NEW APPROACH TO COMBATING THE TRANSMISSION OF THESE VIRUSES BY MOLLUSCAN SHELLFISH K. Henshilwood*, 1. Green** and D. N. Lees* * Centre for Environment. Fisheries and Aquaculture Science. The Nothe, Weymouth DT4 8UB, UK .. Virus Reference Division. Central Public Health Laboratory, 61 Collndale Avenue. London NW95HT. UK ABSTRACf This study investigates human enteric virus contamination of a shellfish harvesting area. Samples were analysed over a J4-month period for Small Round Structured Viruses (SRSVs) using a previously developed nested RT-PCR. A clear seasonal difference was observed with the largest numbers of positive samples obtained during the winter period (October to March). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period. RT-PCR positive amplicons were further characterised by cloning and sequencing. Sequence analysis of the positive samples identified eleven SRSV strains. of both Genogroup I and Genogroup U. occurring throughout the study period. Many shellfish samples contained a mixture of strains with a few samples containing up to three different strains with both Genogroups represented. The observed common occurrence of strain mixtures may have implications for the role of shellfish as a vector for dissemination of SRSV strains. These results show that nested RT-PCR can identify SRSV contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible approach to combating the transmission of SRSVs by molluscan shellfish and could potentially offer significantly enhanced levels of public health protection. Q:) 1998 IAWQ Published by Elsevier Science Ltd. All rights reserved
KEYWORDS Oysters; SRSVs; gastroenteritis; PCR. INTRODUCfION Increasing evidence suggests that SRSVs are a significant cause of gastroenteritis in the adult population (Caul. 1996). Although person-to-person transmission probably accounts for the bulk of cases, spread by the faecal-oral route through food or water vectors is likely to be important in the further dissemination of these viruses into vulnerable populations. Preventing such index cases may prove significant in combating this cause of adult gastroenteritis. Transmission of SRSVs through consumption of molluscan shellfish faecally contaminated in the marine environment is well documented. In developed countries many marine water sources used for shellfisheries and recreational purposes are faecally polluted to some extent. Legislative controls for such areas. based on conventional bacterial indicators. have failed to prevent illness outbreaks 51
52
K. HENSHILWOOD et al.
associated with molluscan shellfish (Kohn et al., 1995). Monitoring shellfish harvesting areas for SRSV contamination may provide an alternative approach for more effective public health controls . We describe the application of a previously developed nested SRSV RT-PCR (Lees et al., 1995; Green et al.• 1998) for monitoring viral contamination in an oyster harvesting area over a one year period. The harvesting area was affected by several sewage discharges, was classified as Category B under EC legislation and had previously been implicated in outbreaks of viral gastroenteritis. All oysters harvested from this area were purified (depurated) before sale for consumption. Following amplification SRSV RT-PCR positive amplicons were characterised by cloning and sequence analysis to gain information about the prevalence and diversity of SRSV strains contaminating this shellfishery. The public health significance of these findings is discussed in relation to the potential of this approach for more effective control of shellfish associated viralgastroenteritis. MATERIALS AND METHODS
Harvesting area studies - samples of 24 oysters were obtained from a commercial producer both before and after depuration on a 1-2 week basis. All samples were received in the laboratory within 48h. On receipt, 10 oysters were assayed for E coli using standard methods and the remaining oysters frozen at -20°C for subsequent analysis for SRSVs.
Shellfish processing, virus extraction and purification, and extraction of viral RNA • these procedures have previously been described in full (Lees et al.; 1994). Nested SRSV RT-PCR - this method and its application to shellfish samples has been fully described previously (Lees et al., 1995; Green et al., 1998). Essentially, the nested RT-PCR uses three primers in the first round amplification and two Genogroup specific primer sets in the nested PCR. They are known to detect approximately 90% of the SRSVs currently circulating in the UK. Cloning and sequencing • all RT-PCR positive amplicons were separated from unincorporated oligonucleotide primers and nucleotides using Chromaspin 100 columns (Clontech Laboratories Inc) ligated into a pGem vector and transformed (pGem-T Vector System, Promega). Colonies from each sample were screened for inserts using colony PCR. A minimum of five positive clones from each sample were further purified using microsep 30k columns (Filtron Technology Corporation, MA) and both DNA strands sequenced using the ABI PRISMTMdye terminator cycle sequencing system (Perkin Elmer) and analysed on an ABI 310 genetic analyser. Sequence analysis - sequence data analysis was performed using the MegaAlign and EditSeq components of the Lasergene software (DNAStar, London UK). RESULTS Table 1 tabulates E coli and SRSV results for all samples tested in this survey. Whilst high levels of E coli were present in the majority of oysters sampled before depuration all samples were subsequently found to contain E coli levels below the end-product standard of <2301loog following depuration. By contrast SRSVs were found to be present in oysters both before (56% positive) and after (38% positive) depuration . This data is consistent with previous observations from viral gastroenteritis outbreaks showing the inadequacy of E coli as an indicator of the presence of viral contamination in depurated shellfish. Figure I shows SRSV RT-PCR results for samples taken throughout the study period presented chronologically together with the known outbreaks of gastroenteric food poisoning associated with oysters from this harvesting area during the study period. A clear seasonal difference can be observed in numbers of SRSV positive oysters with the largest numbers of positive samples (73%) obtained during the winter period (October-March inclusive). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period (Figure I) . It is also noticeable that, for the most part,
Humanenteric virus contamination of a shellfishharvestingarea
53
SRSVs would have been detectable in the harvesting area prior to an outbreak. suggesting that timely monitoring could provide an additional approach to public health controls .
xx
x XXX
~X
XXXX
x
x
XX X X X
Figure I. SRSV RT·PCR results throughoutstudy periodand occurrenceof illness outbreaks associatedwith oysters fromstudy area.
Table I . Summary of E coli and SRSV results during study period Date /995 08 Aug 15 Aug 22 Aug 30 Aug 13 Sep 26Sep 10 Oct 24 Oct 07 Nov 21 Nov /996 09 Jan 16 Jan 23 Jan 31 Jan
Before depuration E coli RT-PCR 2,400 >18,000 >18,000 2,400 3,100 220 2,400 5,400 5,400 1,100
After depuration RT-PCR
Confirmed-
Date
E coli
+ +
Ongoing Ongoing
+
Yes
08 Aug 17 Aug 24 Aug 01 Sep IS Sep 28Sep 13 Oct 26 Oct 09 Nov 28 Nov
<20 <20 <20 <20 <20 <20 <20 <20 <20 <20
12 Jan l81an 25 Jan 02 Feb 08 Feb 16 Feb 22 Feb 01 Mar 08 Mar 14 Mar 22 Mar 29 Mar 18 Apr 02 May 07 Jun 20Jun 19 Jul 15 Aug OS Sep 19 Sep 04 Oct 18 Oct
<20 70 20 <20 <20 <20 <20 <20 ISO 90 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20
1,100 + + 110 + 200 + 10 + 06 Feb 500 14 Feb 700 + 20 Feb 500 + 28 Feb 110 + 06 Mar 5,400 + 12 Mar 750 + 19Mar 310 + <20 27 Mar + 16 Apr 1,300 30 Apr 1,300 05 Jun <20 18 Jun 230 16 Jul <20 13 Aug 22,000 03 Sep 310 + 17 Sep 220 OIOct <20 + 16 Oct 70 + ·confirmed by sequence analysis
Yes Ongoing Yes To be done Yes Yes Yes Yes Yes Yes Yes Yes
To be done To be done To be done
Confirmed-
+ + + + +
To be done Yes Yes Yes Yes
+ + +
Yes Yes Ongoing
+ +
Yes Yes
+ +
To be done To be done
K. HENSHILWOOD et al.
S4
Of the SRSV positive samples during the winter period, virus was successfully eliminated from only two samples during depuration with 63% of samples remaining positive. This concurs with evidence from many outbreaks showing that although bacterial indicators can be removed by depuration human enteric viruses are more problematical. By contrast to the winter period the prevalence of SRSVs in oysters was considerably lower in the summer period (April-September inclusive) with only 31% of samples containing SRSVs. Interestingly depuration also appeared to be more efficient during the summer with SRSVs being successfully eliminated from all four SRSV positive samples following depuration (Table I). This is a novel finding which has potential significance for further studies on virus elimination during depuration. PCR positive amplicons generated during this study were cloned and sequenced to investigate further the type and diversity of strains contaminating oysters. Of the samples completed to date those confirmed as SRSV sequence (the large majority) are shown in Table l. Some samples are still under analysis. Difficulties are mostly associated with obtaining sufficient template DNA for sequencing when virus levels in shellfish are low. Of the 23 SRSV RT-PCR positive samples analysed between September 1995 and April 1996 about 80%t.have currently been confirmed by sequence analysis as SRSV. Sequence from these confirmed positives has been further analysed to investigate the type and diversity of SRSV strains. Sequence comparison using MegaAlign pileup of the 32 sequenced isolates showed isolates could be subdivided into groups sharing genetic relationship. Eleven unique strains were identified with homology at the nucleotide level of 90% or more. These strain relationships are illustrated in Figure 2.
Slrain t Slrain 2 strain 3 S1rIin 4 SII1in 5 SII1in s 08SlIt Shield (U0446lI) SII1in 7
SIt
s..IDga.SEQ SlrIin a
snna
NorwIIl (U87661) ~(lD74'8)
SII1in 10 Lnd* (XIl6557) Snow IbI1laiI (l23831) strain 11 Mexico (U22498)
47.4 4S
40
35
30
25
20
15
10
5
51
0
Figure 2. Phylogenetic tree showingrelationships betweenSRSVisolatesand publishedsequences. [The phylogenetic tree and divergence/similarity plot was generated using the ClustalV algorithm withinMegAlign (DNA Star Inc). The tree is basedon the alignment of approximately 80 nucleotides withinthe RNApolymerase excludingPeR primers.The dendrogram shows the geneticrelationships of II SRSVrepresentative strains. Branch lengthsare indicatedby the unbroken lines.The dotted linesare usedto providea balanceddisplayof the phyJogram and do not representactualbranchlengths.I
Alignment comparison with published sequences enabled determination of strain Genogroup (GI or GIl) and, in some cases, provided an identification. Some strains could not be identified to the 90% or greater level of identity suggesting that other less common strains were also in circulation. Some strains were isolated more frequently than others. Genogroup II stain number 10 in particular. was isolated from many of the positive samples. It was also noticeable that many samples contained more than one SRSV strain often
Human enteric virus contamination of a shellfish harvesting area
.5.5
with a mix of Genogroups (data not shown). Three samples contained both a Genogroup IT strain and two different Genogroup I strains. Of the samples analysed Genogroup I strains were more prevalent than Genogroup II strains although 42% of samples contained a mixture of both Genogroups. It was also noticeable that depuration affected the distribution of strains with only 17% of samples post-depuration containing both Genogroups. These results suggest this harvesting area was contaminated with a variety of SRSV strains during the study period with, somet imes , many different strains occurring during a fairly short time period. Some strains were isolated periodically throughout the study period whereas others represented a single isolation. DISCUSSION This study investigates, for the first time, the feasibility of applying molecular techniques to the monitoring of SRSV contamination in a shellfish harvested area . We show that in the chosen study area many samples were positive for SRSVs, particularly during the winter months, and furthermore that many shellfish samples contained a mixture of SRSV strains. A few samples contained up to three different strains representing both Genogroups. Although mixtures of SRSV strain genogroups in shellfi sh associated outbreaks has been previously reported (Sugieda et al., (996), it is perhaps suprising to find this such a common occurrence in this study. These results may have implications for the importance of shellfish as a vector for the dissemination of SRSV strains into the community. It is interesting to note that, following depuration. fewer samples contained a mixture of strains suggesting at least partial removal of SRSVs during depuration. Our data also suggest that SRSV removal during depuration may be more effective during the summer months. This finding has significance for future studies on virus removal during depuration. Characterisation of contaminating SRSV strains by sequencing showed that virus strains differ in their persistence in the harvesting area. Some strains were isolated on only one occasion whereas others were isolated on many occasions throughout the study period. Strain 10 in particular was repeatedly isolated over at least a 4-month period. It is not clear from this study whether such strains persist in shellfish following an initial contamination event or whether they are repeatedly seeded into the harvesting area from sewage discharges. It would be interesting to correlate SRSV episodes in the community with contamination of harvesting areas to investigate this further. Our results showed that SRSV contamination in this harvesting area followed a clear seasonal trend. Th is correlates both with known outbreaks from the study harvesting area and with the known winter seasonality of illness from shellfish associated outbreaks in the UK. In conclusion these results show that nested RT-PCR can identify virus contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible new approach to combating the transmission of SRSVs by molluscan shellfish and this approach could potentially provide significantly enhanced levels of public health protection. ACKNOWLEDGEMENTS This work was funded by MAFF Food Hygiene Division. UK. REFERENCES Caul, E. O. (1996). Viral Gastroenteritis - small round structured viruses, caliciviruses and astroviruses • the clinical and diagnostic perspective . J. CUn. Path.• 49, 874. Green, J., Henshilwood, K.• Gallimore. C. I.• Brown, D. W. G. and Lees. D. N. (1998). A nested reverse transcriptase PCR assay for detection of small round-structured viruses in environmentally contaminated molluscan shellfish. AppL Environ. Microb iol.• 64, 8.58-863. Lees, D. N., Henshilwood, K .and Dore, W. J. (t994). Development of a method for detection of enterov iruses in shellfish by PeR with poliovirus as a model. ApjJl. Environ.Microbiol.• 60,2999·300.5. Lees. D. N.• Henshilwood, K, Green. J.• Gallimore, C.I. and Brown, D. W. G. (199.5). Detection ofsmall round structured viruses in shellfish by reverse transcription PCR. AppL Environ. Microbial; 61,4418-4424.
56
K. HENSHIL WOOD et al.
Kohn, M. A., Farley. T. A., Ando, T., Curtis, M.• Wilson. S. A., Jin, Q., Monroe, S. S., Baron, R. C., Mcfarland, L. M. and Glass, R. I. (1995). An outbreak of Norwalk virus gastroenteritis associated with eating raw oysters: implications for maintaining safe oyster beds. J. Am. Med. Ass.•273. 466-471. Sugieda, M., Nakajima, K. and Nakajima, S. (1996). Outbreaks of Norwalk-like virus-associated gastroenteritis traced to shellfish - coexistence of two genotypes in one specimen. Epidemiol. Infect., 116. 339-346.
Pergamon
PIT: S0273-1223(98)00799-9
Wat Sci. Tull. Vol. 38. No. 12. pp. SI-S6. 1998. @1998IAWQ Published by ElsevierScienceLId Printed in Greal Britain. All rights reserved 0273-1223/98 S19'00 + 0'00
MONITORING THE MARINE ENVIRONMENT FOR SMALL ROUND STRUCTURED VIRUSES (SRSVS): A NEW APPROACH TO COMBATING THE TRANSMISSION OF THESE VIRUSES BY MOLLUSCAN SHELLFISH K. Henshilwood*, 1. Green** and D. N. Lees* * Centre for Environment. Fisheries and Aquaculture Science. The Nothe, Weymouth DT4 8UB, UK .. Virus Reference Division. Central Public Health Laboratory, 61 Collndale Avenue. London NW95HT. UK ABSTRACf This study investigates human enteric virus contamination of a shellfish harvesting area. Samples were analysed over a J4-month period for Small Round Structured Viruses (SRSVs) using a previously developed nested RT-PCR. A clear seasonal difference was observed with the largest numbers of positive samples obtained during the winter period (October to March). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period. RT-PCR positive amplicons were further characterised by cloning and sequencing. Sequence analysis of the positive samples identified eleven SRSV strains. of both Genogroup I and Genogroup U. occurring throughout the study period. Many shellfish samples contained a mixture of strains with a few samples containing up to three different strains with both Genogroups represented. The observed common occurrence of strain mixtures may have implications for the role of shellfish as a vector for dissemination of SRSV strains. These results show that nested RT-PCR can identify SRSV contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible approach to combating the transmission of SRSVs by molluscan shellfish and could potentially offer significantly enhanced levels of public health protection. Q:) 1998 IAWQ Published by Elsevier Science Ltd. All rights reserved
KEYWORDS Oysters; SRSVs; gastroenteritis; PCR. INTRODUCfION Increasing evidence suggests that SRSVs are a significant cause of gastroenteritis in the adult population (Caul. 1996). Although person-to-person transmission probably accounts for the bulk of cases, spread by the faecal-oral route through food or water vectors is likely to be important in the further dissemination of these viruses into vulnerable populations. Preventing such index cases may prove significant in combating this cause of adult gastroenteritis. Transmission of SRSVs through consumption of molluscan shellfish faecally contaminated in the marine environment is well documented. In developed countries many marine water sources used for shellfisheries and recreational purposes are faecally polluted to some extent. Legislative controls for such areas. based on conventional bacterial indicators. have failed to prevent illness outbreaks 51
52
K. HENSHILWOOD et al.
associated with molluscan shellfish (Kohn et al., 1995). Monitoring shellfish harvesting areas for SRSV contamination may provide an alternative approach for more effective public health controls . We describe the application of a previously developed nested SRSV RT-PCR (Lees et al., 1995; Green et al.• 1998) for monitoring viral contamination in an oyster harvesting area over a one year period. The harvesting area was affected by several sewage discharges, was classified as Category B under EC legislation and had previously been implicated in outbreaks of viral gastroenteritis. All oysters harvested from this area were purified (depurated) before sale for consumption. Following amplification SRSV RT-PCR positive amplicons were characterised by cloning and sequence analysis to gain information about the prevalence and diversity of SRSV strains contaminating this shellfishery. The public health significance of these findings is discussed in relation to the potential of this approach for more effective control of shellfish associated viralgastroenteritis. MATERIALS AND METHODS
Harvesting area studies - samples of 24 oysters were obtained from a commercial producer both before and after depuration on a 1-2 week basis. All samples were received in the laboratory within 48h. On receipt, 10 oysters were assayed for E coli using standard methods and the remaining oysters frozen at -20°C for subsequent analysis for SRSVs.
Shellfish processing, virus extraction and purification, and extraction of viral RNA • these procedures have previously been described in full (Lees et al.; 1994). Nested SRSV RT-PCR - this method and its application to shellfish samples has been fully described previously (Lees et al., 1995; Green et al., 1998). Essentially, the nested RT-PCR uses three primers in the first round amplification and two Genogroup specific primer sets in the nested PCR. They are known to detect approximately 90% of the SRSVs currently circulating in the UK. Cloning and sequencing • all RT-PCR positive amplicons were separated from unincorporated oligonucleotide primers and nucleotides using Chromaspin 100 columns (Clontech Laboratories Inc) ligated into a pGem vector and transformed (pGem-T Vector System, Promega). Colonies from each sample were screened for inserts using colony PCR. A minimum of five positive clones from each sample were further purified using microsep 30k columns (Filtron Technology Corporation, MA) and both DNA strands sequenced using the ABI PRISMTMdye terminator cycle sequencing system (Perkin Elmer) and analysed on an ABI 310 genetic analyser. Sequence analysis - sequence data analysis was performed using the MegaAlign and EditSeq components of the Lasergene software (DNAStar, London UK). RESULTS Table 1 tabulates E coli and SRSV results for all samples tested in this survey. Whilst high levels of E coli were present in the majority of oysters sampled before depuration all samples were subsequently found to contain E coli levels below the end-product standard of <2301loog following depuration. By contrast SRSVs were found to be present in oysters both before (56% positive) and after (38% positive) depuration . This data is consistent with previous observations from viral gastroenteritis outbreaks showing the inadequacy of E coli as an indicator of the presence of viral contamination in depurated shellfish. Figure I shows SRSV RT-PCR results for samples taken throughout the study period presented chronologically together with the known outbreaks of gastroenteric food poisoning associated with oysters from this harvesting area during the study period. A clear seasonal difference can be observed in numbers of SRSV positive oysters with the largest numbers of positive samples (73%) obtained during the winter period (October-March inclusive). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period (Figure I) . It is also noticeable that, for the most part,
Humanenteric virus contamination of a shellfishharvestingarea
53
SRSVs would have been detectable in the harvesting area prior to an outbreak. suggesting that timely monitoring could provide an additional approach to public health controls .
xx
x XXX
~X
XXXX
x
x
XX X X X
Figure I. SRSV RT·PCR results throughoutstudy periodand occurrenceof illness outbreaks associatedwith oysters fromstudy area.
Table I . Summary of E coli and SRSV results during study period Date /995 08 Aug 15 Aug 22 Aug 30 Aug 13 Sep 26Sep 10 Oct 24 Oct 07 Nov 21 Nov /996 09 Jan 16 Jan 23 Jan 31 Jan
Before depuration E coli RT-PCR 2,400 >18,000 >18,000 2,400 3,100 220 2,400 5,400 5,400 1,100
After depuration RT-PCR
Confirmed-
Date
E coli
+ +
Ongoing Ongoing
+
Yes
08 Aug 17 Aug 24 Aug 01 Sep IS Sep 28Sep 13 Oct 26 Oct 09 Nov 28 Nov
<20 <20 <20 <20 <20 <20 <20 <20 <20 <20
12 Jan l81an 25 Jan 02 Feb 08 Feb 16 Feb 22 Feb 01 Mar 08 Mar 14 Mar 22 Mar 29 Mar 18 Apr 02 May 07 Jun 20Jun 19 Jul 15 Aug OS Sep 19 Sep 04 Oct 18 Oct
<20 70 20 <20 <20 <20 <20 <20 ISO 90 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20
1,100 + + 110 + 200 + 10 + 06 Feb 500 14 Feb 700 + 20 Feb 500 + 28 Feb 110 + 06 Mar 5,400 + 12 Mar 750 + 19Mar 310 + <20 27 Mar + 16 Apr 1,300 30 Apr 1,300 05 Jun <20 18 Jun 230 16 Jul <20 13 Aug 22,000 03 Sep 310 + 17 Sep 220 OIOct <20 + 16 Oct 70 + ·confirmed by sequence analysis
Yes Ongoing Yes To be done Yes Yes Yes Yes Yes Yes Yes Yes
To be done To be done To be done
Confirmed-
+ + + + +
To be done Yes Yes Yes Yes
+ + +
Yes Yes Ongoing
+ +
Yes Yes
+ +
To be done To be done
K. HENSHILWOOD et al.
S4
Of the SRSV positive samples during the winter period, virus was successfully eliminated from only two samples during depuration with 63% of samples remaining positive. This concurs with evidence from many outbreaks showing that although bacterial indicators can be removed by depuration human enteric viruses are more problematical. By contrast to the winter period the prevalence of SRSVs in oysters was considerably lower in the summer period (April-September inclusive) with only 31% of samples containing SRSVs. Interestingly depuration also appeared to be more efficient during the summer with SRSVs being successfully eliminated from all four SRSV positive samples following depuration (Table I). This is a novel finding which has potential significance for further studies on virus elimination during depuration. PCR positive amplicons generated during this study were cloned and sequenced to investigate further the type and diversity of strains contaminating oysters. Of the samples completed to date those confirmed as SRSV sequence (the large majority) are shown in Table l. Some samples are still under analysis. Difficulties are mostly associated with obtaining sufficient template DNA for sequencing when virus levels in shellfish are low. Of the 23 SRSV RT-PCR positive samples analysed between September 1995 and April 1996 about 80%t.have currently been confirmed by sequence analysis as SRSV. Sequence from these confirmed positives has been further analysed to investigate the type and diversity of SRSV strains. Sequence comparison using MegaAlign pileup of the 32 sequenced isolates showed isolates could be subdivided into groups sharing genetic relationship. Eleven unique strains were identified with homology at the nucleotide level of 90% or more. These strain relationships are illustrated in Figure 2.
Slrain t Slrain 2 strain 3 S1rIin 4 SII1in 5 SII1in s 08SlIt Shield (U0446lI) SII1in 7
SIt
s..IDga.SEQ SlrIin a
snna
NorwIIl (U87661) ~(lD74'8)
SII1in 10 Lnd* (XIl6557) Snow IbI1laiI (l23831) strain 11 Mexico (U22498)
47.4 4S
40
35
30
25
20
15
10
5
51
0
Figure 2. Phylogenetic tree showingrelationships betweenSRSVisolatesand publishedsequences. [The phylogenetic tree and divergence/similarity plot was generated using the ClustalV algorithm withinMegAlign (DNA Star Inc). The tree is basedon the alignment of approximately 80 nucleotides withinthe RNApolymerase excludingPeR primers.The dendrogram shows the geneticrelationships of II SRSVrepresentative strains. Branch lengthsare indicatedby the unbroken lines.The dotted linesare usedto providea balanceddisplayof the phyJogram and do not representactualbranchlengths.I
Alignment comparison with published sequences enabled determination of strain Genogroup (GI or GIl) and, in some cases, provided an identification. Some strains could not be identified to the 90% or greater level of identity suggesting that other less common strains were also in circulation. Some strains were isolated more frequently than others. Genogroup II stain number 10 in particular. was isolated from many of the positive samples. It was also noticeable that many samples contained more than one SRSV strain often
Human enteric virus contamination of a shellfish harvesting area
.5.5
with a mix of Genogroups (data not shown). Three samples contained both a Genogroup IT strain and two different Genogroup I strains. Of the samples analysed Genogroup I strains were more prevalent than Genogroup II strains although 42% of samples contained a mixture of both Genogroups. It was also noticeable that depuration affected the distribution of strains with only 17% of samples post-depuration containing both Genogroups. These results suggest this harvesting area was contaminated with a variety of SRSV strains during the study period with, somet imes , many different strains occurring during a fairly short time period. Some strains were isolated periodically throughout the study period whereas others represented a single isolation. DISCUSSION This study investigates, for the first time, the feasibility of applying molecular techniques to the monitoring of SRSV contamination in a shellfish harvested area . We show that in the chosen study area many samples were positive for SRSVs, particularly during the winter months, and furthermore that many shellfish samples contained a mixture of SRSV strains. A few samples contained up to three different strains representing both Genogroups. Although mixtures of SRSV strain genogroups in shellfi sh associated outbreaks has been previously reported (Sugieda et al., (996), it is perhaps suprising to find this such a common occurrence in this study. These results may have implications for the importance of shellfish as a vector for the dissemination of SRSV strains into the community. It is interesting to note that, following depuration. fewer samples contained a mixture of strains suggesting at least partial removal of SRSVs during depuration. Our data also suggest that SRSV removal during depuration may be more effective during the summer months. This finding has significance for future studies on virus removal during depuration. Characterisation of contaminating SRSV strains by sequencing showed that virus strains differ in their persistence in the harvesting area. Some strains were isolated on only one occasion whereas others were isolated on many occasions throughout the study period. Strain 10 in particular was repeatedly isolated over at least a 4-month period. It is not clear from this study whether such strains persist in shellfish following an initial contamination event or whether they are repeatedly seeded into the harvesting area from sewage discharges. It would be interesting to correlate SRSV episodes in the community with contamination of harvesting areas to investigate this further. Our results showed that SRSV contamination in this harvesting area followed a clear seasonal trend. Th is correlates both with known outbreaks from the study harvesting area and with the known winter seasonality of illness from shellfish associated outbreaks in the UK. In conclusion these results show that nested RT-PCR can identify virus contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible new approach to combating the transmission of SRSVs by molluscan shellfish and this approach could potentially provide significantly enhanced levels of public health protection. ACKNOWLEDGEMENTS This work was funded by MAFF Food Hygiene Division. UK. REFERENCES Caul, E. O. (1996). Viral Gastroenteritis - small round structured viruses, caliciviruses and astroviruses • the clinical and diagnostic perspective . J. CUn. Path.• 49, 874. Green, J., Henshilwood, K.• Gallimore. C. I.• Brown, D. W. G. and Lees. D. N. (1998). A nested reverse transcriptase PCR assay for detection of small round-structured viruses in environmentally contaminated molluscan shellfish. AppL Environ. Microb iol.• 64, 8.58-863. Lees, D. N., Henshilwood, K .and Dore, W. J. (t994). Development of a method for detection of enterov iruses in shellfish by PeR with poliovirus as a model. ApjJl. Environ.Microbiol.• 60,2999·300.5. Lees. D. N.• Henshilwood, K, Green. J.• Gallimore, C.I. and Brown, D. W. G. (199.5). Detection ofsmall round structured viruses in shellfish by reverse transcription PCR. AppL Environ. Microbial; 61,4418-4424.
56
K. HENSHIL WOOD et al.
Kohn, M. A., Farley. T. A., Ando, T., Curtis, M.• Wilson. S. A., Jin, Q., Monroe, S. S., Baron, R. C., Mcfarland, L. M. and Glass, R. I. (1995). An outbreak of Norwalk virus gastroenteritis associated with eating raw oysters: implications for maintaining safe oyster beds. J. Am. Med. Ass.•273. 466-471. Sugieda, M., Nakajima, K. and Nakajima, S. (1996). Outbreaks of Norwalk-like virus-associated gastroenteritis traced to shellfish - coexistence of two genotypes in one specimen. Epidemiol. Infect., 116. 339-346.