Generation and Characterization of a SARS-CoV-2-Susceptible Mouse Model Using Adeno-Associated Virus (AAV6.2FF)-Mediated Respiratory Delivery of the Human ACE2 Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic with millions of human infections. There continues to be a pressing need to develop potential therapies and vaccines to inhibit SARS-CoV-2 infection to mitigate the ongoing pandemic. Epidemiological data from the current pandemic indicates that there may be sex-dependent differences in disease outcomes. To investigate these differences, we proposed to use common small animal species that are frequently used to model disease with viruses. However, common laboratory strains of mice are not readily infected by SARS-CoV-2 because of differences in the angiotensin-converting enzyme 2 (ACE2), the cellular receptor for the virus. To overcome this limitation, we transduced common laboratory accessible strains of mice of different sexes and age groups with a novel a triple AAV6 mutant, termed AAV6.2FF, encoding either human ACE2 or luciferase via intranasal administration to promote expression in the lung and nasal turbinates. Infection of AAV-hACE2-transduced mice with SARS-CoV-2 resulted in high viral titers in the lungs and nasal turbinates, establishment of an IgM and IgG antibody response, and modulation of lung and nasal turbinate cytokine profiles. There were insignificant differences in infection characteristics between age groups and sex-related differences; however, there were significant strain-related differences between BALB/c vs. C57BL/6 mice. We show that AAV-hACE2-transduced mice are a useful for determining immune responses and for potential evaluation of SARS-CoV-2 vaccines and antiviral therapies, and this study serves as a model for the utility of this approach to rapidly develop small-animal models for emerging viruses.

In Vitro Inactivation of SARS-CoV-2 Using Gamma Radiation.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is classified as a Risk Group 3 pathogen; propagative work with this live virus should be conducted in biosafety level-3 (BSL-3) laboratories. However, inactivated virus can be safely handled in BSL-2 laboratories. Gamma irradiation is one of the methods used to inactivate a variety of pathogens including viruses. Objective: To determine the radiation dose required to inactivate SARS-CoV-2 and its effect, if any, on subsequent polymerase chain reaction (PCR) assay. Methods: Aliquots of SARS-CoV-2 virus culture were subjected to increasing doses of gamma radiation to determine the proper dose required to inactivate the virus. Real-time quantitative polymerase chain reaction (RT-qPCR) data from irradiated samples was compared with that of the non-irradiated samples to assess the effect of gamma radiation on PCR assay. Results: A radiation dose of 1 Mrad was required to completely inactivate 106.5 TCID50/ml of SARS-CoV-2. The influence of gamma radiation on PCR sensitivity was inversely related and dose-dependent up to 0.5 Mrad with no further reduction thereafter. Conclusion: Gamma irradiation can be used as a reliable method to inactivate SARS-CoV-2 with minimal effect on subsequent PCR assay.

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P2-positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P2 production in cells to promote efficient delivery to NPC1.

Immunopathogenesis of severe acute respiratory disease in Zaire ebolavirus-infected pigs.

Ebola viruses (EBOV) are filamentous single-stranded RNA viruses of the family Filoviridae. Zaire ebolavirus (ZEBOV) causes severe haemorrhagic fever in humans, great apes and non-human primates (NHPs) with high fatality rates. In contrast, Reston ebolavirus (REBOV), the only species found outside Africa, is lethal to some NHPs but has never been linked to clinical disease in humans despite documented exposure. REBOV was isolated from pigs in the Philippines and subsequent experiments confirmed the susceptibility of pigs to both REBOV and ZEBOV with predilection for the lungs. However, only ZEBOV caused severe lung pathology in 5-6 weeks old pigs leading to respiratory distress. To further elucidate the mechanisms for lung pathology, microarray analysis of changes in gene expression was performed on lung tissue from ZEBOV-infected pigs. Furthermore, systemic effects were monitored by looking at changes in peripheral blood leukocyte subsets and systemic cytokine responses. Following oro-nasal challenge, ZEBOV replicated mainly in the respiratory tract, causing severe inflammation of the lungs and consequently rapid and difficult breathing. Neutrophils and macrophages infiltrated the lungs but only the latter were positive for ZEBOV antigen. Genes for proinflammatory cytokines, chemokines and acute phase proteins, known to attract immune cells to sites of infection, were upregulated in the lungs, causing the heavy influx of cells into this site. Systemic effects included a decline in the proportion of monocyte/dendritic and B cells and a mild proinflammatory cytokine response. Serum IgM was detected on day 5 and 6 post infection. In conclusion, a dysregulation/over-activation of the pulmonary proinflammatory response may play a crucial role in the pathogenesis of ZEBOV infection in 5-6 weeks old pigs by attracting inflammatory cells to the lungs.

Assessment of the efficacy of commercially available and candidate vaccines against a pandemic H1N1 2009 virus.

BACKGROUND: The emergence and global spread of the pandemic H1N1 2009 influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. METHODS: Ferrets were immunized with the 2008-2009 formulations of commercially available live attenuated (FluMist; MedImmune) or split-inactivated (Fluviral; GlaxoSmithKline) vaccines, a commercial swine vaccine (FluSure; Pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (A/Mexico/InDRE4487/2009). Adaptive immune responses were monitored, and the animals were challenged with A/Mexico/InDRE4487/2009 after 5 weeks. RESULTS: Only animals that received the swine or matched vaccines developed detectable hemagglutination-inhibiting antibodies against the challenge virus, whereas a T cell response was exclusively detected in animals vaccinated with FluMist. After challenge, all animals had high levels of virus replication in the upper respiratory tract. However, preexisting anti-pandemic H1N1 2009 antibodies resulted in reduced clinical signs and improved survival. Surprisingly, FluMist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin-10. CONCLUSIONS: The present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic H1N1 2009 virus, and it suggests that a higher dose or prime-boost regimen may be required. The consequences of mismatched immunity to influenza merit further investigation.