Primers for PCR-based detection of ostreid herpes virus-1 (OsHV-1): Application in a survey of New Zealand molluscs

• Published in: Aquaculture Vol. 272; no. 1-4; pp. 126 - 139
• Main Authors: Webb, Stephen C., Fidler, Andrew, Renault, Tristan
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 26-11-2007; Elsevier Science; Elsevier Sequoia S.A
• Subjects: Animal aquaculture; Animal diseases; Animal productions; Aquaculture; Austrovenus stutchburyi; Biological and medical sciences; Crassostrea gigas; disease detection; DNA primers; Dosinia anus; Fundamental and applied biological sciences. Psychology; Gastropoda; General aspects; Haliotis iris; Herpes viruses; Herpesviridae; Herpesvirus; Histology; Mactra discors; Marine; Microbiology; mollusc culture; Mollusca; Mollusks; Ostrea; Ostrea (Tiostrea) chilensis; Ostrea chilensis; ostreid herpes virus-1; Ostreid herpesvirus; Paphies subtriangulata; PCR; Pecten; Pecten novaezelandiae; Perna canaliculus; polymerase chain reaction; Spisula; Spisula aequilatera; viral diseases of animals and humans; Virology; New Zealand; Oceania; PSE, New Zealand; Pecten novaezelandiae; Spisula aequilatera; Haliotis iris; Dosinia anus; Mactra discors; Ostreid herpesvirus; Austrovenus stutchburyi; Ostrea (Tiostrea) chilensis; PCR; Perna canaliculus; Crassostrea gigas; Paphies subtriangulata; Iridaceae; Monocotyledones; Ostrea; Herpes; Gastropoda; Mytilidae; Infection; Marine environment; Polymerase chain reaction; Virus; Spisula aequilatera; Pecten novaezelandiae; Austrovenus stutchburyi; Crassostrea gigas; Ostrea (Tiostrea) chilensis; Dosinia anus; Mactra discors; Paphies subtriangulata; Haliotis iris; Anus; Bivalvia; Viral disease; Angiospermae; Spermatophyta; Invertebrata; Mollusca; Detection; Haliotis; Aquaculture; Ostreid herpesvirus; PCR; Perna canaliculus
• ISSN: 0044-8486; 1873-5622
• DOI: 10.1016/j.aquaculture.2007.07.224

The bivalves, Perna canaliculus, Spisula aequilatera, Pecten novaezelandiae, Austrovenus stutchburyi, Crassostrea gigas, Ostrea chilensis, Dosinia anus, Mactra discors, Paphies subtriangulata and the gastropod Haliotis iris were surveyed by histology and subsequent PCR for ostreid herpes virus (OsHV-1) infections. Histology was used to screen for indicative nuclear features such as Cowdry type A bodies, pycnosis and chromatin margination. Although no Cowdry bodies were seen in any samples, individuals from all species except M. discors, P. subtriangulata and D. anus exhibited some of the other features (albeit sparsely and atypically) and 75 samples out of 885 examined were deemed worthy of further investigation. DNA was purified successfully from 68 of these 75 samples. DNA was also extracted from fresh oyster tissue for the host oyster positive control. All PCR primers used were designed to amplify small (<300 bp) DNA fragments as would be expected from formalin-fixed tissue. The efficacy of these primer pairs with histology samples was confirmed by detectable PCR amplification of oyster and viral DNA from a known OsHV-1 infected fixed and paraffin embedded histology sample of C. gigas. 68 of the 75 survey samples produced visible PCR amplification with the new Cg18SFor/Cg18SRev primer pair demonstrating successful host DNA extraction. This primer pair targets the C. gigas 18S ribosomal gene (product: 292 bp) and detectable PCR confirms both the successful extraction of DNA fragments large enough for amplification by the other primers and also the absence of PCR inhibitors. As little as 1 ng of C. gigas genomic DNA template generated a visible PCR product. Sequencing and alignment of the amplification product confirmed it as part of the C. gigas 18S rRNA gene. OsHV-1 DNA was sought by PCR in the 68 Cg18SFor/Cg18SRev PCR positives using two primer pairs: the extant C9/C10 (product: 196 bp), and the new pair OsHVDPFor/OsHVDPRev (product: 197 bp). As little as 1 pg and 10 pg (∼5000 and 50,000 OsHV-1 genome copies per PCR reaction) allowed these primer pairs, respectively, to detectably amplify a specific product. Sequencing and alignment with the OsHV-1 genomic sequence confirmed the amplification products as being the anticipated portions of the genome for these primer pairs. The PCR assays failed to detect OsHV-1 in any extracts except for the known positives and it is concluded that OsHV-1 is absent in this survey of New Zealand molluscs. Alternative explanations for the cytological features seen during the histology survey leading to provisional positives for OsHV-1 infection are discussed.