Adipose Tissues from Human and Bat-Derived Cell Lines Support Ebola Virus Infection.

Ebola virus is a zoonotic pathogen with a geographic range covering diverse ecosystems that are home to many potential reservoir species. Although researchers have detected Ebola virus RNA and serological evidence of previous infection in different rodents and bats, the infectious virus has not been isolated. The field is missing critical knowledge about where the virus is maintained between outbreaks, either because the virus is rarely encountered, overlooked during sampling, and/or requires specific unknown conditions that regulate viral expression. This study assessed adipose tissue as a previously overlooked tissue capable of supporting Ebola virus infection. Adipose tissue is a dynamic endocrine organ helping to regulate and coordinate homeostasis, energy metabolism, and neuroendocrine and immune functions. Through in vitro infection of human and bat (Eptesicus fuscus) brown adipose tissue cultures using wild-type Ebola virus, this study showed high levels of viral replication for 28 days with no qualitative indicators of cytopathic effects. In addition, alterations in adipocyte metabolism following long-term infection were qualitatively observed through an increase in lipid droplet number while decreasing in size, a harbinger of lipolysis or adipocyte browning. The finding that bat and human adipocytes are susceptible to Ebola virus infection has important implications for potential tissue tropisms that have not yet been investigated. Additionally, the findings suggest how the metabolism of this tissue may play a role in pathogenesis, viral transmission, and/or zoonotic spillover events.

The Inability of Marburg Virus to Cause Disease in Ferrets Is Not Solely Linked to the Virus Glycoprotein.

Ebola virus (EBOV) causes lethal disease in ferrets, whereas Marburg virus (MARV) does not. To investigate this difference, we first evaluated viral entry by infecting ferret spleen cells with vesicular stomatitis viruses pseudotyped with either MARV or EBOV glycoprotein (GP). Both viruses were capable of infecting ferret spleen cells, suggesting that lack of disease is not due to a block in MARV entry. Next, we evaluated replication kinetics of authentic MARV and EBOV in ferret cell lines and demonstrated that, unlike EBOV, MARV was only capable of low levels of replication. Finally, we inoculated ferrets with a recombinant EBOV expressing MARV GP in place of EBOV GP. Infection resulted in uniformly lethal disease within 7-9 days postinfection, while MARV-inoculated animals survived until study endpoint. Together these data suggest that the inability of MARV to cause disease in ferrets is not entirely linked to GP.

The Ferret as a Model for Filovirus Pathogenesis and Countermeasure Evaluation.

The domestic ferret (Mustela putorius furo) has long been a popular animal model for evaluating viral pathogenesis and transmission as well as the efficacy of candidate countermeasures. Without question, the ferret has been most widely implemented for modeling respiratory viruses, particularly influenza viruses; however, in recent years, it has gained attention as a novel animal model for characterizing filovirus infections. Although ferrets appear resistant to infection and disease caused by Marburg and Ravn viruses, they are highly susceptible to lethal disease caused by Ebola, Sudan, Bundibugyo, and Reston viruses. Notably, unlike the immunocompetent rodent models of filovirus infection, ferrets are susceptible to lethal disease caused by wild-type viruses, and they recapitulate many aspects of human filovirus disease, including systemic virus replication, coagulation abnormalities, and a dysregulated immune response. Along with the stringency with which they reproduce Ebola disease, their relatively small size and availability make ferrets an attractive choice for countermeasure evaluation and pathogenesis modeling. Indeed, they are so far the only small animal model available for Bundibugyo virus. Nevertheless, ferrets do have their limitations, including the lack of commercially available reagents to dissect host responses and their unproven predictive value in therapeutic evaluation. Although the use of the ferret model in ebolavirus research has been consistent over the last few years, its widespread use and utility remains to be fully proven. This review provides a comprehensive overview of the ferret models of filovirus infection and perspective on their ongoing use in pathogenesis modeling and countermeasure evaluation.

Simulated sunlight decreases the viability of SARS-CoV-2 in mucus.

The novel coronavirus, SARS-CoV-2, has spread into a pandemic since its emergence in Wuhan, China in December of 2019. This has been facilitated by its high transmissibility within the human population and its ability to remain viable on inanimate surfaces for an extended period. To address the latter, we examined the effect of simulated sunlight on the viability of SARS-CoV-2 spiked into tissue culture medium or mucus. The study revealed that inactivation took 37 minutes in medium and 107 minutes in mucus. These times-to-inactivation were unexpected since they are longer than have been observed in other studies. From this work, we demonstrate that sunlight represents an effective decontamination method but the speed of decontamination is variable based on the underlying matrix. This information has an important impact on the development of infection prevention and control protocols to reduce the spread of this deadly pathogen.

Evaluation of a commercially-available surrogate virus neutralization test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.

Taï Forest Virus Does Not Cause Lethal Disease in Ferrets.

Filoviruses are zoonotic, negative-sense RNA viruses, most of which are capable of causing severe disease in humans and nonhuman primates, often with high case fatality rates. Among these viruses, those belonging to the Ebolavirus genus-particularly Ebola virus, Sudan virus, and Bundibugyo virus-represent some of the most pathogenic to humans. Taï Forest virus (TAFV) is thought to be among the least pathogenic ebolaviruses; however, only a single non-fatal case has been documented in humans, in 1994. With the recent success of the ferret as a lethal model for a number of ebolaviruses, we set out to evaluate its suitability as a model for TAFV. Our results demonstrate that, unlike other ebolaviruses, TAFV infection in ferrets does not result in lethal disease. None of the intramuscularly inoculated animals demonstrated any overt signs of disease, whereas the intranasally inoculated animals exhibited mild to moderate weight loss during the early stage of infection but recovered quickly. Low levels of viral RNA were detected in the blood and tissues of several animals, particularly the intranasally inoculated animals, and all animals mounted a humoral immune response, with high titers of GP-specific IgG detectable as early as 14 days post-infection. These data provide additional insight into the pathogenesis of TAFV.

Comparison analysis of different swabs and transport mediums suitable for SARS-CoV-2 testing following shortages.

On March 11, 2020, the World Health Organization (WHO) assessed COVID-19, caused by SARS-CoV-2, as a pandemic. As of June 1, 2020, SARS-CoV-2 has had a documented effect of over 6 million cases world-wide, amounting to over 370,000 deaths (World Health Organization, 2020. Novel Coronavirus (COVID-19) Situation. http://https://covid19.who.int/). Consequently, the high demand for testing has resulted in a depletion of commercially available consumables, including the recommended swabs and viral transport media (VTM) required for nasopharyngeal sampling. Therefore, the potential use of unvalidated alternatives must be explored to address the global shortage of testing supplies. To tackle this issue, we evaluated the utility of different swabs and transport mediums for the molecular detection of SARS-CoV-2. This study compared the performance of six swabs commonly found in primary and tertiary health care settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6" cotton tipped applicators, and HOLOGIC Aptima) for their efficacy in testing for SARS-CoV-2. Separately, the molecular detection of SARS-CoV-2 was completed from different transport mediums (DMEM, PBS, 100 % ethanol, 0.9 % normal saline and VTM), which were kept up to three days at room temperature (RT). The results indicate that there is no meaningful difference in viral yield from different swabs and most transport mediums for the collection and detection of SARS-CoV-2, indicating swab and medium alternatives could be used if supplies run out.

Predicting Infectious Severe Acute Respiratory Syndrome Coronavirus 2 From Diagnostic Samples.

BACKGROUND: Reverse-transcription polymerase chain reaction (RT-PCR) has become the primary method to diagnose viral diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RT-PCR detects RNA, not infectious virus; thus, its ability to determine duration of infectivity of patients is limited. Infectivity is a critical determinant in informing public health guidelines/interventions. Our goal was to determine the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT), and infectivity in cell culture. METHODS: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR-confirmed positive samples and determined their ability to infect Vero cell lines. RESULTS: Ninety RT-PCR SARS-CoV-2-positive samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median tissue culture infectious dose/mL was 1780 (interquartile range, 282-8511). There was no growth in samples with a Ct > 24 or STT > 8 days. Multivariate logistic regression using positive viral culture as a binary predictor variable, STT, and Ct demonstrated an odds ratio (OR) for positive viral culture of 0.64 (95% confidence interval [CI], .49-.84; P < .001) for every 1-unit increase in Ct. Area under the receiver operating characteristic curve for Ct vs positive culture was OR, 0.91 (95% CI, .85-.97; P < .001), with 97% specificity obtained at a Ct of > 24. CONCLUSIONS: SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ct < 24 and STT < 8 days. Infectivity of patients with Ct > 24 and duration of symptoms > 8 days may be low. This information can inform public health policy and guide clinical, infection control, and occupational health decisions. Further studies of larger size are needed.

Virus-Like Particles Derived From a Virulent Strain of Pest des Petits Ruminants Virus Elicit a More Vigorous Immune Response in Mice and Small Ruminants Than Those From a Vaccine Strain.

Peste des petits ruminants (PPRs) is highly contagious, acute or subacute disease of small ruminants caused by peste des petits ruminants virus (PPRV). To date, several studies have designed and evaluated PPRV-like particles (VLPs) as a vaccine candidate for the prevention and control of PPR, with the majority of these VLPs constructed using sequences derived from a PPRV vaccine strain due to its high immunogenicity. However, because of the lack of available genetic material and certain structural proteins and/or the alteration of posttranslational glycosylation modifications, the immunogenicity of VLPs derived from a vaccine strain is not always optimal. In this study, two PPRV VLP candidates, derived from either the lineage IV Tibet/30 virulent strain or the lineage II Nigeria 75/1 vaccine strain, were generated using a baculovirus system through the coexpression of the PPRV matrix (M), hemagglutinin (H), and fusion (F) proteins in the high expression level cell line High Five. These VLPs were then used to immunize mice, goats, and sheep followed by two boosts after primary immunization. Both VLPs were found to induce a potent humoral immune response as demonstrated by the high ratio of immunoglobulin G1 (IgG1) to IgG2a. In all animals, both VLPs induced high titers of virus-neutralizing antibodies (VNAs), as well as H- and F-specific antibodies, with the Tibet/30 VLPs yielding higher antibody titers by comparison to the Nigeria 75/1 VLPs. Studies in mice also demonstrated that the Tibet/30 VLPs induced a more robust interleukin 4 and interferon γ response than the Nigeria 75/1 VLPs. Goats and sheep immunized with both VLPs exhibited a robust humoral and cell-mediated immune response. Furthermore, our results demonstrated that the VLPs derived from the virulent lineage IV Tibet/30 strain were more immunogenic, inducing a more potent and robust humoral and cell-mediated immune response in vaccinated animals by comparison to the lineage II Nigeria 75/1 vaccine strain VLPs. In addition, VNA titers were significantly higher among animals vaccinated with the Tibet/30 VLPs by comparison to the Nigeria 75/1 VLPs. Taken together, these findings suggest that VLPs derived from the virulent lineage IV Tibet/30 strain are more immunogenic by comparison to those derived from the lineage II Nigeria 75/1 vaccine strain and thus represent a promising vaccine candidate for the control and eradication of PPR.

Peste des Petits Ruminants Virus-Like Particles Induce a Potent Humoral and Cellular Immune Response in Goats.

Peste des petits ruminants is a highly contagious acute or subacute disease of small ruminants caused by the peste des petits ruminants virus (PPRV), and it is responsible for significant economic losses in animal husbandry. Vaccination represents the most effective means of controlling this disease, with virus-like particle (VLP) vaccines offering promising vaccine candidates. In this study, a PPRV VLP-based vaccine was developed using a baculovirus expression system, allowing for the simultaneous expression of the PPRV matrix (M), hemagglutinin (H), fusion (F) and nucleocapsid (N) proteins in insect cells. Immunization of mice and goats with PPRV VLPs elicited a robust neutralization response and a potent cellular immune response. Mouse studies demonstrated that VLPs induced a more robust IFN-γ response in CD4+ and CD8+ T cells than PPRV Nigeria 75/1 and recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. In addition, PPRV VLPs induced a strong Th1 class response in mice, as indicated by a high IgG2a to IgG1 ratio. Goat studies demonstrated that PPRV VLPs can induce the production of antibodies specific for F and H proteins and can also stimulate the production of virus neutralizing antibodies to the same magnitude as the PPRV Nigeria 75/1 vaccine. Higher amounts of IFN-γ in VLP-immunized animal serum suggested that VLPs also elicited a cellular immune response in goats. These results demonstrated that VLPs elicit a potent immune response against PPRV infection in small ruminants, making PPRV VLPs a potential candidate for PPRV vaccine development.

Virus inoculation and treatment regimens for evaluating anti-filovirus monoclonal antibody efficacy in vivo.

The development of monoclonal antibodies to treat disease caused by filoviruses, particularly Ebola virus, has risen steeply in recent years thanks to several key studies demonstrating their remarkable therapeutic potential. The increased drive to develop new and better monoclonal antibodies has necessarily seen an increase in animal model efficacy testing, which is critical to the pre-clinical development of any novel countermeasure. Primary and secondary efficacy testing against filoviruses typically makes use of one or more rodent models (mice, guinea pigs, and occasionally hamsters) or the more recently described ferret model, although the exact choice of model depends on the specific filovirus being evaluated. Indeed, no single small animal model exists for all filoviruses, and the use of any given model must consider the nature of that model as well as the nature of the therapeutic and the experimental objectives. Confirmatory evaluation, on the other hand, is performed in nonhuman primates (rhesus or cynomolgus macaques) regardless of the filovirus. In light of the number of different animal models that are currently used in monoclonal antibody efficacy testing, we sought to better understand how these efficacy tests are being performed by numerous different laboratories around the world. To this end, we review the animal models that are being used for antibody efficacy testing against filoviruses, and we highlight the challenge doses and routes of infection that are used. We also describe the various antibody treatment regimens, including antibody dose, route, and schedule of administration, that are used in these model systems. We do not identify any single best model or treatment regimen, and we do not advocate for field-wide protocol standardization. Instead, we hope to provide a comprehensive resource that will facilitate and enhance the continued pre-clinical development of novel monoclonal antibody therapeutics.