Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen.

We report the whole-genome sequence of monkeypox virus obtained using MinION technology (Oxford Nanopore Technologies) from a French clinical specimen during the 2022 epidemic. Amplicon-based sequencing and shotgun metagenomic approaches were directly applied to the sample.

Complement-dependent mpox-virus-neutralizing antibodies in infected and vaccinated individuals.

Mpox virus (MPXV) caused a multi-country outbreak in non-endemic areas in 2022. Following historic success of smallpox vaccination with vaccinia virus (VACV)-based vaccines, the third generation modified vaccinia Ankara (MVA)-based vaccine was used as prophylaxis for MPXV, but its effectiveness remains poorly characterized. Here, we applied two assays to quantify neutralizing antibodies (NAbs) in sera from control, MPXV-infected, or MVA-vaccinated individuals. Various levels of MVA NAbs were detected after infection, historic smallpox, or recent MVA vaccination. MPXV was minimally sensitive to neutralization. However, addition of complement enhanced detection of responsive individuals and NAb levels. Anti-MVA and -MPXV NAbs were observed in 94% and 82% of infected individuals, respectively, and 92% and 56% of MVA vaccinees, respectively. NAb titers were higher in individuals born before 1980, highlighting the impact of historic smallpox vaccination on humoral immunity. Altogether, our results indicate that MPXV neutralization is complement dependent and uncover mechanisms underlying vaccine effectiveness.

Complete Genome Sequences of Monkeypox Virus from a French Clinical Sample and the Corresponding Isolated Strain, Obtained Using Nanopore Sequencing.

We report the whole-genome sequences of a monkeypox virus from the skin lesion of a French patient and the corresponding isolated viral strain. Both viral genomic sequences were successfully obtained by applying shotgun metagenomics using the Oxford Nanopore Technologies sequencing approach.

Heat inactivation of monkeypox virus.

Different kinds of media spiked with monkeypox virus (MPXV) were subjected to heat inactivation at different temperatures for various periods of time. The results showed that MPXV was inactivated in less than 5 min at 70 °C and less than 15 min at 60 °C, with no difference between viruses from the West African and Central African clades. The present findings could help laboratory workers to manipulate MPXV in optimal biosafety conditions and improve their protocols.

Database of SARS-CoV-2 and coronaviruses kinetics relevant for assessing persistence in food processing plants.

SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a virus causing severe acute respiratory disease in humans, emerged in late 2019. This respiratory virus can spread via aerosols, fomites, contaminated hands or surfaces as for other coronaviruses. Studying their persistence under different environmental conditions represents a key step for better understanding the virus transmission. This work aimed to present a reproducible procedure for collecting data of stability and inactivation kinetics from the scientific literature. The aim was to identify data useful for characterizing the persistence of viruses in the food production plants. As a result, a large dataset related to persistence on matrices or in liquid media under different environmental conditions is presented. This procedure, combining bibliographic survey, data digitalization techniques and predictive microbiological modelling, identified 65 research articles providing 455 coronaviruses kinetics. A ranking step as well as a technical validation with a Gage Repeatability & Reproducibility process were performed to check the quality of the kinetics. All data were deposited in public repositories for future uses by other researchers.

ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads.

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as "non-positive" (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Heat inactivation of the severe acute respiratory syndrome coronavirus 2.

Cell culture medium, nasopharyngeal and sera samples spiked with SARS-CoV-2 were subjected to heat inactivation for various periods of time, ranging from 30 s to 60 min. Our results showed that SARS-CoV-2 could be inactivated in less than 30 min, 15 min, and 3 min at 56 °C, 65 °C, and 95 °C, respectively. These data could help laboratory workers to improve their protocols by handling the virus in biosafety conditions.

Historical Discoveries on Viruses in the Environment and Their Impact on Public Health.

BACKGROUND: Transmission of many viruses occurs by direct transmission during a close contact between two hosts, or by an indirect transmission through the environment. Several and often interconnected factors, both abiotic and biotic, determine the persistence of these viruses released in the environment, which can last from a few seconds to several years. Moreover, viruses in the environment are able to travel short to very long distances, especially in the air or in water. SUMMARY: Although well described now, the role of these environments as intermediaries or as reservoirs in virus transmission has been extensively studied and debated in the last century. The majority of these discoveries, such as the pioneer work on bacteria transmission, the progressive discoveries of viruses, as well as the persistence of the influenza virus in the air varying along with droplet sizes, or the role of water in the transmission of poliovirus, have contributed to the improvement of public health. Recent outbreaks of human coronavirus, influenza virus, and Ebola virus have also demonstrated the contemporaneity of these research studies and the need to study virus persistence in the environment. Key Messages: In this review, we discuss historical discoveries that contributed to describe biotic and abiotic factors determining viral persistence in the environment.

Rapid Genomic Characterization of SARS-CoV-2 by Direct Amplicon-Based Sequencing Through Comparison of MinION and Illumina iSeq100TM System.

Global human health is increasingly challenged by emerging viral threats, especially those observed over the last 20 years with coronavirus-related human diseases, such as the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Recently, in late December 2019, a novel Betacoronavirus, SARS-CoV-2, originating from the Chinese city of Wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, COVID-19. Real-time genome sequencing in such viral outbreaks is a key issue to confirm identification and characterization of the involved pathogen and to help establish public health measures. Here, we implemented an amplicon-based sequencing approach combined with easily deployable next-generation sequencers, the small and hand-held MinION sequencer and the latest most compact Illumina sequencer, the iSeq100TM system. Our results highlighted the great potential of the amplicon-based approach to obtain consensus genomes of SARS-CoV-2 from clinical samples in just a few hours. Both these mobile next-generation sequencers are proven to be efficient to obtain viral sequences and easy to implement, with a minimal laboratory environment requirement, providing useful opportunities in the field and in remote areas.

Modeling the Inactivation of Viruses from the Coronaviridae Family in Response to Temperature and Relative Humidity in Suspensions or on Surfaces.

Temperature and relative humidity are major factors determining virus inactivation in the environment. This article reviews inactivation data regarding coronaviruses on surfaces and in liquids from published studies and develops secondary models to predict coronaviruses inactivation as a function of temperature and relative humidity. A total of 102 D values (i.e., the time to obtain a log10 reduction of virus infectivity), including values for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were collected from 26 published studies. The values obtained from the different coronaviruses and studies were found to be generally consistent. Five different models were fitted to the global data set of D values. The most appropriate model considered temperature and relative humidity. A spreadsheet predicting the inactivation of coronaviruses and the associated uncertainty is presented and can be used to predict virus inactivation for untested temperatures, time points, or any coronavirus strains belonging to Alphacoronavirus and Betacoronavirus genera.IMPORTANCE The prediction of the persistence of SARS-CoV-2 on fomites is essential in investigating the importance of contact transmission. This study collects available information on inactivation kinetics of coronaviruses in both solid and liquid fomites and creates a mathematical model for the impact of temperature and relative humidity on virus persistence. The predictions of the model can support more robust decision-making and could be useful in various public health contexts. A calculator for the natural clearance of SARS-CoV-2 depending on temperature and relative humidity could be a valuable operational tool for public authorities.

Molecular characterization of measles virus strains circulating in Cameroon during the 2013-2016 epidemics.

The first genotyping data on measles virus (MeV) strains in Cameroon dates from 1994, while other studies were realized in 2001 and 2011 with the establishment of MeV virological surveillance. However, the genetic data of MeV strains circulating in Cameroon remains fragmented and concentrated in certain regions, hence the need for an update. The objective of this study was to have recent data on MeV genotypes circulating in Cameroon. Ninety throat swabs collected during recent measles outbreaks were analyzed by MeV genotyping RT-PCR using the nucleoprotein gene N. The resulting sequences were analyzed on the basis of 450 nucleotides with MEGA 7 software. Overall genome analysis was performed on 40/90 sequences. The strains were from all ten regions and all belonged to cluster 1 of genotype B3. The genotype B3 has been circulating in Cameroon for long periods of time; efforts must be made in immunization for its elimination.

Monkeypox virus phylogenetic similarities between a human case detected in Cameroon in 2018 and the 2017-2018 outbreak in Nigeria.

A monkeypox virus was detected from a human clinical case in 2018 in Cameroon; a country where no human cases were reported since 1989. The virus exhibited close genetic relatedness with another monkeypox virus isolated in Nigeria during the 2017-2018 outbreak. Although our molecular findings argue in favor of an extension of the monkeypox outbreak from Nigeria into Cameroon, the possibility that the monkeypox virus detected could be indigenous to Cameroon cannot be ruled out.

Influenza Virus Segment Composition Influences Viral Stability in the Environment.

The transmission routes of Influenza A viruses (IAVs) submit virus particles to a wide range of environmental conditions that affect their transmission. In water, temperature, salinity, and pH are important factors modulating viral persistence in a strain-dependent manner, and the viral factors driving IAV persistence remain to be described. We used an innovative method based on a real-time cell system analysis to quantify viral decay in an environmental model. Thus, we identified the viral hemagglutinin (HA) and neuraminidase (NA) as the main proteins driving the environmental persistence by comparing the inactivation slopes of several reassortant viruses. We also introduced synonymous and non-synonymous mutations in the HA or in the NA that modulated IAV persistence. Our results demonstrate that HA stability and expression level, as well as calcium-binding sites of the NA protein, are molecular determinants of viral persistence. Finally, IAV particles could not trigger membrane fusion after environmental exposure, stressing the importance of the HA and the NA for environmental persistence.

Avian Influenza Virus Surveillance in High Arctic Breeding Geese, Greenland.

The connectedness in Arctic regions between migratory waterbird populations originating from different continents and the potential for virus exchange at their shared Arctic breeding ground point to the need to explore the largely unstudied circumpolar circulation of avian influenza viruses (AIV). We here report the investigation of AIV in wild birds and lakes in a high Arctic area of Northeast Greenland. No AIV could be detected in the fecal, feather, and water samples collected from large flocks of pink-footed geese Anser brachyrhynchus and barnacle geese Branta leucopsis in and around refuge lakes, where they congregate at high density during their flightless molting period in summer.

Development of Mobile Laboratory for Viral Hemorrhagic Fever Detection in Africa.

Background: A mobile laboratory transportable on commercial flights was developed to enable local response to viral hemorrhagic fever outbreaks. Methods: The development progressed from use of mobile real-time reverse-transcription polymerase chain reaction to mobile real-time recombinase polymerase amplification. In this study, we describe various stages of the mobile laboratory development. Results: A brief overview of mobile laboratory deployments, which culminated in the first on-site detection of Ebola virus disease (EVD) in March 2014, and their successful use in a campaign to roll back EVD cases in Conakry in the West Africa Ebola virus outbreak are described. Conclusions: The developed mobile laboratory successfully enabled local teams to perform rapid disgnostic testing for viral hemorrhagic fever.

Cleavage of hemagglutinin-bearing lentiviral pseudotypes and their use in the study of influenza virus persistence.

Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability.

Heat inactivation of the Middle East respiratory syndrome coronavirus.

The culture supernatants of the emerging Middle East respiratory syndrome coronavirus (MERS-CoV) were submitted to three temperatures over time and tested for infectivity by TCID50 method on Vero E6 cells. At 56°C, almost 25 minutes were necessary to reduce the initial titre by 4 log10 . Increasing temperature to 65°C had a strong negative effect on viral infectivity as virucidy dropped significantly to 1 minute. On the contrary, no significant decrease in titre was observed after 2 hours at 25°C. These data might be useful in establishing biosafety measures in laboratories against MERS-CoV.

Influenza A virus survival in water is influenced by the origin species of the host cell.

BACKGROUND: Influenza A viruses have an envelope made of a lipid bilayer and two surface glycoproteins, the hemagglutinin and the neuraminidase. The structure of the virus is directly dependent on the genetic makeup of the viral genome except the glycosylation moieties and the composition of the lipid bilayer. They both depend on the host cell and are in direct contact with the environment, such as air or water. Virus survival is important for virus transmission from contaminated waters in the case of wild aquatic birds or from contaminated surface or air for humans. OBJECTIVE: The objective of this study was to check whether the origin species of the host cell has an influence on influenza A virus survival. METHOD: The persistence in water at 35°C of viruses grown on either mammalian cells or avian cells and belonging to two different subtypes H1N1 and H5N1 was compared. RESULTS: Both H5N1 and H1N1 viruses remained infectious for periods of time as long as 19-25 days, respectively. However, within the same subtype, viruses grown on mammalian cells were more stable in water at 35°C than their counterparts grown on avian cells, even for viruses sharing the same genetic background. CONCLUSIONS: This difference in virus stability outside the host is probably connected to the nature of the lipid bilayer taken from the cell or to the carbohydrate side chains of the virus surface glycoproteins. Moreover, the long-lasting survival time might have a critical role in the ecology of influenza viruses, especially for avian viruses.

Persistence of the 2009 pandemic influenza A (H1N1) virus in water and on non-porous surface.

Knowledge of influenza A virus survival in different environmental conditions is a key element for the implementation of hygiene and personal protection measures by health authorities. As it is dependent on virus isolates even within the same subtype, we studied the survival of the 2009 H1N1 pandemic (H1N1pdm) virus in water and on non-porous surface. The H1N1pdm virus was subjected to various environmental parameters over time and tested for infectivity. In water, at low and medium salinity levels and 4°C, virus survived at least 200 days. Increasing temperature and salinity had a strong negative effect on the survival of the virus which remained infectious no more than 1 day at 35°C and 270 parts per thousand (ppt) of salt. Based on modeled data, the H1N1pdm virus retained its infectivity on smooth non-porous surface for at least 7 days at 35°C and up to 66 days at 4°C. The H1N1pdm virus has thus the ability to persist in water and on glass surface for extended periods of time, even at 35°C. Additional experiments suggest that external viral structures in direct contact with the environment are mostly involved in loss of virus infectivity.