title: Molecular identification of three novel herpesviruses found in Australian farmed saltwater crocodiles (Crocodylus porosus) and Australian captive freshwater crocodiles (Crocodylus johnstoni).
abstract: As part of a larger investigation into three emerging disease syndromes highlighted by conjunctivitis and pharyngitis, systemic lymphoid proliferation and encephalitis, and lymphonodular skin infiltrates in farmed saltwater crocodiles (Crocodylus porosus) and one emerging syndrome of systemic lymphoid proliferation in captive freshwater crocodiles (Crocodylus johnstoni), cytopathic effects (CPE), including syncytial cell formation, were observed in primary crocodile cell lines exposed to clarified tissue homogenates from affected crocodiles. Ten cell cultures with CPE were then screened for herpesviruses using two broadly-reactive herpesvirus PCRs. Amplicons were obtained from 9 of 10 cell cultures and were sequenced. Three novel herpesviruses were discovered and the phylogenetic analysis of these viruses showed there was a 63% Bayesian posterior probability value supporting these viruses clustering with the subfamily Alphaherpesvirinae, and 100% posterior probability of clustering with a clade containing the Alphaherpesvirinae and other unassigned reptile herpesviruses. It is proposed that they are named Crocodyline herpesvirus (CrHV) 1, 2 and 3. CrHV1 and 2 were only isolated from saltwater crocodiles and CrHV3 was only isolated from freshwater crocodiles. A duplex PCR was designed that was able to detect these herpesviruses in formalin-fixed paraffin-embedded tissues, a sample type that neither of the broadly-reactive PCRs was able to detect these herpesviruses in. This work describes the isolation, molecular detection and phylogeny of these novel herpesviruses but the association that they have with the emerging disease syndromes requires further investigation.
authors.affiliation: Murdoch University, School of Veterinary and Life Sciences, Murdoch, Western Australia 6150, Australia. Electronic address: t.hyndman@murdoch.edu.au.
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Hyndman
authors.affiliation: Berrimah Veterinary Laboratories, Northern Territory Government, GPO Box 3000, Darwin, Northern Territory 0801, Australia. Electronic address: cathy.shilton@nt.gov.au.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Shilton
authors.affiliation: Department of Small Animal Clinical Sciences, University of Florida College of Veterinary Medicine, Gainesville, Florida 32610, USA. Electronic address: wellehanj@ufl.edu.
authors.forename: James F X
authors.initials: JF
authors.lastname: Wellehan
authors.affiliation: Berrimah Veterinary Laboratories, Northern Territory Government, GPO Box 3000, Darwin, Northern Territory 0801, Australia. Electronic address: steven.davis@nt.gov.au.
authors.forename: Steven
authors.initials: S
authors.lastname: Davis
authors.affiliation: The University of Sydney, Sydney, New South Wales 2006, Australia; Centre for Crocodile Research, Noonamah, Northern Territory 0837, Australia. Electronic address: sally@crocresearch.com.au.
authors.forename: Sally R
authors.initials: SR
authors.lastname: Isberg
authors.affiliation: The University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: david.phalen@sydney.edu.au.
authors.forename: David
authors.initials: D
authors.lastname: Phalen
authors.affiliation: Berrimah Veterinary Laboratories, Northern Territory Government, GPO Box 3000, Darwin, Northern Territory 0801, Australia. Electronic address: lorna.melville@nt.gov.au.
authors.forename: Lorna
authors.initials: L
authors.lastname: Melville
mesh_terms: D000489:Alligators and Crocodiles / Q000821:virology*; D000818:Animals; D000829:Animals, Domestic; D001315:Australia; D001499:Bayes Theorem; D002478:Cells, Cultured; D004279:DNA, Viral / Q000032:analysis*; D006564:Herpesviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000662:veterinary* / Q000821:virology; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000662:veterinary; D014871:Water Microbiology
chemical_list: D004279:DNA, Viral
keywords: Alphaherpesvirus; PCR; Phylogeny; Reptile; Virus isolation