Exposure Route Influences Disease Severity in the COVID-19 Cynomolgus Macaque Model.

The emergence of SARS-CoV-2 and the subsequent pandemic has highlighted the need for animal models that faithfully replicate the salient features of COVID-19 disease in humans. These models are necessary for the rapid selection, testing, and evaluation of potential medical countermeasures. Here, we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure-combined intratracheal/intranasal and small particle aerosol-in two nonhuman primate species, rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models produced suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.

Modeling the stability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on skin, currency, and clothing.

A new coronavirus (SARS-CoV-2) emerged in the winter of 2019 in Wuhan, China, and rapidly spread around the world. The extent and efficiency of SARS-CoV-2 pandemic is far greater than previous coronaviruses that emerged in the 21st Century. Here, we modeled stability of SARS-CoV-2 on skin, paper currency, and clothing to determine if these surfaces may factor in the fomite transmission dynamics of SARS-CoV-2. Skin, currency, and clothing samples were exposed to SARS-CoV-2 under laboratory conditions and incubated at three different temperatures (4°C± 2°C, 22°C± 2°C, and 37°C ± 2°C). We evaluated stability at 0 hours (h), 4 h, 8 h, 24 h, 72 h, 96 h, 7 days, and 14 days post-exposure. SARS-CoV-2 was stable on skin through the duration of the experiment at 4°C (14 days). Virus remained stable on skin for at least 96 h at 22°C and for at least 8h at 37°C. There were minimal differences between the tested currency samples. The virus remained stable on the $1 U.S.A. Bank Note for at least 96 h at 4°C while we did not detect viable virus on the $20 U.S.A. Bank Note samples beyond 72 h. The virus remained stable on both Bank Notes for at least 8 h at 22°C and 4 h at 37°C. Clothing samples were similar in stability to the currency. Viable virus remained for at least 96 h at 4°C and at least 4 h at 22°C. We did not detect viable virus on clothing samples at 37°C after initial exposure. This study confirms the inverse relationship between virus stability and temperature. Furthermore, virus stability on skin demonstrates the need for continued hand hygiene practices to minimize fomite transmission both in the general population as well as in workplaces where close contact is common.

Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than one million cases and tens of thousands of deaths. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.

Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.

Sequence Optimized Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus within the family Bunyaviridae. Infection can result in general myalgia, fever, and headache with some patients developing hemorrhagic fever with mortality rates ranging from 5% to 30%. CCHFV has a wide geographic range that includes Africa, Asia, the Middle East, and Europe with nucleotide sequence variation approaching 20% across the three negative-sense RNA genome segments. While phylogenetic clustering generally aligns with geographic origin of individual strains, distribution can be wide due to tick/CCHFV dispersion via migrating birds. This sequence diversity negatively impacts existing molecular diagnostic assays, leading to false negative diagnostic results. Here, we updated a previously developed CCHFV real-time reverse transcription polymerase chain reaction (RT-PCR) assay to include strains not detected using that original assay. Deep sequencing of eight different CCHFV strains, including three that were not detectable using the original assay, identified sequence variants within this assay target region. New primers and probe based on the sequencing results and newly deposited sequences in GenBank greatly improved assay sensitivity and inclusivity with the exception of the genetically diverse strain AP92. For example, we observed a four log improvement in IbAr10200 detection with a new limit of detection of 256 PFU/mL. Subsequent comparison of this assay to another commonly used CCHFV real-time RT-PCR assay targeting a different region of the viral genome showed improved detection, and both assays could be used to mitigate CCHFV diversity for diagnostics. Overall, this work demonstrated the importance of continued viral sequencing efforts for robust diagnostic assay development.

Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope.

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter.

Corning HYPERFlask® for viral amplification and production of diagnostic reagents.

Viral preparations are essential components in diagnostic research and development. The production of large quantities of virus traditionally is done by infecting numerous tissue culture flasks or roller bottles, which require large incubators and/or roller bottle racks. The Corning HYPERFlask® is a multilayer flask that uses a gas permeable film to provide gas exchange between the cells and culture medium and the atmospheric environment. This study evaluated the suitability of the HYPERFlask for production of Lassa, Ebola, Bundibugyo, Reston, and Marburg viruses and compared it to more traditional methods using tissue culture flasks and roller bottles. The HYPERFlask produced cultures were equivalent in virus titer and indistinguishable in immunodiagnostic assays. The use of the Corning HYPERFlask for viral production is a viable alternative to traditional tissue culture flasks and roller bottles. HYPERFlasks allow for large volumes of virus to be produced in a small space without specialized equipment.

Evaluation of ViroCyt® Virus Counter for rapid filovirus quantitation.

Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM) for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR). The ViroCyt® Virus Counter (VC) 2100 (ViroCyt, Boulder, CO, USA) is a flow-based instrument capable of quantifying virus particles in solution. Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.