Developing transmissible vaccines for animal infections.

Intrinsically safe designs and a staged transparent development process will be essential.

Infection with mpox virus via the genital mucosae increases shedding and transmission in the multimammate rat (Mastomys natalensis).

The 2022 mpox virus (MPXV) outbreak was sustained by human-to-human transmission; however, it is currently unclear which factors lead to sustained transmission of MPXV. Here we present Mastomys natalensis as a model for MPXV transmission after intraperitoneal, rectal, vaginal, aerosol and transdermal inoculation with an early 2022 human outbreak isolate (Clade IIb). Virus shedding and tissue replication were route dependent and occurred in the presence of self-resolving localized skin, lung, reproductive tract or rectal lesions. Mucosal inoculation via the rectal, vaginal and aerosol routes led to increased shedding, replication and a pro-inflammatory T cell profile compared with skin inoculation. Contact transmission was higher from rectally inoculated animals. This suggests that transmission might be sustained by increased susceptibility of the anal and genital mucosae for infection and subsequent virus release.

An atlas of gross and histologic lesions and immunohistochemical immunoreactivity during the temporal progression of aerosolized Lassa virus induced hemorrhagic fever in cynomolgus macaques.

Lassa virus (LASV) causes an acute multisystemic hemorrhagic fever in humans known as Lassa fever, which is endemic in several African countries. This manuscript focuses on the progression of disease in cynomolgus macaques challenged with aerosolized LASV and serially sampled for the development and progression of gross and histopathologic lesions. Gross lesions were first noted in tissues on day 6 and persisted throughout day 12. Viremia and histologic lesions were first noted on day 6 commencing with the pulmonary system and hemolymphatic system and progressing at later time points to include all systems. Immunoreactivity to LASV antigen was first observed in the lungs of one macaque on day 3 and appeared localized to macrophages with an increase at later time points to include immunoreactivity in all organ systems. Additionally, this manuscript will serve as a detailed atlas of histopathologic lesions and disease progression for comparison to other animal models of aerosolized Arenaviral disease.

Bivalent VSV Vectors Mediate Rapid and Potent Protection from Andes Virus Challenge in Hamsters.

Orthohantaviruses may cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Andes virus (ANDV) is the only orthohantavirus associated with human-human transmission. Therefore, emergency vaccination would be a valuable public health measure to combat ANDV-derived infection clusters. Here, we utilized a promising vesicular stomatitis virus (VSV)-based vaccine to advance the approach for emergency applications. We compared monovalent and bivalent VSV vectors containing the Ebola virus (EBOV), glycoprotein (GP), and ANDV glycoprotein precursor (GPC) for protective efficacy in pre-, peri- and post-exposure immunization by the intraperitoneal and intranasal routes. Inclusion of the EBOV GP was based on its favorable immune cell targeting and the strong innate responses elicited by the VSV-EBOV vaccine. Our data indicates no difference of ANDV GPC expressing VSV vectors in pre-exposure immunization independent of route, but a potential benefit of the bivalent VSVs following peri- and post-exposure intraperitoneal vaccination.

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Histopathologic Characterization of Experimental Peracute SARS-CoV-2 Infection in the Syrian Hamster.

Coronavirus Infectious Disease 2019 (COVID-19) initiated a global pandemic that thus far has resulted in the death of over 6.5 million people internationally. Understanding the viral tropism during the initial, subclinical phase of infection is critical to develop targeted vaccines and therapeutics. With the continued emergence of variants of concern, particularly those that appear to have a tropism for the upper respiratory tract, understanding the complete pathogenesis is critical to develop more effective interventions. Thus far, the Syrian hamster has served as the most consistent small animal model of SARS-CoV-2 infection for mild to moderate respiratory disease. Herein, we utilize histopathology and immunohistochemistry to characterize the peracute phase of disease initiating at 6-h-post-inoculation in the intranasal inoculation route Syrian hamster model. Inflammation and viral replication initiates in the respiratory epithelium of nasal turbinates as early as 12-h-post-inoculation and moves caudally through the nasal cavity by 36-h-post inoculation. Lower respiratory involvement can be detected as early as 12-h-post inoculation in the intranasal inoculated hamster model. These data highlight the importance of rostral nasal cavity sampling at early timepoints for detection of SARS-CoV-2 in the Syrian hamster model.

Isolation and genome sequencing of cytomegaloviruses from Natal multimammate mice (Mastomys natalensis).

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

Combined molnupiravir-nirmatrelvir treatment improves the inhibitory effect on SARS-CoV-2 in macaques.

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have recently been approved as monotherapy for use in high-risk patients with COVID-19. As preclinical data are only available for rodent and ferret models, here we assessed the efficacy of MK-4482 and PF-07321332 alone and in combination against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Macaques were infected with the SARS-CoV-2 Delta variant and treated with vehicle, MK-4482, PF-07321332, or a combination of MK-4482 and PF-07321332. Clinical exams were performed at 1, 2, and 4 days postinfection to assess disease and virological parameters. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs, resulting in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated in the closest COVID-19 surrogate model of human infection.

SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta.

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Because of confounding factors in the human population, such as preexisting immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were up-regulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggest that, in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.

Combined Molnupiravir and Nirmatrelvir Treatment Improves the Inhibitory Effect on SARS-CoV-2 in Rhesus Macaques.

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have each recently been approved as monotherapy for use in high risk COVID-19 patients. As preclinical data are only available for rodent and ferret models, we originally assessed the efficacy of MK-4482 and PF-07321332 alone and then in combination Against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs. Combined treatment resulted in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated here in the closest COVID-19 surrogate model. One Sentence Summary: The combination of molnupiravir and nirmatrelvir inhibits SARS-CoV-2 replication and shedding more effectively than individual treatments in the rhesus macaque model.

Temporal analysis of Lassa virus infection and transmission in experimentally infected Mastomys natalensis.

Little is known about the temporal patterns of infection and transmission of Lassa virus (LASV) within its natural reservoir (Mastomys natalensis). Here, we characterize infection dynamics and transmissibility of a LASV isolate (Soromba-R) in adult lab-reared M. natalensis originating from Mali. The lab-reared M. natalenesis proved to be highly susceptible to LASV isolates from geographically distinct regions of West Africa via multiple routes of exposure, with 50% infectious doses of < 1 TCID50. Postinoculation, LASV Soromba-R established a systemic infection with no signs of clinical disease. Viral RNA was detected in all nine tissues examined with peak concentrations detected between days 7 and 14 postinfection within most organs. There was an overall trend toward clearance of virus within 40 days of infection in most organs. The exception is lung specimens, which retained positivity throughout the course of the 85-day study. Direct (contact) and indirect (fomite) transmission experiments demonstrated 40% of experimentally infected M. natalensis were capable of transmitting LASV to naïve animals, with peak transmissibility occurring between 28 and 42 days post-inoculation. No differences in patterns of infection or transmission were noted between male and female experimentally infected rodents. Adult lab-reared M. natalensis are highly susceptible to genetically distinct LASV strains developing a temporary asymptomatic infection associated with virus shedding resulting in contact and fomite transmission within a cohort.

SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta.

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Due to confounding factors in the human population, such as pre-existing immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were upregulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggests that in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

Molnupiravir inhibits SARS-CoV-2 variants including Omicron in the hamster model.

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC), which contains a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here, we assessed the efficacy of MK-4482 against the earlier Alpha, Beta, and Delta VOCs and Omicron in the hamster COVID-19 model. Omicron replication and associated lung disease in vehicle-treated hamsters was reduced compared with replication and lung disease associated with earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of hamsters infected with Alpha, Beta, or Delta VOCs. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious titers compared with viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

Evaluation of viral load in patients with Ebola virus disease in Liberia: a retrospective observational study.

BACKGROUND: Viral load in patients with Ebola virus disease affects case fatality rate and is an important parameter used for diagnostic cutoffs, stratification in randomised controlled trials, and epidemiological studies. However, viral load in Ebola virus disease is currently estimated using numerous different assays and protocols that were not developed or validated for this purpose. Here, our aim was to conduct a laboratory-based re-evaluation of the viral loads of a large cohort of Liberian patients with Ebola virus disease and analyse these data in the broader context of the west Africa epidemic. METHODS: In this retrospective observational study, whole blood samples from patients at the Eternal Love Winning Africa Ebola treatment unit (Monrovia, Liberia) were re-extracted with an optimised protocol and analysed by droplet digital PCR (ddPCR) using a novel semi-strand specific assay to measure viral load. To allow for more direct comparisons, the ddPCR viral loads were also back-calculated to cycle threshold (Ct) values. The new viral load data were then compared with the Ct values from the original diagnostic quantitative RT-PCR (qRT-PCR) testing to identify differing trends and discrepancies. FINDINGS: Between Aug 28 and Dec 18, 2014, 727 whole blood samples from 528 individuals were collected. 463 (64%) were first-draw samples and 409 (56%) were from patients positive for Ebola virus (EBOV), species Zaire ebolavirus. Of the 307 first-draw EBOV-positive samples, 127 (41%) were from survivors and 180 (59%) were from non-survivors; 155 (50%) were women, 145 (47%) were men, and seven (2%) were not recorded, and the mean age was 29·3 (SD 15·0) years for women and 31·8 (SD 14·8) years for men. Survivors had significantly lower mean viral loads at presentation than non-survivors in both the reanalysed dataset (5·61 [95% CI 5·34-5·87] vs 7·19 [6·99-7·38] log10 EBOV RNA copies per mL; p<0·0001) and diagnostic dataset (Ct value 28·72 [27·97-29·47] vs 26·26 [25·72-26·81]; p<0·0001). However, the prognostic capacity of viral load increased with the reanalysed dataset (odds ratio [OR] of death 8·06 [95% CI 4·81-13·53], p<0·0001 for viral loads above 6·71 log10 EBOV RNA copies per mL vs OR of death 2·02 [1·27-3·20], p=0·0028 for Ct values below 27·37). Diagnostic qRT-PCR significantly (p<0·0001) underestimated viral load in both survivors and non-survivors (difference in diagnostic Ct value minus laboratory Ct value of 1·79 [95% CI 1·16-2·43] for survivors and 5·15 [4·43-5·87] for non-survivors). Six samples that were reported negative by diagnostic testing were found to be positive upon reanalysis and had high viral loads. INTERPRETATION: Inaccurate viral load estimation from diagnostic Ct values is probably multifactorial; however, unaddressed PCR inhibition from tissue damage in patients with fulminant Ebola virus disease could largely account for the discrepancies observed in our study. Testing protocols for Ebola virus disease require further standardisation and validation to produce accurate viral load estimates, minimise false negatives, and allow for reliable epidemiological investigation. FUNDING: Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

SARS-CoV-2 reinfection prevents acute respiratory disease in Syrian hamsters but not replication in the upper respiratory tract.

Human cases of SARS-CoV-2 reinfection have been documented throughout the pandemic, but are likely under-reported. In the current study, we use the Syrian hamster SARS-CoV-2 model to assess reinfection with homologous WA1 and heterologous B.1.1.7 (Alpha) and B.1.351 (Beta) SARS-CoV-2 variants over time. Upon primary infection with SARS-CoV-2 WA1, hamsters rapidly develop a strong and long-lasting humoral immune response. After reinfection with homologous and heterologous SARS-CoV-2 variants, this immune response protects hamsters from clinical disease, virus replication in the lower respiratory tract, and acute lung pathology. However, reinfection leads to SARS-CoV-2 replication in the upper respiratory tract with the potential for virus shedding. Our findings indicate that reinfection results in restricted SARS-CoV-2 replication despite substantial levels of humoral immunity, denoting the potential for transmission through reinfected asymptomatic individuals.

Molnupiravir (MK-4482) is efficacious against Omicron and other SARS-CoV-2 variants in the Syrian hamster COVID-19 model.

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the Syrian hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

UK B.1.1.7 (Alpha) variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, we compared African green monkeys infected intranasally with either the UK B.1.1.7 (Alpha) variant or its contemporary D614G progenitor. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases.

Continuing Orthohantavirus Circulation in Deer Mice in Western Montana.

Hantavirus pulmonary syndrome (HPS) is an often-fatal disease caused by New World hantaviruses, such as Sin Nombre orthohantavirus (SNV). In the US, >800 cases of HPS have been confirmed since it was first discovered in 1993, of which 43 were reported from the state of Montana. The primary cause of HPS in the US is SNV, which is primarily found in the reservoir host Peromyscus maniculatus (deer mouse). The reservoir host covers most of the US, including Montana, where multiple studies found SNV in local deer mouse populations. This study aimed to check the prevalence of SNV in the deer mice at popular recreation sites throughout the Bitterroot Valley in Western Montana as compared to previous studies in western Montana. We found high prevalence (up to 20%) of deer mice positive for SNV RNA in the lungs. We were unable to obtain a SNV tissue culture isolate from the lungs but could passage SNV from lung tissue into naïve deer mice. Our findings demonstrate continuing circulation of SNV in western Montana.

Ebola Virus IgG Seroprevalence in Southern Mali.

Mali had 2 reported introductions of Ebola virus (EBOV) during the 2013-2016 West Africa epidemic. Previously, no evidence for EBOV circulation was reported in Mali. We performed an EBOV serosurvey study in southern Mali. We found low seroprevalence in the population, indicating local exposure to EBOV or closely related ebola viruses.