The Genome of a Tortoise Herpesvirus (Testudinid Herpesvirus 3) Has a Novel Structure and Contains a Large Region That Is Not Required for Replication In Vitro or Virulence In Vivo.
Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it.


OBJECTIVE
Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome and used this information to take steps toward developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are that (i) TeHV-3 has a novel genome structure, (ii) its closest relative is a turtle herpesvirus, (iii) it contains interleukin-10 and semaphorin genes (the first time these have been reported in an alphaherpesvirus), (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo, and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.
Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium Clinic for Birds, Rabbits and Rodents, Department of Clinical Sciences, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
Frédéric
F
Gandar
MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
Gavin S
GS
Wilkie
Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, United Kingdom.
Derek
D
Gatherer
MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
Karen
K
Kerr
Clinic for Birds, Rabbits and Rodents, Department of Clinical Sciences, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
Didier
D
Marlier
Nutrition of Companion Animals, Department of Animal Production, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
Marianne
M
Diez
Laboratory for Clinical Diagnostics, Laboklin GmbH & Co. KG, Bad Kissingen, Germany.
Rachel E
RE
Marschang
Biocontrol Department, Research Unit Electron Microscopy, Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Ukkel, Belgium.
Jan
J
Mast
Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
Benjamin G
BG
Dewals
MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom davison@glasgow.ac.uk a.vdplasschen@ulg.ac.be.
Andrew J
AJ
Davison
Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium davison@glasgow.ac.uk a.vdplasschen@ulg.ac.be.
Alain F C
AF
Vanderplasschen
D018138:Alphaherpesvirinae / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity*; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000821:virology*; D002460:Cell Line; D002874:Chromosome Mapping; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D006566:Herpesviridae Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D016753:Interleukin-10 / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D039961:Semaphorins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D014426:Turtles / Q000821:virology*; D014765:Viral Vaccines / Q000276:immunology*
D004279:DNA, Viral; D039961:Semaphorins; D014765:Viral Vaccines; D016753:Interleukin-10