A biosafety level-2 mouse model for studying betacoronavirus-induced acute lung damage and systemic manifestations

The emergence of life-threatening zoonotic diseases caused by betacoronavirus, including the ongoing COVID-19 pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus MHV-3 develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired the respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic and a high virus burden could be detected in multiple organs alongside with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the pro-inflammatory cytokines IL-1β, IL-6, IL-12, IFN-γ, and TNF, thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line, TNF blockage also diminished the infection-mediated release of pro-inflammatory cytokines and virus replication of human epithelial lung cells infected with SARS-CoV-2. Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower cost, biosafety level-2 in vivo platform for evaluating the respiratory and multi-organ involvement of betacoronavirus infections. Importance Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race towards the characterization of SARS-CoV-2 infection in other animals (e.g. hamsters, cats, ferrets, bats, and monkeys) as well as the adaptation of the mouse model, by either modifying the host or the virus. In the present study, we utilized the natural pathogen of mice MHV as a prototype to model betacoronavirus-induced acute lung injure and multi—organ involvement under biosafety level 2 condition. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops a severe disease which includes acute lung damage and respiratory distress preceding systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Rapid and Flexible Platform To Assess Anti-SARS-CoV-2 Antibody Neutralization and Spike Protein-Specific Antivirals

Assays for rapid biosafety level 2 (BSL2) evaluation of neutralizing properties of antibodies acquired via natural infection or through vaccination is urgently needed. Here, we propose a combinatorial approach in which sera are screened for SARS-CoV-2 spike protein (S) binding using a cell-based immunofluorescent (CBI) assay, and positive samples are further evaluated in a pseudotyped viral multicycle infection-mimicking protocol under BSL2 conditions.

Development and Validation of Two In-house, Low-Cost SARS-CoV-2 Detection Assays

Background One major challenge for detecting the virus that causes COVID19 is commercial SARSCoV2 testing kit or reagent availability. To allow every laboratory or hospital access to an inhouse assay, we developed two low cost SARSCoV2 detection assay protocols using inhouse primers and reagents equipment on hand in most biology or diagnostic laboratories a SYBR Green based RTPCR and PCR assays. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated alternative RNA extraction protocols. Methods SARSCoV2 genome sequences deposited into the GISAID database were retrieved to design and synthesize inhouse primers. Forty patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocols. Both assays used TRIzol and heat-processing techniques to extract RNA from patient samples and to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results The sensitivity and specificity of the primers were evaluated using samples previously confirmed positive for SARSCoV2. The positive amplicons were sequenced to confirm the results. The assay protocols were developed, and the specificity of each PCR product was confirmed using melting curve analyses. The most accurate heat processing technique for primers with short amplicon lengths was 95C for 15 mins. Of 40 samples, both the SYBR Green based quantitative RTPCR assay and the PCR assay detected SARSCoV2 target genes in 28 samples, with no false positive or false-negative results. These findings were concordant with those of the diagnostic laboratory that tested the same samples using a Rotor Gene PCR cycler with an Altona Diagnostics SARSCoV2 kit (R2=0.889). Conclusions These approaches are reliable, repeatable, specific, sensitive, simple, and low cost tools for the detection of SARSCoV2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or cost ineffective.

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories

Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different levels of biosafety protection. Protocol originally designed for a biosafety level 2 facility was modified for biosafety level 3 facility and infection parameters were compared. Particularly, specially designed plastic containers were used for blood feeding; feeders were placed outside the sand fly cage, on the top of the mesh; feeding was performed inside the climatic chamber; separation of engorged females was done in Petri dishes kept on ice; engorged females were kept in the cardboard containers until dissection. All experiments, conducted in both laboratories, resulted in fully developed late stage infections with high number of parasites and colonization of the stomodeal valve. We demonstrated that protocol originally designed for biosafety level 2 facilities can be successfully modified for other biosafety facilities, depending on the special requirements of the individual institution/laboratory.

Validating Autoclave Cycles for Carcass Disposal in Animal Biosafety Level 2/3 Containment Laboratories

Introduction:Animal carcasses differ in composition from other types of solid waste, and through prior testing it was determined that cycle parameters applied to general, solid biohazardous waste did not ensure proper sterilization of ferret carcasses.Objectives:The goals of this study were to develop and validate an autoclave cycle that would ensure the decontamination of infectious animal carcasses before removal from an animal biosafety level 2/3 containment suite for downstream disposal and to test different ways to prepare and package animal carcasses for autoclaving.Methods:Intact ferret carcasses were implanted with biological indicators, and the carcasses were placed in biohazard bags, then into metal pans. To test the efficacy of the autoclave cycle on larger biomasses, 1, 2, or 4 ferret carcasses were placed in a biohazard bag. A total of 4 carcasses were placed in each pan. An autoclave cycle was created to begin the study. After initial tests, minor modifications to the initial test cycle parameters were made, and a new cycle was validated for ferret carcasses up to 2 kg each. Parameters for the validated cycle were as follows: sterilization time 240 minutes, temperature 125°C, 5 prevacuum pulses, and chamber pressure 15 psi.Results:The results of this study indicate that an extended sterilization time is required to successfully decontaminate animal carcasses compared with regular, solid, and biohazardous waste.Conclusions:This study demonstrates that it is possible to sterilize multiple intact ferret carcasses per load under validated autoclave cycle conditions.