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, 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 towards characterizing TeHV-3 and developing prophylactic methods against it. IMPORTANCE: 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 towards 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: (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

In vitro and in vivo assessment of eprociclovir as antiviral treatment against testudinid herpesvirus 3 in Hermann's tortoise (Testudo hermanni)

Tortoises belonging to the Testudinidae family are infected by Testudinid herpesviruses. Testudinid herpesvirus 3 (TeHV-3) is considered the most pathogenic and affects several tortoise species, particularly those from the Testudo genus. As most species of this genus are endangered contribute to ecological concerns over this virus. Here, we aimed to explore the rational development of an antiviral treatment against TeHV-3 using Hermann's tortoise (Testudo hermanni) as a host model. Ten antiviral compounds were tested in cell culture for their toxicity and their activity against TeHV-3. Eight compounds exhibited different levels of activity against TeHV-3 with either no or only minor cytotoxic effects on cells. Next, eprociclovir (EPV, ciprovir) was selected for further investigations in vivo. Its pharmacokinetic properties were investigated after a single sub-cutaneous administration at 5 or 10 mg/kg. Plasma concentrations remained above half maximal effective concentration (EC 50 ) for 2.2 and 4.4 h after administration at 5 and 10 mg/kg, respectively. Finally, EPV toxicity was investigated after administration at the dose of 10 mg/kg, BID for seven consecutive days. As early as one day after initiation of the treatment up to its end, EPV plasma concentration remained under the EC 50. Apathy and anorexia developed after 7 days. Biochemical and anatomopathological examinations revealed nephrotoxic effects of EPV. Altogether, these data suggest that EPV is not a suitable molecule for the treatment of TeHV-3. Further studies are required to determine whether the other molecules identified here for their anti-TeHV-3 activity represent potential candidates for the development of efficacious treatments. © 2019 Elsevier Lt

Influence of a single dose of buprenorphine on rabbit (Oryctolagus cuniculus) gastrointestinal motility

OBJECTIVE: To establish a noninvasive imaging protocol for rabbit gastrointestinal transit evaluation. To assess the effect of a single injection of buprenorphine on the digestive transit of rabbits via this new technique. STUDY DESIGN: Prospective, parallel study. ANIMALS: Fifteen specific pathogen-free male New Zealand White rabbits weighing 2.68 ± 0.28 kg. METHODS: A 10 mL kg-1 barium meal was administered and the rabbits were subjected to serial radiographic and ultrasound examinations without treatment and 1 week later following a single intramuscular dose of 100 μg kg-1 of buprenorphine. Radiographic data from the stomach and caecum were collected and assigned a retention score ranging from 0 (no barium) to 3 (large amount of barium). The resulting scores and pyloric and duodenal contraction counts were analysed using a mixed linear model and are expressed as least square mean (lsm) ± standard error. Transit was estimated based on the apparition time of faeces in the pelvic area and analysed using a Wilcoxon test. A p < 0.05 was considered significant. RESULTS: Buprenorphine treatment induced a higher lsm number of pyloric (1.73 ± 0.19 versus 0.78 ± 0.19, p < 0.01) and lsm duodenal contractions (17.35 ± 1.04 versus 13.44 ± 1.04, p < 0.01). Buprenorphine administration decreased the lsm barium retention score in the stomach (2.44 ± 0.05 versus 2.64 ± 0.05, p < 0.01), but had no effect on the lsm barium retention score in the caecum. The time to apparition of faeces in the pelvic area was not influenced by buprenorphine administration (p = 0.66). CONCLUSIONS AND CLINICAL RELEVANCE: A single high dose of buprenorphine appears to have no adverse effect on gastrointestinal motility in healthy rabbits

Serological and molecular reagents for the detection and characterization of the bacterium-like organism (BLO) of citrus greening disease

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