Francesco C. Origgi

Structural Rearrangements of the Central Region of the Morbillivirus Attachment Protein Stalk Domain Trigger F Protein Refolding for Membrane Fusion

Background: Attachment (H) and fusion glycoproteins of morbilliviruses co-operate to induce membrane fusion for cell entry. Results: Reversible membrane fusion inhibition by engineered disulfide bonds within the central region of the tetrameric H-stalk domain. Conclusion: Structural rearrangements of the H-stalk domain contribute to fusion triggering. Significance: Provides a basis for novel strategies targeting the central region of the attachment protein-stalk domain to prevent Morbillivirus cell entry. It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91–115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111–118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Experimental Transmission of a Herpesvirus in Greek Tortoises (Testudo graeca)

An experimental transmission study aimed at fulfilling Kochs postulates for a herpesvirus-associated stomatitis-rhinitis in Mediterranean tortoises is presented. Clinical, pathologic, serologic, and molecular studies were performed linking tortoise herpesvirus with the pathogenesis of stomatitis-rhinitis. Four adult Greek tortoises received either intranasally or intramuscularly two tortoise herpesvirus isolates by primary experimental infection and secondary challenge 11 months later. After the primary experimental infection and the secondary challenge, clinical signs of illness developed, which included conjunctivitis, diphtheritic oral plaques, and oral discharge. At 4 weeks after the secondary challenge, all tortoises were humanely euthanatized and evaluated. Although neutralizing antibodies developed after the primary experimental infection, they apparently did not prevent the later development of recurrent clinical signs. Polymerase chain reaction (PCR) and reverse transcription-PCR analyses allowed sensitive characterization of the systemic distribution of the herpesvirus DNA sequences and their presence in the cranial nerves and brains of the infected tortoises. Despite the failure to recover the herpesviruses used in the transmission study, the findings support the premise that tortoise herpes-virus is a primary pathogen of Greek tortoises.

Enzyme-Linked Immunosorbent Assay for Detecting Herpesvirus Exposure in Mediterranean Tortoises (Spur-Thighed Tortoise [Testudo graeca] and Hermann's Tortoise [Testudo hermanni])

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to a herpesvirus associated with an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermanns tortoise (Testudo hermanni)]. This serodiagnostic test was validated through a hyperimmunization study. The mean of theA405 readings of the plasma samples collected at time zero of the hyperimmunization study plus three times the standard deviation was used as the cutoff for seropositivity in tortoises. ELISA results were compared to serum neutralization (SN) values for the same samples by using the McNemar test. The results obtained by SN and ELISA were not significantly different (P > 0.05). This new ELISA could be used as an important diagnostic tool for screening wild populations and private and zoo collections of Mediterranean tortoises.

Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4–7.8) but none of the 269 red deer (0%; 0–1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

Emergence of Canine Distemper Virus Strains With Modified Molecular Signature and Enhanced Neuronal Tropism Leading to High Mortality in Wild Carnivores

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination—the classic presentation of CDV infection—was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Paramyxovirus infection in caiman lizards (Draecena guianensis)

Three separate epidemics occurred in caiman lizards (Dracaena guianensis) that were imported into the USA from Peru in late 1998 and early 1999. Histologic evaluation of tissues from necropsied lizards demonstrated a proliferative pneumonia. Electron microscopic examination of lung tissue revealed a virus that was consistent with members of the family Paramyxoviridae. Using a rabbit polyclonal antibody against an isolate of ophidian (snake) paramyxovirus, an immunoperoxidase staining technique demonstrated immunoreactivity within pulmonary epithelial cells of 1 lizard. Homogenates of lung, brain, liver, or kidney from affected lizards were placed in flasks containing monolayers of either terrapene heart cells or viper heart cells. Five to 10 days later, syncytial cells formed. When Vero cells were inoculated with supernatant of infected terrapene heart cells, similar syncytial cells developed. Electron microscopic evaluation of infected terrapene heart cells revealed intracyto-plasmic inclusions consisting of nucleocapsid strands. Using negative-staining electron microscopy, abundant filamentous nucleocapsid material with a herringbone structure typical of the Paramyxoviridae was observed in culture medium of infected viper heart cells. Seven months following the initial epizootic, blood samples were collected from surviving group 1 lizards, and a hemagglutination inhibition assay was performed to determine presence of specific antibody against the caiman lizard isolate. Of the 17 lizards sampled, 7 had titers of <1:20 and 10 had titers of >1:20 and <1:80. This report is only the second of a paramyxovirus identified in a lizard and is the first to snow the relationship between histologic and ultrastructural findings and virus isolation.

Immunohistochemical staining of chlamydial antigen in emerald tree boas (Corallus caninus)

Of 120 privately owned captive-bred and wild-collected emerald tree boas (ETBs) (Corallus caninus), 97 died or were euthanatized. Eighteen snakes were necropsied, and tissues were collected from all major organs and processed for light microscopy. Histologic examination demonstrated histiocytic granulomas in the small intestine, heart, and esophageal tonsils of one ETB, small intestine of a second ETB, and in an esophageal tonsil of a third ETB. Within the center of these granulomas, small, basophilic, punctate organisms were demonstrated using hematoxylin and eosin staining. Transmission electron microscopic examination of an intestinal granuloma demonstrated developmental stages of organisms consistent with members of the family Chlamydiaceae. An immunoperoxidase staining technique and 2 different commercially available monoclonal antibodies against chlamydial lipopolysaccharide antigen was used to identify chlamydial antigen in these lesions. Liver of a puff adder (Bitis arietans) with previously reported systemic chlamydiosis served as the positive control. Both monoclonal antibodies stained antigen in these granulomas. Additionally, macrophages within aggregates of lymphoplasmacytic cells in the colon, small intestine, and esophageal tonsils of 3 other ETBs contained antigen. Although both antibodies labeled antigen in serial sections of tissue, a difference in staining intensity was noted.

Diseases of the respiratory tract of chelonians.

Diseases of the respiratory tract commonly occur in captive chelonians, and several diseases also have occurred in wild chelonians. Infectious causes include viruses, bacteria, fungi, and parasites. Herpesviruses have surfaced as important pathogens of the oral cavity and respiratory tract in Hermanns tortoise (Testudo hermanii), spur-thighed tortoise (Testudo graeca), and other tortoises in Europe and the United States. Herpesvirus-associated respiratory diseases also have been reported in the green turtle, Chelonia mydas, in mariculture in the Cayman Islands. Of diseases caused by bacteria, an upper respiratory tract disease caused by Mycoplasma sp has been reported in free-hanging and captive gopher tortoises in the southeastern United States and in desert tortoises in the Mojave Desert of the southwestern United States. Mycotic pulmonary disease is commonly reported in captive chelonians, especially in those maintained at suboptimal temperatures. An intranuclear coccidia has been seen in several species of captive tortoises in the United States, and, in one case, a severe proliferative pneumonia was associated with organisms in the lung. The most common noninfectious cause of respiratory disease in chelonians results from trauma to the carapace. Although pulmonary fibromas commonly occur in green turtles with fibropapillomatosis, for the most part, tumors of the respiratory tract are uncommon in chelonians.

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii.

Following field observations of wild Agassizs desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.

Characterisation of a new group of Francisella tularensis subsp. holarctica in Switzerland with altered antimicrobial susceptibilities, 1996 to 2013

Molecular analysis of Francisella tularensis subsp. holarctica isolates from humans and animals revealed the presence of two subgroups belonging to the phylogenetic groups B.FTNF002-00 and B.13 in Switzerland. This finding suggests a broader spread of this group in Europe than previously reported. Until recently, only strains belonging to the Western European cluster (group B.FTNF002-00) had been isolated from tularaemia cases in Switzerland. The endemic strains belonging to group B.FTNF002-00 are sensitive to erythromycin, in contrast to the strains of the newly detected group B.13 that are resistant to this antibiotic. All the strains tested were susceptible to ciprofloxacin, streptomycin, gentamicin, nalidixic acid and chloramphenicol but showed reduced susceptibility to tetracycline when tested in a growth medium supplemented with divalent cations. The data show a previously undetected spread of group B.13 westwards in Europe, associated with changes in the antibiotic resistance profile relevant to treatment of tularaemia.