SARS-CoV-2 infection and transmission in the North American deer mouse.

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.

Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans.

The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events.

Marburg and Ravn Virus Infections Do Not Cause Observable Disease in Ferrets.

Ferrets are used for studying infections with wild-type Ebola virus isolates. Here, we investigated whether these animals are also susceptible to wild-type isolates of Marburg virus (MARV). Ferrets were challenged intramuscularly or intranasally with MARV strain Angola and monitored for 3 weeks. Unexpectedly, the animals neither showed observable signs of disease nor died of infection, and viremia was not detected after challenge. All animals were seropositive for MARV-specific immunoglobulin antibodies. Confirmatory studies with MARV strain Musoke and Ravn virus yielded the same outcomes. Therefore, ferrets may be of limited usefulness for studying the pathogenesis of MARV and Ravn virus infections.

Pathogenicity Comparison Between the Kikwit and Makona Ebola Virus Variants in Rhesus Macaques.

Enhanced virulence and/or transmission of West African Ebola virus (EBOV) variants, which are divergent from their Central African counterparts, are suspected to have contributed to the sizable toll of the recent Ebola virus disease (EVD) outbreak. This study evaluated the pathogenicity and shedding in rhesus macaques infected with 1 of 2 West African isolates (EBOV-C05 or EBOV-C07) or a Central African isolate (EBOV-K). All animals infected with EBOV-C05 or EBOV-C07 died of EVD, whereas 2 of 3 EBOV-K-infected animals died. The viremia level was elevated 10-fold in EBOV-C05-infected animals, compared with EBOV-C07- or EBOV-K-infected animals. More-severe lung pathology was observed in 2 of 6 EBOV-C05/C07-infected macaques. This is the first detailed analysis of the recently circulating EBOV-C05/C07 in direct comparison to EBOV-K with 6 animals per group, and it showed that EBOV-C05 but not EBOV-C07 can replicate at higher levels and cause more tissue damage in some animals. Increased virus shedding from individuals who are especially susceptible to EBOV replication is possibly one of the many challenges facing the community of healthcare and policy-making responders since the beginning of the outbreak.

An Adenovirus Vaccine Expressing Ebola Virus Variant Makona Glycoprotein Is Efficacious in Guinea Pigs and Nonhuman Primates.

A licensed vaccine against Ebola virus (EBOV) remains unavailable, despite >11 000 deaths from the 2014-2016 outbreak of EBOV disease in West Africa. Past studies have shown that recombinant vaccine viruses expressing EBOV glycoprotein (GP) are able to protect nonhuman primates (NHPs) from a lethal EBOV challenge. However, these vaccines express the viral GP-based EBOV variants found in Central Africa, which has 97.3% amino acid homology to the Makona variant found in West Africa. Our previous study showed that a recombinant adenovirus serotype 5 (Ad5)-vectored vaccine expressing the Makona EBOV GP (MakGP) was safe and immunogenic during clinical trials in China, but it is unknown whether the vaccine protects against EBOV infection. Here, we demonstrate that guinea pigs immunized with Ad5-MakGP developed robust humoral responses and were protected against exposure to guinea pig-adapted EBOV. Ad5-MakGP also elicited specific B- and T-cell immunity in NHPs and conferred 100% protection when animals were challenged 4 weeks after immunization. These results support further clinical development of this candidate and highlight the utility of Ad5-MakGP as a prophylactic measure in future outbreaks of EBOV disease.

Two-mAb cocktail protects macaques against the Makona variant of Ebola virus.

The 2014-2015 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for specific therapeutic interventions for infected patients. The human-mouse chimeric monoclonal antibody (mAb) cocktail ZMapp, previously shown to be efficacious in EBOV (variant Kikwit) lethally infected nonhuman primates (NHPs) when administration was initiated up to 5 days, was used in some patients during the outbreak. We show that a two-antibody cocktail, MIL77E, is fully protective in NHPs when administered at 50 mg/kg 3 days after challenge with a lethal dose of EBOV variant Makona, the virus responsible for the ongoing 2014-2015 outbreak, whereas a similar formulation of ZMapp protected two of three NHPs. The chimeric MIL77E mAb cocktail is produced in engineered Chinese hamster ovary cells and is based on mAbs c13C6 and c2G4 from ZMapp. The use of only two antibodies in MIL77E opens the door to a pan-ebolavirus cocktail.

Immune Response to Marburg Virus Angola Infection in Nonhuman Primates.

BACKGROUND: The 2005 outbreak of Marburg virus (MARV) infection in Angola was the most lethal MARV infection outbreak in history, with a case-fatality rate (90%) similar to that for Zaire ebolavirus (EBOV) infection. However, very little is known about the pathogenicity of MARV Angola, as few studies have been conducted to date. Therefore, the immune response was examined in MARV Angola-infected nonhuman primates. METHODS: Cynomolgus macaques were infected with MARV Angola and monitored for survival. The effect of MARV Angola on the immune system was examined by immunophenotyping whole-blood and by analyzing cytokine and chemokine levels in plasma and spleen specimens, using flow cytometry. RESULTS: The prominent clinical findings were rapid onset of disease and death (mean time after infection, 6.7 days), fever, depression, anorexia, petechial rash, and lymphopenia. Specifically, T, B, and natural killer cells were severely depleted in the blood by day 6. The typical cytokine storm was present, with levels of interferon γ, tumor necrosis factor, interleukin 6, and CCL2 rising in the blood early during infection. CONCLUSIONS: MARV Angola displayed the same virulence and disease pathology as EBOV. MARV Angola appears to cause a more rapid onset and severe outcome of infection than other MARV strains.

A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection.

As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.

VSVΔG/EBOV GP-induced innate protection enhances natural killer cell activity to increase survival in a lethal mouse adapted Ebola virus infection.

Members of the species Zaire ebolavirus cause severe hemorrhagic fever with up to a 90% mortality rate in humans. The VSVΔG/EBOV GP vaccine has provided 100% protection in the mouse, guinea pig, and nonhuman primate (NHP) models, and has also been utilized as a post-exposure therapeutic to protect mice, guinea pigs, and NHPs from a lethal challenge of Ebola virus (EBOV). EBOV infection causes rapid mortality in human and animal models, with death occurring as early as 6 days after infection, suggesting a vital role for the innate immune system to control the infection before cells of the adaptive immune system can assume control. Natural killer (NK) cells are the predominant cell of the innate immune response, which has been shown to expand with VSVΔG/EBOV GP treatment. In the current study, an in vivo mouse model of the VSVΔG/EBOV GP post-exposure treatment was used for a mouse adapted (MA)-EBOV infection, to determine the putative VSVΔG/EBOV GP-induced protective mechanism of NK cells. NK depletion studies demonstrated that mice with NK cells survive longer in a MA-EBOV infection, which is further enhanced with VSVΔG/EBOV GP treatment. NK cell mediated cytotoxicity and IFN-γ secretion was significantly higher with VSVΔG/EBOV GP treatment. Cell mediated cytotoxicity assays and perforin knockout mice experiments suggest that there are perforin-dependent and -independent mechanisms involved. Together, these data suggest that NK cells play an important role in VSVΔG/EBOV GP-induced protection of EBOV by increasing NK cytotoxicity, and IFN-γ secretion.

Immunization with vesicular stomatitis virus vaccine expressing the Ebola glycoprotein provides sustained long-term protection in rodents.

Ebola virus (EBOV) infections cause lethal hemorrhagic fever in humans, resulting in up to 90% mortality. EBOV outbreaks are sporadic and unpredictable in nature; therefore, a vaccine that is able to provide durable immunity is needed to protect those who are at risk of exposure to the virus. This study assesses the long-term efficacy of the vesicular stomatitis virus (VSV)-based vaccine (VSVΔG/EBOVGP) in two rodent models of EBOV infection. Mice and guinea pigs were first immunized with 2×10(4) or 2×10(5) plaque forming units (PFU) of VSVΔG/EBOVGP, respectively. Challenge of mice with a lethal dose of mouse-adapted EBOV (MA-EBOV) at 6.5 and 9 months after vaccination provided complete protection, and 80% (12 of 15 survivors) protection at 12 months after vaccination. Challenge of guinea pigs with a lethal dose of guinea pig-adapted EBOV (GA-EBOV) at 7, 12 and 18 months after vaccination resulted in 83% (5 of 6 survivors) at 7 months after vaccination, and 100% survival at 12 and 18 months after vaccination. No weight loss or clinical signs were observed in the surviving animals. Antibody responses were analyzed using sera from individual rodents. Levels of EBOV glycoprotein-specific IgG antibody measured immediately before challenge appeared to correlate with protection. These studies confirm that vaccination with VSVΔG/EBOVGP is able to confer long-term protection against Ebola infection in mice and guinea pigs, and support follow-up studies in non-human primates.

Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.

Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viraemia and abnormalities in blood count and blood chemistry were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clinical use.

Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory.

Containment level 4 (CL4) laboratories studying biosafety level 4 viruses are under strict regulations to conduct nonhuman primate (NHP) studies in compliance of both animal welfare and biosafety requirements. NHPs housed in open-barred cages raise concerns about cross-contamination between animals, and accidental exposure of personnel to infectious materials. To address these concerns, two NHP experiments were performed. One examined the simultaneous infection of 6 groups of NHPs with 6 different viruses (Machupo, Junin, Rift Valley Fever, Crimean-Congo Hemorrhagic Fever, Nipah and Hendra viruses). Washing personnel between handling each NHP group, floor to ceiling biobubble with HEPA filter, and plexiglass between cages were employed for partial primary containment. The second experiment employed no primary containment around open barred cages with Ebola virus infected NHPs 0.3 meters from naïve NHPs. Viral antigen-specific ELISAs, qRT-PCR and TCID50 infectious assays were utilized to determine antibody levels and viral loads. No transmission of virus to neighbouring NHPs was observed suggesting limited containment protocols are sufficient for multi-viral CL4 experiments within one room. The results support the concept that Ebola virus infection is self-contained in NHPs infected intramuscularly, at least in the present experimental conditions, and is not transmitted to naïve NHPs via an airborne route.

Sustained protection against Ebola virus infection following treatment of infected nonhuman primates with ZMAb.

Ebola virus (EBOV) is one of the most lethal filoviruses, with mortality rates of up to 90% in humans. Previously, we demonstrated 100% and 50% survival of EBOV-infected cynomologus macaques with a combination of 3 EBOV-GP-specific monoclonal antibodies (ZMAb) administered at 24 or 48 hours post-exposure, respectively. The survivors demonstrated EBOV-GP-specific humoral and cell-mediated immune responses. In order to evaluate whether the immune response induced in NHPs during the ZMAb treatment and EBOV challenge is sufficient to protect survivors against a subsequent exposure, animals that survived the initial challenge were rechallenged at 10 or 13 weeks after the initial challenge. The animals rechallenged at 10 weeks all survived whereas 4 of 6 animals survived a rechallenge at 13 weeks. The data indicate that a robust immune response was generated during the successful treatment of EBOV-infected NHPs with EBOV, which resulted in sustained protection against a second lethal exposure.

mAbs and Ad-vectored IFN-α therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms.

ZMAb is a promising treatment against Ebola virus (EBOV) disease that has been shown to protect 50% (two of four) of nonhuman primates (NHPs) when administered 2 days post-infection (dpi). To extend the treatment window and improve protection, we combined ZMAb with adenovirus-vectored interferon-α (Ad-IFN) and evaluated efficacy in EBOV-infected NHPs. Seventy-five percent (three of four) and 100% (four of four) of cynomolgus and rhesus macaques survived, respectively, when treatment was initiated after detection of viremia at 3 dpi. Fifty percent (two of four) of the cynomolgus macaques survived when Ad-IFN was given at 1 dpi, followed by ZMAb starting at 4 dpi, after positive diagnosis. The treatment was able to suppress viremia reaching ~10(5) TCID50 (median tissue culture infectious dose) per milliliter, leading to survival and robust specific immune responses. This study describes conditions capable of saving 100% of EBOV-infected NHPs when initiated after the presence of detectable viremia along with symptoms.

The million mutation project: a new approach to genetics in Caenorhabditis elegans.

We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

Monoclonal antibodies combined with adenovirus-vectored interferon significantly extend the treatment window in Ebola virus-infected guinea pigs.

Monoclonal antibodies (MAbs) are currently a promising treatment strategy against Ebola virus infection. This study combined MAbs with an adenovirus-vectored interferon (DEF201) to evaluate the efficacy in guinea pigs and extend the treatment window obtained with MAbs alone. Initiating the combination therapy at 3 days postinfection (d.p.i.) provided 100% survival, a significant improvement over survival with either treatment alone. The administration of DEF201 within 2 d.p.i. permits later MAb use, with protective efficacy observed up to 8 d.p.i.

Ebola GP-specific monoclonal antibodies protect mice and guinea pigs from lethal Ebola virus infection.

Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as pools of 3-4 MAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted EBOV. Each of the 8 MAbs (100 µg) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven MAbs were effective 2 days post-infection (dpi), with 1 MAb demonstrating partial protection 3 dpi. In the guinea pigs each MAb showed partial protection at 1 dpi, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3-4 MAbs completely protected the majority of animals, while administration at 2-3 dpi achieved 50-100% protection. This data suggests that the MAbs generated are capable of protecting both animal species against lethal Ebola virus challenge. These results indicate that MAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection.

Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies.

Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.

The use of a mobile laboratory unit in support of patient management and epidemiological surveillance during the 2005 Marburg Outbreak in Angola.

BACKGROUND: Marburg virus (MARV), a zoonotic pathogen causing severe hemorrhagic fever in man, has emerged in Angola resulting in the largest outbreak of Marburg hemorrhagic fever (MHF) with the highest case fatality rate to date. METHODOLOGY/PRINCIPAL FINDINGS: A mobile laboratory unit (MLU) was deployed as part of the World Health Organization outbreak response. Utilizing quantitative real-time PCR assays, this laboratory provided specific MARV diagnostics in Uige, the epicentre of the outbreak. The MLU operated over a period of 88 days and tested 620 specimens from 388 individuals. Specimens included mainly oral swabs and EDTA blood. Following establishing on site, the MLU operation allowed a diagnostic response in <4 hours from sample receiving. Most cases were found among females in the child-bearing age and in children less than five years of age. The outbreak had a high number of paediatric cases and breastfeeding may have been a factor in MARV transmission as indicated by the epidemiology and MARV positive breast milk specimens. Oral swabs were a useful alternative specimen source to whole blood/serum allowing testing of patients in circumstances of resistance to invasive procedures but limited diagnostic testing to molecular approaches. There was a high concordance in test results between the MLU and the reference laboratory in Luanda operated by the US Centers for Disease Control and Prevention. CONCLUSIONS/SIGNIFICANCE: The MLU was an important outbreak response asset providing support in patient management and epidemiological surveillance. Field laboratory capacity should be expanded and made an essential part of any future outbreak investigation.