Monkeypox virus: emerging of an old neglected virus.

Since April 2022, cases of simian orthopoxvirosis (commonly known as monkeypox) have been reported in more than hundred non-endemic countries. The causative agent, the Monkeypox virus (MPXV), is a virus of the family Poxviridae belonging to the genus Orthopoxvirus (OPXV). The sudden and unusual emergence of this virus mainly in Europe and in the United States has highlighted a previously neglected infectious disease. This virus has been endemic in Africa for at least several decades, since its discovery in 1958 in captive monkeys. MPXV, because of its proximity to the smallpox virus, is part of the list of Microorganisms and Toxins (MOT), which includes all human pathogens considered to be potentially misused for malicious purposes (biological weapons proliferation, bioterrorism) or susceptible to provoke laboratory accidents. As such, its use is subjected to strict regulations in level-3 biosafety laboratories, which de facto limits the possibilities of its study in France. The objective of this article is to review the current knowledge about OPXV in general, and then to focus on the virus responsible for the 2022 MPXV outbreak.

[Monkeypox virus: emerging of an old neglected virus]. / Virus Monkeypox : l'émergence d'un virus ancien jusque-là négligé.

Since April 2022, cases of simian orthopoxvirosis (commonly known as monkeypox) have been reported in more than hundred non-endemic countries. The causative agent, the Monkeypox virus (MPXV), is a virus of the family Poxviridae belonging to the genus Orthopoxvirus (OPXV). The sudden and unusual emergence of this virus mainly in Europe and in the United States has highlighted a previously neglected infectious disease. This virus has been endemic in Africa for at least several decades, since its discovery in 1958 in captive monkeys. MPXV, because of its proximity to the smallpox virus, is part of the list of Microorganisms and Toxins (MOT), which includes all human pathogens considered to be potentially misused for malicious purposes (biological weapons proliferation, bioterrorism) or susceptible to provoke laboratory accidents. As such, its use is subjected to strict regulations in level-3 biosafety laboratories, which de facto limits the possibilities of its study in France. The objective of this article is to review the current knowledge about OPXV in general, and then to focus on the virus responsible for the 2022 MPXV outbreak.

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.

Microarrays in virology: basic concepts and applications for virus detection.

For a few decades, the introduction and development of molecular methods in microbiology have shaped the detection and characterization of pathogens. Although serological and, more punctually, viral culture methods remain basic tools for viral diagnosis, molecular advances based on qPCR have brought a number of novel advantages, in terms of speed, specificity and costs. On the other hand, microarrays have demonstrated their own advantages by increasing drastically the capabilities of detection and characterization of a large range of viruses in a unique step. Nowadays, several microarray-based platforms exist that can be classified in different families according to the type of matrix (solid or liquid), the size and density of probes, the method used for visualizing hybridization results with the target and finally relative costs. The aims of this review will be to overview (i) basic concepts of the different technologies used and to enlighten differences, advantages and drawbacks of each type of platform and (ii) the applications in virology for the detection and characterization of viral agents.

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.

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.

Use of consensus sequences for the design of high density resequencing microarrays: the influenza virus paradigm.

BACKGROUND: A resequencing microarray called PathogenID v2.0 has been developed and used to explore various strategies of sequence selection for its design. The part dedicated to influenza viruses was based on consensus sequences specific for one gene generated from global alignments of a large number of influenza virus sequences available in databanks. RESULTS: For each HA (H1, H2, H3, H5, H7 and H9) and NA (N1, N2 and N7) molecular type chosen to be tested, 1 to 3 consensus sequences were computed and tiled on the microarray. A total of 12 influenza virus samples from different host origins (humans, pigs, horses and birds) and isolated over a period of about 50 years were used in this study. Influenza viruses were correctly identified, and in most cases with the accurate information of the time of their emergence. CONCLUSIONS: PathogenID v2.0 microarray demonstrated its ability to type and subtype influenza viruses, often to the level of viral variants, with a minimum number of tiled sequences. This validated the strategy of using consensus sequences, which do not exist in nature, for our microarray design. The versatility, rapidity and high discriminatory power of the PathogenID v2.0 microarray could prove critical to detect and identify viral genome reassortment events resulting in a novel virus with epidemic or pandemic potential and therefore assist health authorities to make efficient decisions about patient treatment and outbreak management.

Codon conservation in the influenza A virus genome defines RNA packaging signals.

Genome segmentation facilitates reassortment and rapid evolution of influenza A virus. However, segmentation complicates particle assembly as virions must contain all eight vRNA species to be infectious. Specific packaging signals exist that extend into the coding regions of most if not all segments, but these RNA motifs are poorly defined. We measured codon variability in a large dataset of sequences to identify areas of low nucleotide sequence variation independent of amino acid conservation in each segment. Most clusters of codons showing very little synonymous variation were located at segment termini, consistent with previous experimental data mapping packaging signals. Certain internal regions of conservation, most notably in the PA gene, may however signify previously unidentified functions in the virus genome. To experimentally test the bioinformatics analysis, we introduced synonymous mutations into conserved codons within known packaging signals and measured incorporation of the mutant segment into virus particles. Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging. Thus our analysis identifies cis-acting sequences in the influenza virus genome at the nucleotide level. Furthermore, we propose that strain-specific differences exist in certain packaging signals, most notably the haemagglutinin gene; this finding has major implications for the evolution of pandemic viruses.

Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR.

BACKGROUND: Phi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments (<2000 bases) obtained by reverse transcription of certain viral RNA genomes. Therefore, ligation of cDNA fragments is necessary prior phi29 polymerase based amplification. We adapted the QuantiTect Whole Transcriptome Kit (Qiagen) to our purposes and designated the method as Whole Transcriptome Amplification (WTA). RESULTS: WTA successfully amplified cDNA from a panel of RNA viruses representing the diversity of ribovirus genome sizes. We amplified a range of genome copy numbers from 15 to 4 x 10(7) using WTA, which yielded quantities of amplified DNA as high as 1.2 microg/microl or 10(10) target copies. The amplification factor varied between 10(9) and 10(6). We also demonstrated that co-amplification occurred when viral RNA was mixed with bacterial DNA. CONCLUSION: This is the first report in the scientific literature showing that a modified WGA (WTA) approach can be successfully applied to viral genomic RNA of all sizes. Amplifying viral RNA by WTA provides considerably better sensitivity and accuracy of detection compared to random RT-PCR.

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.

Naturally occurring substitutions of the human T-cell leukemia virus type 1 3' LTR influence strand-transfer reaction.

Having isolated somatically mutated HTLV-1 3' LTR sequences from six infected individuals, the effect of these mutations on the integration process in vitro was investigated. Double-strand pre-processed HTLV-1 3' LTR ends (53-54 bp) were used in an in vitro strand-transfer reaction, together with HTLV-1 purified integrase and using a synthetic double-strand naked DNA oligonucleotide as target. Integration efficiency was measured by a fluorescent PCR assay. No significant difference in the pattern of strand transfer was observed between the distinct patients consensus sequences. For each patient, the effect of acquired somatic mutations was then assessed by comparing the strand-transfer efficiency of the mutated sequences (n=8, each harboring one to two substitutions) with that of the corresponding patient consensus sequence. Five somatic mutations or deletions at positions 7, 10, 21, 30, and 53 from the proviral 3' end did not alter the reaction efficiency. By contrast, a single G-->A transition at position 52 was found to result in 33% gain of function. Furthermore, a C-->T transition at 41 bp from the provirus 3' end decreased the reaction efficiency by 80%. This is the first study investigating the effect of naturally acquired substitutions on the strand-transfer capacity of long LTR sequences in vitro. Disproving the hitherto assumed opinion that integration specificity is restricted to the extreme boundary of the LTR end, i.e. the last 12-20 bp of the unintegrated provirus, the present results demonstrate that naturally occurred substitutions of the HTLV-1 LTR can alter significantly its strand-transfer capacity.

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.

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.

Recurrent Breast Abscesses due to Corynebacterium kroppenstedtii, a Human Pathogen Uncommon in Caucasian Women.

Background. Corynebacterium kroppenstedtii (Ck) was first described in 1998 from human sputum. Contrary to what is observed in ethnic groups such as Maori, Ck is rarely isolated from breast abscesses and granulomatous mastitis in Caucasian women. Case Presentation. We herein report a case of recurrent breast abscesses in a 46-year-old Caucasian woman. Conclusion. In the case of recurrent breast abscesses, even in Caucasian women, the possible involvement of Ck should be investigated. The current lack of such investigations, probably due to the difficulty to detect Ck, may cause the underestimation of such an aetiology.