Combined Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 Reduces Molnupiravir-Induced Mutagenicity and Prevents Selection for Nirmatrelvir/Ritonavir Resistance Mutations.

We investigated the mutation profiles of severe acute respiratory syndrome coronavirus 2 in samples collected from a molnupiravir and nirmatrelvir/ritonavir combination therapy in macaques. We found that molnupiravir induced several nirmatrelvir resistance mutations at low abundance that were not further selected in combination therapy. Coadministration of nirmatrelvir/ritonavir lowered the magnitude of the mutagenetic effect of molnupiravir.

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.

Lassa viral dynamics in non-human primates treated with favipiravir or ribavirin.

Lassa fever is an haemorrhagic fever caused by Lassa virus (LASV). There is no vaccine approved against LASV and the only recommended antiviral treatment relies on ribavirin, despite limited evidence of efficacy. Recently, the nucleotide analogue favipiravir showed a high antiviral efficacy, with 100% survival obtained in an otherwise fully lethal non-human primate (NHP) model of Lassa fever. However the mechanism of action of the drug is not known and the absence of pharmacokinetic data limits the translation of these results to the human setting. Here we aimed to better understand the antiviral effect of favipiravir by developping the first mathematical model recapitulating Lassa viral dynamics and treatment. We analyzed the viral dynamics in 24 NHPs left untreated or treated with ribavirin or favipiravir, and we put the results in perspective with those obtained with the same drugs in the context of Ebola infection. Our model estimates favipiravir EC50 in vivo to 2.89 µg.mL-1, which is much lower than what was found against Ebola virus. The main mechanism of action of favipiravir was to decrease virus infectivity, with an efficacy of 91% at the highest dose. Based on our knowledge acquired on the drug pharmacokinetics in humans, our model predicts that favipiravir doses larger than 1200 mg twice a day should have the capability to strongly reduce the production infectious virus and provide a milestone towards a future use in humans.

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.

Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers.

BACKGROUND: As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. METHODS: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time. RESULTS: PPMO designed to base-pair with sequence in the 5' terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4-6 log10 in cell cultures at 48-72 h post-infection, in a non-toxic and dose-responsive manner. CONCLUSIONS: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

Multiple DNA viruses identified in multimammate mouse (Mastomys natalensis) populations from across regions of sub-Saharan Africa.

The multimammate mouse (Mastomys natalensis; M. natalensis) serves as the main reservoir for the zoonotic arenavirus Lassa virus (LASV), and this has led to considerable investigation into the distribution of LASV and other related arenaviruses in this host species. In contrast to the situation with arenaviruses, the presence of other viruses in M. natalensis remains largely unexplored. In this study, herpesviruses and polyomaviruses were identified and partially characterized by PCR methods, sequencing, and phylogenetic analysis. In tissues sampled from M. natalensis populations in Côte d'Ivoire and Mali, six new DNA viruses (four betaherpesviruses, one gammaherpesvirus and one polyomavirus) were identified. Phylogenetic analysis based on glycoprotein B amino acid sequences showed that the herpesviruses clustered with cytomegaloviruses and rhadinoviruses of multiple rodent species. The complete circular genome of the newly identified polyomavirus was amplified by PCR. Amino acid sequence analysis of the large T antigen or VP1 showed that this virus clustered with a known polyomavirus from a house mouse (species Mus musculus polyomavirus 1). These two polyomaviruses form a clade with other rodent polyomaviruses, and the newly identified virus represents the third known polyomavirus of M. natalensis. This study represents the first identification of herpesviruses and the discovery of a novel polyomavirus in M. natalensis. In contrast to arenaviruses, we anticipate that these newly identified viruses represent a low zoonotic risk due to the normally highly restricted specificity of members of these two DNA virus families to their individual mammalian host species.

The Effect of Plasmodium on the Outcome of Ebola Virus Infection in a Mouse Model.

Following the Ebola virus epidemic in West Africa, several studies investigated whether there was an effect of Plasmodium coinfection on survival in Ebola virus (EBOV) disease patients. Different effects of coinfection were found in different patient cohorts. To determine whether an effect of Plasmodium coinfection on EBOV survival may exist, we modeled coinfection of Plasmodium yoelii and mouse-adapted EBOV (MA-EBOV) in CD1 mice. Subsequent infection with MA-EBOV at different time points after P. yoelii infection did not have any significant effect on survival.

Human Polyclonal Antibodies Produced by Transchromosomal Cattle Provide Partial Protection Against Lethal Zaire Ebolavirus Challenge in Rhesus Macaques.

Antibody therapy has been used to treat a variety of diseases and the success of ZMapp and other monoclonal antibody-based therapies during the 2014-2016 West African Ebola outbreak has shown this countermeasure can be a successful therapy for Ebola hemorrhagic fever. This study utilized transchromosomal bovines (TcB) vaccinated with a DNA plasmid encoding Ebola virus glycoprotein sequence to produce human polyclonal antibodies directed against Ebola virus glycoprotein. When administered 1 day postinfection, these TcB polyclonal antibodies provided partial protection and resulted in a 50% survival rate following a lethal challenge of Ebola virus Makona in rhesus macaques.

Use of Favipiravir to Treat Lassa Virus Infection in Macaques.

Lassa virus, the cause of Lassa fever in humans, is endemic to West Africa. Treatment of Lassa fever is primarily supportive, although ribavirin has shown limited efficacy if administered early during infection. We tested favipiravir in Lassa virus-viremic macaques and found that 300 mg/kg daily for 2 weeks successfully treated infection.

Domestic Pig Unlikely Reservoir for MERS-CoV.

We tested the suitability of the domestic pig as a model for Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Inoculation did not cause disease, but a low level of virus replication, shedding, and seroconversion were observed. Pigs do not recapitulate human MERS-CoV and are unlikely to constitute a reservoir in nature.

Laboratory Response to 2014 Ebola Virus Outbreak in Mali.

Aware of the rapid spread of Ebola virus (EBOV) during the current West African epidemic, Mali took several proactive steps to rapidly identify cases within its borders. Under the Mali International Center for Excellence in Research program, a collaboration between the National Institute of Allergy and Infectious Diseases and the Malian Ministry of Higher Education and Scientific Research established a national EBOV diagnostic site at the University of Sciences, Techniques and Technologies of Bamako in the SEREFO Laboratory. Two separate introductions of EBOV occurred in Mali from neighboring Guinea, but both chains of transmission were quickly halted, and Mali was declared "Ebola free" on 18 January 2015 and has remained so since. The SEREFO Laboratory was instrumental in the success of Mali's Ebola response by providing timely and accurate diagnostics. As of today, the SEREFO Laboratory has tested 103 samples from 88 suspected cases, 10 of which were EBOV positive, since the Ebola diagnostics unit started in April 2014. The establishment of Ebola diagnostics in the SEREFO Laboratory, safety precautions, and diagnostics are described.

Clinical Chemistry of Patients With Ebola in Monrovia, Liberia.

The development of point-of-care clinical chemistry analyzers has enabled the implementation of these ancillary tests in field laboratories in resource-limited outbreak areas. The Eternal Love Winning Africa (ELWA) outbreak diagnostic laboratory, established in Monrovia, Liberia, to provide Ebola virus and Plasmodium spp. diagnostics during the Ebola epidemic, implemented clinical chemistry analyzers in December 2014. Clinical chemistry testing was performed for 68 patients in triage, including 12 patients infected with Ebola virus and 18 infected with Plasmodium spp. The main distinguishing feature in clinical chemistry of Ebola virus-infected patients was the elevation in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyltransferase levels and the decrease in calcium. The implementation of clinical chemistry is probably most helpful when the medical supportive care implemented at the Ebola treatment unit allows for correction of biochemistry derangements and on-site clinical chemistry analyzers can be used to monitor electrolyte balance.

Ebola Laboratory Response at the Eternal Love Winning Africa Campus, Monrovia, Liberia, 2014-2015.

West Africa experienced the first epidemic of Ebola virus infection, with by far the greatest number of cases in Guinea, Sierra Leone, and Liberia. The unprecedented epidemic triggered an unparalleled response, including the deployment of multiple Ebola treatment units and mobile/field diagnostic laboratories. The National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention deployed a joint laboratory to Monrovia, Liberia, in August 2014 to support the newly founded Ebola treatment unit at the Eternal Love Winning Africa (ELWA) campus. The laboratory operated initially out of a tent structure but quickly moved into a fixed-wall building owing to severe weather conditions, the need for increased security, and the high sample volume. Until May 2015, when the laboratory closed, the site handled close to 6000 clinical specimens for Ebola virus diagnosis and supported the medical staff in case patient management. Laboratory operation and safety, as well as Ebola virus diagnostic assays, are described and discussed; in addition, lessons learned for future deployments are reviewed.

Plasmodium Parasitemia Associated With Increased Survival in Ebola Virus-Infected Patients.

BACKGROUND: The ongoing Ebola outbreak in West Africa has resulted in 28 646 suspected, probable, and confirmed Ebola virus infections. Nevertheless, malaria remains a large public health burden in the region affected by the outbreak. A joint Centers for Disease Control and Prevention/National Institutes of Health diagnostic laboratory was established in Monrovia, Liberia, in August 2014, to provide laboratory diagnostics for Ebola virus. METHODS: All blood samples from suspected Ebola virus-infected patients admitted to the Médecins Sans Frontières ELWA3 Ebola treatment unit in Monrovia were tested by quantitative real-time polymerase chain reaction for the presence of Ebola virus and Plasmodium species RNA. Clinical outcome in laboratory-confirmed Ebola virus-infected patients was analyzed as a function of age, sex, Ebola viremia, and Plasmodium species parasitemia. RESULTS: The case fatality rate of 1182 patients with laboratory-confirmed Ebola virus infections was 52%. The probability of surviving decreased with increasing age and decreased with increasing Ebola viral load. Ebola virus-infected patients were 20% more likely to survive when Plasmodium species parasitemia was detected, even after controlling for Ebola viral load and age; those with the highest levels of parasitemia had a survival rate of 83%. This effect was independent of treatment with antimalarials, as this was provided to all patients. Moreover, treatment with antimalarials did not affect survival in the Ebola virus mouse model. CONCLUSIONS: Plasmodium species parasitemia is associated with an increase in the probability of surviving Ebola virus infection. More research is needed to understand the molecular mechanism underlying this remarkable phenomenon and translate it into treatment options for Ebola virus infection.

Lassa Virus Seroprevalence in Sibirilia Commune, Bougouni District, Southern Mali.

Lassa virus (LASV) is endemic to several nations in West Africa. In Mali, LASV was unknown until an exported case of Lassa fever was reported in 2009. Since that time, rodent surveys have found evidence of LASV-infected Mastomys natalensis rats in several communities in southern Mali, near the border with Côte d'Ivoire. Despite increased awareness, to date only a single case of Lassa fever has been confirmed in Mali. We conducted a survey to determine the prevalence of LASV exposure among persons in 3 villages in southern Mali where the presence of infected rodents has been documented. LASV IgG seroprevalence ranged from 14.5% to 44% per village. No sex bias was noted; however, seropositivity rates increased with participant age. These findings confirm human LASV exposure in Mali and suggest that LASV infection/Lassa fever is a potential public health concern in southern Mali.

Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool.

Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.

The Merits of Malaria Diagnostics during an Ebola Virus Disease Outbreak.

Malaria is a major public health concern in the countries affected by the Ebola virus disease epidemic in West Africa. We determined the feasibility of using molecular malaria diagnostics during an Ebola virus disease outbreak and report the incidence of Plasmodium spp. parasitemia in persons with suspected Ebola virus infection.

Incorporation of mouse APOBEC3 into murine leukemia virus virions decreases the activity and fidelity of reverse transcriptase.

APOBEC3 proteins are restriction factors that induce G→A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G→A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.

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.

Seroprevalence of Arboviruses in a Malaria Hyperendemic Area in Southern Mali.

Diagnostics for febrile illnesses other than malaria are not readily available in rural sub-Saharan Africa. This study assessed exposure to three mosquito-borne arboviruses-dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV)-in southern Mali. Seroprevalence for DENV, CHIKV, and ZIKV was analyzed by detection of IgG antibodies and determined to be 77.2%, 31.2%, and 25.8%, respectively. Among study participants, 11.3% were IgG-positive for all three arboviruses. DENV had the highest seroprevalence rate at all sites; the highest seroprevalence of CHIKV and ZIKV was observed in Bamba. The seroprevalence for all three arboviruses increased with age, and the highest seroprevalence was observed among adults older than 50 years. The prevalence of Plasmodium spp. in the cohort was analyzed by microscopy and determined to be 44.5% (N = 600) with Plasmodium falciparum representing 95.1% of all infections. This study demonstrates the co-circulation of arboviruses in a region hyperendemic for malaria and highlights the needs for arbovirus diagnostics in rural sub-Saharan Africa.