Ostreid herpes virus (OsHV-1) research at Cawthron Detection of the virus, and its characterization by PCR and DNA sequencing

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1 Ostreid herpes virus (OsHV-1) research at Cawthron Detection of the virus, and its characterization by PCR and DNA sequencing A Joint Cawthron/IFREMER Project 11 November 2011

2 Background The ostreid herpes virus poses no human health threat. Herpes viruses are associated with mortalities worldwide in bivalves, including the Pacific oyster (Crassostrea gigas). A herpes virus from naturally infected larval C. gigas collected in 1995 in a French commercial hatchery has been purified and its genome sequenced. It was classified as Ostreid herpesvirus 1 (OsHV-1) within the family Malacoherpesviridae. Variants of this genome have since been described including an apparently more virulent OsHV-1 µvar. A collaboration between Cawthron and Ifremer is using molecular methods to shed light on the affinities of the New Zealand strain of OsHV-1. H:\STEVEW\Resilience\Resilience talks

3 Detection of OsHV-1 Gross examination of dead oysters Histopathology Molecular techniques, e.g. PCR, ISH Histopathology Broad spectrum No direct observation of viruses - only nuclear and other changes Samples also usable for molecular methods such as PCR

4 Typical Result

5 Ostrea chilensis Bonamia exitiosa 20µm

6 Histopathology in virus research

7 Nuclear features chromatin margination Normal Abnormal

8 Histopathology: limitations False positives ~5% of histology samples show some altered nuclear features. Healthy palps and muscle tissues often show chromatin margination. Moribund, but uninfected tissues, often exhibit features such as chromatin margination. False negatives Heavily infected oysters sometimes show no indicative nuclear features. Cowdrey bodies are not common in OsHV-1 infected oysters. HISTOLOGY ALONE IS UNRELIABLE

9 Polymerase Chain Reaction (PCR) Amplification of DNA Can be highly specific - strength and weakness. Requires careful interpretation - false positives and false negatives.

10 PCR Procedure Sample preparation - snippet of gill/mantle tissue, whole larvae DNA purification Fresh samples best Less DNA from formalin fixed specimens Identification of specific DNA in sample Detection of Host DNA - to demonstrate successful purification Detection of Pathogen DNA Interpretation of results DNA +ve for pathogen is positive DNA -ve for pathogen is probably negative if host DNA has been amplified Subject to sensitivity limit of assay

11 PCR methods End point PCR Good for assessment of amplification product size and for production of amplified copies of DNA for sequencing Real time PCR Rapid screening of large sample numbers for OsHV-1 detection Can be adapted to QPCR for quantification of virus

12 The current study Analysis of PCR-amplified DNA fragments from three virus genome areas. Identification of specific mutations that appear to be related to genogroups. Inferences about the affinity between the geographical sources of OsHV-1.

13 ORFs in this study ORF4 - most variable of the 3 studied genome areas distinguishing several genotypes or genogroups. ORFs 35/36/37/38 OsHV-1 µvar and related virus isolates showed a characteristic deletion of 605 bp corresponding to the lack of the entire ORF36 and ORF37 and to a partial deletion of ORF38. ORFs 42/ ORFs ignored

14 PCRs for short fragment DNA: formalin-fixed paraffin-embedded archival specimens Sample Primers C9/C10 Primers CF/CR Primers 37 TER F/R November 2010 PCR PCR PCR Adult 80 + oshv µvar 75 - µvar Adult 81 + oshv µvar 76 - µvar March 2005 PCR PCR PCR Spat 54 + oshv µvar 50 - µvar Spat 56 + oshv µvar 52 - µvar

15 OsHV-1 characteristics NZ 2005 archival samples: Primers ORF Result C9/C10 4 CF/CR 4 Same sequence as OsHV-1 µvar (GenBank n HQ842610) Same size as OsHV-1 µvar (2008/055/France) Del 36-37F2/Del 36-37R 35/36/37/38 Same size as OsHV-1 µvar (2008/055/France) Corresponds with OsHV-1µVar reported from France

16 PCRs suitable for good quality DNA (24 Nov 2010) Primers C2/C6 ORF 4 Primers IA1/IA2 ORF 43 Sample Source PCR Adult Fresh 31 + oshv µvar PCR Adult Fresh 32 + oshv µvar Larvae Fresh 35 + oshv µvar Larvae Alcohol fixed 36 + oshv µvar Larvae Alcohol fixed 37 + oshv µvar Larvae Alcohol fixed 38 + oshv µvar Larvae Alcohol fixed 39 + oshv µvar Larvae Alcohol fixed 40 + oshv µvar Larvae Alcohol fixed 41 + oshv µvar Sub adult Alcohol fixed 42 + oshv µvar

17 DNA sequence results NZ 2010 samples Primers ORF Result C2/C6 4 Del 36-37F2/Del 36-37R 4 OsHV-1 µvar: similar to Chinese and Japanese samples but with one bp addition OsHV-1 µvar: Close to Japanese, Chinese and some French OsHV-1 µvar with similar deletions IA2/IA1 42/43 Same deletions as OsHV-1 µvar

18 Summary OsHV-1 µvar positives detected in New Zealand larvae and adults from OsHV-1 µvar positives from archival (2005) paraffin-embedded NZ oysters. The NZ virus appears (by sequencing) to be a single strain of µvar with affinities to that from China, Japan and some French samples. These are interim findings: plans for further sequence analyses of NZ OsHV-1 positives.

19 Other work Retrospective analysis of archival samples from other oyster mortalities. Further sequencing of a wider range of NZ OsHV-1 positives. Surveying of wild populations. Retrospective analysis of archival samples from NZ sand clam mortalities - particularly Spisula sp. Further comparisons with OsHV-1 from other parts of the world In situ hybridization - staining of OsHV-1 infected areas to allow location of the viral DNA in specific parts of the section. Selective breeding.

20 Acknowledgements Dumont D'urville NZ/France Support Programme International Mobility Fund. Collaboration supported by Staff of Cawthron Institute and IFREMER, (Institut Français de Recherche pour l Exploitation de la Mer), Laboratoire de Génétique et Pathologie La Tremblade, France. Taranaki Medlab for histology services. Ministry of Agriculture and Forestry (MAF) Investigation and Diagnostic Centre, Animal Health Laboratory Wallaceville.

21 References Webb, SC Studies on ostreid herpes virus 1: a causal agent implicated in summer mortality in the oyster Crassostrea gigas, by PCR of archival histology specimens. Prepared for the Royal Society of New Zealand. Cawthron Report No p. Renault T, Moreau P, Faury N, Pepin J-F, Segarra A, Webb SC. (In prep). Phylogenetic analysis of clinical ostreid herpesvirus 1 isolates using amplified fragments from three virus genome areas.