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Including Quotes: No Yes. Including Pitches: No Yes. Larry S. Schlesinger, MD is an internationally recognized authority in infectious diseases with a particular interest in tuberculosis and lung biology. During his tenure he founded the Center for Microbial Interface Biology, a Board of Trustees approved university-wide center with a focus on infectious diseases of concern to public health. Schlesinger is a leading physician scientist whose studies focus on the pathogenesis of tuberculosis and other airborne infectious agents that subvert lung immune mechanisms.

A generalist nested PCR directed to the herpesviral DNA polymerase that allows the detection of herpesviruses of different subfamilies by Van Devanter et al. The glycoprotein B gene was partially amplified using the GH1 system described previously [ 17 ]. Nucleotide sequences were analysed and assembled into consensus sequences using the BioEdit version 7. Nucleotide sequences were translated using Mega X Partial nucleotide bp sequences of the viral DNA polymerase gene were aligned using the Clustal W with gap opening penalty and a gap extend penalty of 30 and 15, respectively.

The evolutionary history of 28 partial DNA polymerase protein sequences of gammaherpesviruses was inferred by Maximum Likelihood. A discrete Gamma distribution G was used to model evolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable I. For more accurate phylogenetic analysis of the hare herpesvirus described is this manuscript, the partial nucleotide bp sequences of the viral DNA polymerase gene catalytic subunit was concatenated with the partial bp nucleotide sequences of the viral Glycoprotein B gene belonging to the same strain.

The sequences were translated and aligned using the Clustal W with gap opening penalty and a gap extend penalty of 30 and 15, respectively. The final alignment was edited to include all the sequences, corresponding to nucleotides and amino acids of length. The analysis involved 45 amino acid sequences. Isolation of herpesvirus was attempted from organs of hares coinfected with MYXV and LeHV-5, namely from liver and spleen, penile and vulva samples. In addition, liver and spleen samples from two hares with single herpesvirus infection, were also used. Following centrifugation, the supernatant was filtered through a 0.

Three passages were carried out. The supernatant and cell pellet of each passage were tested for the presence of herpesvirus by PCR [ 17 ]. This lesion was more evident in males, affecting the penile glans, but was also observed in females inthe vulva. Other lesions observed in these hares included the presence of herpetic-like skin vesicles, uncommon in rabbits with myxomatosis.

Further investigation of the macroscopic lesions and histopathological patterns was carried out in hares co-infected with LeHV-5 and MYXV. Hare-1 presented with eyelids thickened by the accumulation of mucopurulent exudate and marked enlargement of the penis measuring 1. C orpus cavernosum CC ; penile uretra U. At the necropsy, hare-2 showed ulcerated multinodular thickening of the eyelids and lips. Accumulation of mucopurulent exudate in both eyes was also registered and a small vesicle was present in the lower lip Fig 3.

A vesicle can be seen in the lower lip arrow. The dermis of hare-1 showed fusiform or stellate mesenchymal cell proliferation, surrounded by abundant extracellular matrix, scattered infiltration by lymphocytes and macrophages, and small aggregates of heterophiles, consistent with myxomatosis. The penile epithelium of this hare was mostly necrotic and replaced by a thick band of necrotic cells, heterophils and red blood cells Fig 2. Severe heterophile infiltrations of the stroma, in either a diffuse pattern or multifocal aggregates, were also seen.

In the stroma, there was also proliferation of pleomorphic spindle cells, with some nuclei almost filled with slightly eosinophilic inclusion bodies Cowdry type A inclusions Fig 4 , suggesting a nuclear replicating virus. These lesions, unexpected in myxomatosis, are compatible with herpesvirus. In the skin of hare-2, a ballooning degeneration of keratinocytes was registered. Coalescent intra-epidermal and subepidermal vesicopustules Fig 5 filled with fibrin and necrotic cells debris and multifocal detachment of the eyelids, lips and foreskin epidermis were seen.

In the underlying dermis, multifocal haemorrhages, intense infiltration by heterophils and necrotic cells with accumulation of chromatin debris were present Fig 5.

Below the dermis, accumulation of myxoid tissue with pleomorphic spindle cells, some of which showing rounded or oval and slightly eosinophilic intranuclear inclusion bodies, was observed Fig 6. An infiltrate of mononucleated inflammatory cells and heterophils was present in skeletal muscle tissue.

Oedema and inflammatory cell infiltration with signs of necrosis. An intranuclear inclusion body in a mesenchymal cell can be seen arrow. Samples from hare-1 and hare-2 were further processed and analysed for TEM allowing the confirmation of the presence of herpesvirus in different tissues. In the penile soft tissue of hare-1, spherical virions, with structure and dimensions compatible with herpesviruses, comprising an inner core packed into an icosahedral capsid, were observed in the nucleus of stromal cells Fig 7 , indicating nuclear replication Fig 7B , which is an attribute of herpesviruses.

The viral capsid contained a relatively small, asymmetrical, electron-dense region that probably represents the condensed DNA core. In this animal, also positive to myxomatosis, no MYXV particles were found in the samples processed. Fifty percent of the hares were positive to LeHV-5, of which Six hares showed doubtful results from which four were MYXV-negative. Parasitological and bacteriological examinations did not reveal any infections that could justify the death of these animals.