Widespread recombination, reassortment, and transmission of unbalanced compound viral genotypes in natural arenavirus infections.

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.

BILATERALLY SYMMETRICAL ORAL AMELANOTIC MELANOMA IN A BOA CONSTRICTOR (BOA CONSTRICTOR CONSTRICTOR).

A 17-yr-old boa constrictor (Boa constrictor constrictor) presented initially with diffuse gingival swelling, loose teeth, and loss of body condition. Examination under anesthesia revealed two firm pink masses within the oral cavity. The largest mass was removed for biopsy. Histopathology and Melan-A-positive immunohistochemistry labeling confirmed a diagnosis of amelanotic melanoma. Secondary stomatitis was treated with antibiotics to improve quality of life, but the snake's condition deteriorated quickly over the next 2 mo. Euthanasia was elected and a gross postmortem examination was performed. Gross postmortem examination and histopathology results demonstrated that the neoplastic cells had spread in an unusual symmetrical pattern along all four dental arcades: the right and left sides of both the mandible and maxilla. Histopathology confirmed metastasis throughout the liver and spleen, despite the lack of gross lesions.

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis).

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine-zolazepam (7 mg kg(-1) bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilize viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen-thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilize viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.