Search

The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method.

The 1918 H1N1 influenza pandemic was among the most severe in history, taking the lives of approximately 50 million people worldwide, and novel prophylactic vaccines are urgently needed to prevent another pandemic. Given that macaques are physiologically relevant preclinical models of human immunology that have advanced the clinical treatment of infectious diseases, a lethal pandemic influenza challenge model would provide a stringent platform for testing new influenza vaccine concepts. To this end, we infected rhesus macaques and Mauritian cynomolgus macaques with highly pathogenic 1918 H1N1 influenza virus and assessed pathogenesis and disease severity. Despite infection with a high dose of 1918 influenza delivered via multiple routes, rhesus macaques demonstrated minimal signs of disease, with only intermittent viral shedding. Cynomolgus macaques infected via intrabronchial instillation demonstrated mild symptoms, with disease severity depending on the infection dose. Cynomolgus macaques infected with a high dose of 1918 influenza delivered via multiple routes experienced moderate disease characterized by consistent viral shedding, pulmonary infiltrates, and elevated inflammatory cytokine levels. However, 1918 influenza was uniformly nonlethal in these two species, demonstrating that this isolate is insufficiently pathogenic in rhesus and Mauritian cynomolgus macaques to support testing novel prophylactic influenza approaches where protection from severe disease combined with a lethal outcome is desired as a highly stringent indication of vaccine efficacy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent responsible for the ongoing global pandemic. With over 500 million cases and more than 6 million deaths reported globally, the need for access to effective vaccines is clear. An ideal SARS-CoV-2 vaccine will prevent pathology in the lungs and prevent virus replication in the upper respiratory tract, thus reducing transmission. Here, we assessed the efficacy of an adjuvanted SARS-CoV-2 S1 subunit vaccine, called COVAC-1, in an African green monkey (AGM) model. AGMs immunized and boosted with COVAC-1 were protected from SARS-CoV-2 challenge compared to unvaccinated controls based on reduced pathology and reduced viral RNA levels and infectious virus in the respiratory tract. Both neutralizing antibodies and antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) were observed in vaccinated animals prior to the challenge. COVAC-1 induced effective protection, including in the upper respiratory tract, thus supporting further development and utility for determining the mechanism that confers this protection.AUTHOR SUMMARYVaccines that can prevent the onward transmission of SARS-CoV-2 and prevent disease are highly desirable. Whether this can be accomplished without mucosal immunization by a parenterally administered subunit vaccine is not well established. Here we demonstrate that following two vaccinations, a protein subunit vaccine containing the S1 portion of the SARS-CoV-2 spike glycoprotein and the novel adjuvant TriAdj significantly reduces the amount of virus in the lungs and also mediates rapid clearance of the virus from the upper respiratory tract. Further support of the effectiveness of COVAC-1 was the observation of reduced pathology in the lungs and viral RNA being largely absent from tissues, blood, and rectal swabs. Thus COVAC-1 appears promising at mediating protection in both the upper and lower respiratory tract and may be capable of reducing subsequent transmission of SARS-CoV-2. Further investigation into the mechanism of protection in the upper respiratory tract and the initial immune response that supports this would be warranted.

Increasing cases of SARS-CoV-2 breakthrough infections from immunization with current spike protein-based COVID-19 vaccines highlight the need to develop alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins based on a novel vaccinia virus (VACV) ACAM2000 platform (rACAM2000). In this platform, the vaccinia virus host range and immunoregulatory gene E3L was deleted to make the virus attenuated and to enhance innate immune responses, and another host range gene, K3L, was replaced with a poxvirus ortholog gene, taterapox virus 037 (TATV037), to make virus replication competent in both hamster and human cells. Following a single intramuscular immunization, the rACAM2000 coexpressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and shorter recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titer, and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate, and further studies will investigate if the rACAM2000 vaccine candidate can induce a long-lasting immunity against infection by SARS-CoV-2 variants of concern.

SUMMARYIncreasing cases of SARS-CoV-2 breakthrough infections from immunization with predominantly spike protein based COVID-19 vaccines highlight the need for alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins in a novel vaccinia virus ACAM2000 platform (rACAM2000). Following a single intramuscular immunization, the rACAM2000 co-expressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and quicker recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titre and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that the rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate and further studies will investigate if the rACAM2000 vaccine candidate can induce a long lasting immunity against infection of SARS-CoV-2 variants of concern.

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown., Deer mice are natural hosts for a number of human pathogens. Here, Griffin et al. report that intranasal exposure of the North American deer mouse to SARS-CoV-2 results in virus replication and shedding, despite causing only mild or asymptomatic illness. Additionally, infected deer mice can transmit SARS-CoV-2 to naïve deer mice.

The emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant pandemic of coronavirus disease 2019 (COVID-19) has led to over one hundred million confirmed infections, greater than three million deaths, and severe economic and social disruption. Animal models of SARS-CoV-2 are critical tools for the pre-clinical evaluation of antivirals, vaccines, and candidate therapeutics currently under urgent development to curb COVID-19-associated morbidity and mortality. The golden (Syrian) hamster model of SARS-CoV-2 infection recapitulates key characteristics of severe COVID-19, including high-titer viral replication in the upper and lower respiratory tract and the development of pathogenic lesions in the lungs. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in golden hamsters. We report that delivery of SARS-CoV-2 primarily to the nasal passages (low-volume intranasal), the upper and lower respiratory tract (high-volume intranasal), or the digestive tract (intragastric) results in comparable viral titers in the lung tissue and similar levels of viral shedding during acute infection. However, low-volume intranasal exposure results in milder weight loss during acute infection while intragastric exposure leads to a diminished capacity to regain body weight following the period of acute illness. Further, we examined both sex and age differences in response to SARS-CoV-2 infection. Male hamsters, and to a greater extent older male hamsters, display an impaired capacity to recover from illness and a delay in viral clearance compared to females. Lastly, route of exposure, sex, and age were found to influence the nature of the host inflammatory cytokine response, but they had a minimal effect on both the quality and durability of the humoral immune response as well as the susceptibility of hamsters to SARS-CoV-2 re-infection. Together, these data indicate that the route of exposure, sex, and age have a meaningful impact SARS-CoV-2 pathogenesis in hamsters and that these variables should be considered when designing pre-clinical challenge studies.

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs, but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19., Graphical Abstract

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

The zoonotic spillover of the pandemic SARS-coronavirus 2 (SARS-CoV-2) from an animal reservoir, currently presumed to be the Chinese horseshoe bat, into a naïve human population has rapidly resulted in a significant global public health emergency. Worldwide circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive non-domesticated animals, potentially seeding new host reservoir species and geographic regions in which bat SARS-like coronaviruses have not historically been endemic. Here we report that North American deer mice (Peromyscus maniculatus) and some closely related members of theCricetidaefamily of rodents possess key amino acid residues within the angiotensin-converting enzyme 2 (ACE2) receptor known to confer SARS-CoV-2 spike protein binding.Peromyscusrodent species are widely distributed across North America and are the primary host reservoirs of several emerging pathogens that repeatedly spill over into humans includingBorrelia burgdorferi, the causative agent of Lyme disease, deer tick virus, and Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome (HPS). We demonstrate that adult deer mice are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment ofPeromyscusrodents as a North American reservoir for SARS-CoV-2 is unknown. Nevertheless, efforts to monitor wild, peri-domesticPeromyscusrodent populations are likely warranted as the SARS-CoV-2 pandemic progresses.

Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease, however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions., Graphical abstract, Immunology; Virology

BackgroundThere are currently limited data for the use of specific antiviral therapies for the treatment of Ebola virus disease (EVD). While there is anecdotal evidence that supportive care may be effective, there is a paucity of direct experimental data to demonstrate a role for supportive care in EVD. We studied the impact of ICU-level supportive care interventions including fluid resuscitation, vasoactive medications, blood transfusion, hydrocortisone, and ventilator support on the pathophysiology of EVD in rhesus macaques infected with a universally lethal dose of Ebola virus strain Makona C07.MethodsFour NHPs were infected with a universally lethal dose Ebola virus strain Makona, in accordance with the gold standard lethal Ebola NHP challenge model. Following infection, the following therapeutic interventions were employed: continuous bedside supportive care, ventilator support, judicious fluid resuscitation, vasoactive medications, blood transfusion, and hydrocortisone as needed to treat cardiovascular compromise. A range of physiological parameters were continuously monitored to gage any response to the interventions.ResultsAll four NHPs developed EVD and demonstrated a similar clinical course. All animals reached a terminal endpoint, which occurred at an average time of 166.5 ± 14.8 h post-infection. Fluid administration may have temporarily blunted a rise in lactate, but the effect was short lived. Vasoactive medications resulted in short-lived improvements in mean arterial pressure. Blood transfusion and hydrocortisone did not appear to have a significant positive impact on the course of the disease.ConclusionsThe model employed for this study is reflective of an intramuscular infection in humans (e.g., needle stick) and is highly lethal to NHPs. Using this model, we found that the animals developed progressive severe organ dysfunction and profound shock preceding death. While the overall impact of supportive care on the observed pathophysiology was limited, we did observe some time-dependent positive responses. Since this model is highly lethal, it does not reflect the full spectrum of human EVD. Our findings support the need for continued development of animal models that replicate the spectrum of human disease as well as ongoing development of anti-Ebola therapies to complement supportive care.

Background: The 2014–2016 Ebola outbreak in West Africa recapitulated that nosocomial spread of Ebola virus could occur and that health care workers were at particular risk including notable cases in Europe and North America. These instances highlighted the need for centers to better prepare for potential Ebola virus cases, including understanding how the virus spreads and which interventions pose the greatest risk. Methods: We created a fully equipped intensive care unit (ICU), within a Biosafety Level 4 (BSL4) laboratory, and infected multiple sedated non-human primates (NHPs) with Ebola virus. While providing bedside care, we sampled blood, urine, and gastric residuals, as well as buccal, ocular, nasal, rectal, and skin swabs, to assess the risks associated with routine care. We also assessed the physical environment at end-point. Results: Although viral RNA was detectable in blood as early as three days post-infection, it was not detectable in the urine, gastric fluid, or swabs until late-stage disease. While droplet spread and fomite contamination were present on a few of the surfaces that were routinely touched while providing care in the ICU for the infected animal, these may have been abrogated through good routine hygiene practices. Conclusions: Overall this study has helped further our understanding of which procedures may pose the highest risk to healthcare providers and provides temporal evidence of this over the clinical course of disease.

Pre-mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre-mRNA by splicing factors, histone deacetylases (HDACs) and K-acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA-MB-231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA-MB-231 cells. We show that H3K4me3 steady-state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA-MB-231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre-mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre-mRNA. J. Cell. Physiol. 231: 2196-2204, 2016. © 2016 Wiley Periodicals, Inc.

Highlights • High demand for diagnostic testing for COVID-19 has depleted commercially available consumables for diagnostic testing. • Alternative swabs that are not approved for Nasopharyngeal sampling work well for COVID-19 diagnostics. • Alternative fluids that are readily available in hospital settings can function as alternatives to Viral Transport Media. • 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., 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.

Pre-mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre-mRNA by splicing factors, histone deacetylases (HDACs) and K-acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA-MB-231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA-MB-231 cells. We show that H3K4me3 steady-state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA-MB-231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre-mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre-mRNA. J. Cell. Physiol. 231: 2196-2204, 2016. © 2016 Wiley Periodicals, Inc.

Table S1. Time to death in rhesus macaques infected with EBOV variant Makona C07 while cared for in a caged setting. (DOCX 13 kb)

Table S1. Time to death in rhesus macaques infected with EBOV variant Makona C07 while cared for in a caged setting. (DOCX 13 kb)