Characterisation of a Live-Attenuated Rabies Virus Expressing a Secreted scFv for the Treatment of Rabies.

Rabies virus (RABV) causes possibly the oldest disease and is responsible for an estimated >59,000 human fatalities/year. Post exposure prophylaxis (PEP), the administration of vaccine and rabies immunoglobulin, is a highly effective tool which is frequently unavailable in RABV endemic areas. Furthermore, due to the constraints of the blood-brain barrier, current PEP regimes are ineffective after the onset of clinical symptoms which invariably result in death. To circumvent this barrier, a live-attenuated recombinant RABV expressing a highly RABV-neutralising scFv antibody (62-71-3) linked to the fluorescent marker mCherry was designed. Once rescued, the resulting construct (named RABV-62scFv) was grown to high titres, its growth and cellular dissemination kinetics characterised, and the functionality of the recombinant 62-71-3 scFv assessed. Encouraging scFv production and subsequent virus neutralisation results demonstrate the potential for development of a therapeutic live-attenuated virus-based post-infection treatment (PIT) for RABV infection.

Taiwan Bat Lyssavirus: In Vitro and In Vivo Assessment of the Ability of Rabies Vaccine-Derived Antibodies to Neutralise a Novel Lyssavirus.

Rabies is a neglected tropical disease. The prototype virus, the rabies virus, still causes tens of thousands of human fatalities annually. Rabies is one member of the genus Lyssavirus. The burden of other lyssaviruses is unclear. The continued emergence of novel lyssaviruses means that assessment of vaccine efficacy against these viruses is critical, as standard rabies vaccines are not efficacious against all lyssaviruses. Taiwan bat lyssavirus (TWBLV) was first reported in 2018 following isolation from Japanese house bats. Since the initial detection and genetic characterisation, no attempts have been made to antigenically define this virus. Due to the inaccessibility of the wildtype isolate, the successful generation of a live recombinant virus, cSN-TWBLV, is described, where the full-length genome clone of the RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of TWBLV. In vitro and in vivo characterization of cSN-TWBLV was undertaken and demonstrated evidence for cross-neutralisation of cSN-TWBLV with phylogroup I -specific sera and rabies virus standard sera. For neutralisation equivalent to 0.5 IU/mL of WHO and World Organisation of Animal Health (WOAH) sera against CVS, 0.5 IU/mL of WOAH sera and 2.5 IU/mL of WHO sera were required to neutralise cSN-TWBLV. In addition, specific sera for ARAV and EBLV-1 exhibited the highest neutralising antibody titres against cSN-TWBLV, compared to other phylogroup I-specific sera.

Comparison of Serological Assays for the Detection of SARS-CoV-2 Antibodies.

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced 'COVID-19 like-illness', and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.

Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens.

The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.

Genome-wide association analysis uncovers variants for reproductive variation across dog breeds and links to domestication.

BACKGROUND AND OBJECTIVES: The diversity of eutherian reproductive strategies has led to variation in many traits, such as number of offspring, age of reproductive maturity and gestation length. While reproductive trait variation has been extensively investigated and is well established in mammals, the genetic loci contributing to this variation remain largely unknown. The domestic dog, Canis lupus familiaris is a powerful model for studies of the genetics of inherited disease due to its unique history of domestication. To gain insight into the genetic basis of reproductive traits across domestic dog breeds, we collected phenotypic data for four traits, cesarean section rate, litter size, stillbirth rate and gestation length, from primary literature and breeders' handbooks. METHODOLOGY: By matching our phenotypic data to genomic data from the Cornell Veterinary Biobank, we performed genome-wide association analyses for these four reproductive traits, using body mass and kinship among breeds as covariates. RESULTS: We identified 12 genome-wide significant associations between these traits and genetic loci, including variants near CACNA2D3 with gestation length, MSRB3 and MSANTD1 with litter size, SMOC2 with cesarean section rate and UFM1 with stillbirth rate. A few of these loci, such as CACNA2D3 and MSRB3, have been previously implicated in human reproductive pathologies, whereas others have been associated with domestication-related traits, including brachycephaly (SMOC2) and coat curl (KRT71). CONCLUSIONS AND IMPLICATIONS: We hypothesize that the artificial selection that gave rise to dog breeds also influenced the observed variation in their reproductive traits. Overall, our work establishes the domestic dog as a system for studying the genetics of reproductive biology and disease. LAY SUMMARY: The genetic contributors to variation in mammalian reproductive traits remain largely unknown. We took advantage of the domestic dog, a powerful model system, to test for associations between genome-wide variants and four reproductive traits (cesarean section rate, litter size, stillbirth rate and gestation length) that vary extensively across breeds. We identified associations at a dozen loci, including ones previously associated with domestication-related traits, suggesting that selection on dog breeds also influenced their reproductive traits.

Trying to treat the untreatable: experimental approaches to clear rabies virus infection from the CNS.

Rabies virus causes an invariably fatal encephalitis following the onset of clinical disease. Despite the availability of safe and effective vaccines, the clinical stages of rabies encephalitis remain untreatable, with few survivors being documented. A principal obstacle to the treatment of rabies is the neurotropic nature of the virus, with the blood-brain barrier size exclusion limit rendering the delivery of antiviral drugs and molecules to the central nervous system inherently problematic. This review focuses on efforts to try and overcome barriers to molecule delivery to treat clinical rabies and overviews current progress in the development of experimental live rabies virus vaccines that may have future applications in the treatment of clinical rabies, including the attenuation of rabies virus vectors through either the duplication or mutation of existing genes or the incorporation of non-viral elements within the genome. Rabies post-infection treatment (PIT) remains the holy grail of rabies research.