Clinical and environmental culturomics and genomics of respiratory viruses

UR D-258, Microbes, Evolution, Phylogénie et Infection (MEPHI)
Aix-Marseille Université (AMU)

Team leaders :
COLSON Philippe (PU-PH, AMU/AP-HM)

Members
AZZA Said
BANCOD Audrey
BOSCHI Céline
CHABRIERE Eric
DUFAU Marion
LA SCOLA  Bernard
LE MOINE Johanna
MILITELLO Muriel
MOAL Valérie
WURTZ Nathalie

General background of the team’s thematics and project

Viral infections are major threats for humans (Lipkin and Firth, 2013; Lipkin, 2013; Nathanson, 2016; Luo and Gao, 2020). They cause acute and chronic diseases, are responsible for multiple epidemics of various extents and of pandemics, and are associated with tremendous burdens of morbidity and mortality at the global and country scales. They emerge and evolve according to changes in human behavior and in the biosphere, and many are zoonoses. Unfortunately, weapons to fight viral infections are limited in number and efficacy (De Clercq, 2016).

Viruses are major agents of respiratory infections and diseases in humans (GBD 2019 Diseases and Injuries Collaborators, 2020). Upper respiratory viral infections are highly common in children and adults worldwide (Goolsby, 2001). Lower respiratory infections were in 2010-2019, before the SARS-CoV-2 pandemic, the fourth, second and sixth leading causes of death globally, in low-, mediddle- and high-income countries, respectively, and viruses were major causes of these infections (https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death). Coinfections involving viruses as well as viruses and bacteria or fungi could be critical in the development and severity of virus-associated diseases (Lim et al., 2016; Boschi et al., 2021; Almand et al., 2017). The incidence of respiratory viral infections is notably substantial among solid organ transplant recipients, which are prone to protracted and chronic infections and diseases (Mombelli et al., 2021).

Advances in existing technologies and new technologies have enhanced the detection and characterization of known, re-emerging and newly-emerging viral diseases worldwide (Mokili et al., 2012; Lipkin and Firth, 2013:). Viral culture has been central in virus discoveries then tended to be neglected, being replaced as first line tool by metagenomics that has expanded concurrently with next-generation sequencing (NGS) technologies (Leland and Ginocchio, 2007; Mokili et al., 2012). Currently, the SARS-CoV-2 pandemic is illustrating how much we need and gain knowledge and understanding from performing viral culture and viral genomics to detect and characterize new viral lineages and variants and study their infections and diseases (La Scola et al., 2020; Wurtz et al., 2021; Pires De Souza et al., 2022; Pires De Souza et al., 2022; Jaafar et al., 2022; Cao et al., 2022; Lv et al., 2020; Li et al., 2021; Lemey et al., 2021; Aksamentov et al., 2021; Colson et al., 2022). However, although thousands of SARS-CoV-2 have been cultured and approximately 15 million SARS-CoV-2 genomes have been deposited in sequence databases (https://gisaid.org/; https://www.ncbi.nlm.nih.gov/genbank/), the numbers of isolates and genomes available for a majority of the other respiratory viruses are dramatically weak.

Furthermore, as for the majority of infectious syndromes, we observe through our exhaustive weekly epidemiological surveillance (Colson et al., 2016) that among the respiratory samples (mostly nasopharyngeal samples collected for the diagnosis of viruses) sent to our diagnostic laboratory, no infectious etiology is found for approximately 50% of them; this despite the fact that the syndromic diagnostic approach by multiplex real-time PCR has been used since several years in our laboratory, and showed co-infections with respiratory viruses in some cases (Colson et al., 2016). We suspect that other viruses are present in the nasopharyngeal samples that remain undiagnosed because they are untargeted, genetically divergent, recombinant, or new. Several newly-emerging viruses for instance occurred in recent decades among solid organ transplant recipients (Moal et al., 2013).

Background of the team

The fairly tight team will comprise: Pr Valérie Moal, MD PhD (ISI (http://www.webofknowledge.com/): h-index= 32, 170 publications, citations= 3,951, SAMPRA (https://ihu.sampra.fr/): score= 1648), nephrologist and in charge of the cohort of kidney transplant patients in Marseille public hospitals; Professor Bernard La Scola, MD PhD (ISI: h-index= 71, 460 publications, citations= 24145, highly cited 2018, 2021; SAMPRA: score= 6496), former research team leader, medical bacteriologist and virologist, scientific manager of the NSB3 laboratory, head of virology department at IHU Méditerranée infection, and specialist in viral cultures; Dr Sarah Aherfi, PharmD PhD (ISI: h-index= 15, 57 publications, citations= 759, SAMPRA: score= 807), medical virologist, currently specializing in the monitoring of viral epidemics through analysis of wastewater by real-time PCR and metagenomics on a 1-year internship in Pascal Barbry’s laboratory in Nice that is expert in this field (https://www.ipmc.cnrs.fr/cgi-bin/site.cgi?page=barbry; Rios et al., 2021); Dr. Céline Boschi, PharmD PhD (ISI: h-index= 7, 19 publications, citations= 193), trained in medical virology, who has specialized since the beginning of her PhD thesis in viral culture and associated tests, in particular tests of sensitivity to antivirals and antibodies; Pr Eric Chabrière, PhD (ISI: h-index= 31, 141 publications, citations= 6242, SAMPRA: score= 807), expert in structural biology and proteomics; and finally Professor Philippe Colson, PharmD PhD (ISI: h-index= 52, 424 publications, citations= 14368, highly cited 2021; SAMPRA: score= 6155), medical virologist, expert in viral genomics, who will lead the team. These six researchers with various backgrounds and skills are 27-59 years of age. They published pairwise between 13 and 116 articles, and four are teammates in a former team.

B. La Scola is primarily a bacteriologist expert in the field of culture of intracellular bacteria and giant viruses. He heads the virology and infectious diseases crises departments at IHU Méditerranée infection and Marseille university and public hospitals, which includes supervision of respiratory infections. He discovered mimiviruses and other families of giant viruses, as well as virophages that infect some of them (La Scola et al., 2003; La Scola et al., 2008; Rolland et al., 2019). He has been used and successful to improve culture assays for the purpose of diagnosis and for research, through modifications in medium culture compositions, pre- and per-culture treatments, methods of lecture of the cytopathic effect, and through automatization of processes and miniaturization of culture devices (see for instance: Bou Khalil et al., 2016). His skills have proven being highly efficient in culturing conventional viruses as he isolated about 6000 SARS-CoV-2 and was able to test for dozens of them their sensitivities to antivirals and antibodies from infected patients or used as treatments (Boschi et al., 2022; Jaafar et al., 2021a). He notably showed with his teammates and students’ significant differences in results according to the methods used, the cells used as culture support, and the viral isolate including within a same lineage. In this setting he standardized viral culture protocols notably regarding viral titers, and time to viral cytopathic effect and compound cytotoxicity assessments. He also isolated several other conventional viruses among which endemic coronaviruses, parainfluenza viruses, influenza viruses, adenoviruses, but also measles viruses, enteroviruses, poxviruses, or HIV (Jaafar et al., 2021b). P. Colson is responsible for the virological diagnosis unit on HIV, viral hepatitis, human papillomaviruses, and respiratory viruses at IHU Méditerranée infection and Marseille university and public hospitals. He notably substantially contributed to the description of the emergence of autochthonous hepatitis E virus infections in developed countries (56 articles, 2268 citations; for instance: Colson et al., 2010; Gerolami et al., 2008). He has been trained to bioinformatics and viral genomics by Dr Eugene V. Koonin, a leading researcher in viral genomics an evolutionary biology, and his team in the Evolutionary Genomics Research Group at the NCBI (https://www.ncbi.nlm.nih.gov/research/groups/koonin/), and contributed dozens of articles in the field of giant viruses in teams head by B. La Scola (for instance: Colson et al., 2011; Colson et al., 2017; Colson et al., 2018). More recently, he contributed 89 publications on SARS-CoV-2 including 43 on SARS-CoV-2 variants, and 9 publications on other respiratory viruses. S. Aherfi and C. Boschi have both been trained in virology, viral culture and sequence analyses by B. La Scola and/or P. Colson. S. They are involved in viral diagnosis of HIV, viral hepatitis, human papillomaviruses, and respiratory viruses. S. Aherfi has notably worked in research in the field of giant viruses using genomics and proteomics (Aherfi et al., 2022; Aherfi et al., 2018) and recently on the detection of SARS-CoV-2 variants in sewage (Le Targa et al., 2022). C. Boschi has been deeply invested during her PhD thesis on the activity of drugs and neutralizing and monoclonal antibodies on SARS-CoV-2 replication (Boschi et al., 2022). V. Moal is in charge of a clinical cohort of approximately 500 kidney transplant recipients at Marseille university hospitals. She deeply collaborated since 2009 with P. Colson as a PhD fellow on the thematic of hepatitis E virus in kidney transplant recipients (16 articles) and since 2018 with B. La Scola on the thematic of giant viruses (Moal et al., 2018) and most recently SARS-CoV-2 (Boschi et al., 2022). Finally, E. Chabrière is an expert in structural biology and proteomics. He discovered a human plasma phosphate binding protein that stabilizes human paraoxonase 1 enzyme (Morales et al., 2007; Gonzalez et al., 2014), and notably worked on quorum quenching enzymes (Bzdrenga et al., 2017:) and AHL-interfering enzymes (Billot et al., 2020:). He particularly interacted deeply with B. La Scola, P. Colson and S. Aherfi for the study of giant virus enzymes, other proteins, and proteomic data (Levasseur et al., 2016; Colson et al., 2020; Aherfi et al., 2022; Boratto et al., 2020).

Goals of the future team

The overall goal of the team is relatively simple: to invest in viral culture to make it more sensitive and suitable for high-throughput isolation of known respiratory viruses, whether easily cultured, tediously culktured or not cultured to date, and in the genomics of these respiratory viruses either directly from respiratory samples or from culture supernatants. The ultimate objective is that the improvement of viral culture techniques combined with metagenomics will make it possible to isolate and discover from clinical respiratory samples new viral variants and genetically-divergent known viruses not covered by current single or multiplex targeted systems of molecular biology detection, or unknown viruses in symptomatic patients with unexplained respiratory infections. As a matter of fact, any clinical syndrome and clinical specimen will be potentially eligible for investigations with the strategies we will improve and implement, but we will essentially focus on respiratory specimens and diseases. The monitoring of the prevalence, incidence, outcome and genotypic and phenotypic evolutions of these viruses, and of their epidemics or diseases, in particular for the new viral genotypes and/or species, will be carried out both on the cohort of immunocompromised patients of Pr Moal, but also in the environment, in particular the sewers of our city, in collaboration with the Battalion of Marseille firefighters with whom we have been working since the start of the COVID-19 crisis.

Axis 1: Viral culturomics

While viral culture and serology, based on viruses initially observed by electron microscopy and then cultured, have been the basis of virology and virus discovery, the appearance and then the development of molecular biology techniques some forty years ago have led to an almost total transfer of all the skills of virologists to these latter methods. Viral culture became therefore neglected as a virus diagnosis and discovery tool. This transfer was very understandable given the constraints of viral culture (Leland et al., 2007). Performed initially on embryonated eggs then primary cells, and despite the appearance of immortalized lines, the procedures remain complex, long and not very productive. In addition, the necessary equipment, especially when we are going to be interested in class 3 agents, is extremely expensive, both in terms of infrastructure costs and in experienced personnel. Moreover, during the past decade, with the advent and extent of viral metagenomics techniques (Mokili et al., 2012), the appeal of viral culture has still further declined. However, culture remains the basis for obtaining and studying new viral strains and for carrying out seroneutralization techniques as we experienced during the COVID-19 crisis (Boschi et al., 2022; Jaafar et al., 2021a). Maintaining and improving culture capacities are therefore strong challenges that can allow the future team to keep a unique expertise while generating collections that will encourage national and international collaborations. For example, our team has since the COVID-19 crisis provided many strains at their request to official collections (ATCC (https://www.atcc.org/), NICBS (https://www.nibsc.org/) but also to manufacturers (Valneva (https://valneva.com/?lang=fr), Biosellal (https://biosellal.com/)). Our culture capacities are based on the surfaces available in the NSB3 laboratory of the IHU for which we are responsible for the hospital sector and for MEPHI research activities, ie. more than 500 m2 available for viral culture. We are going to relaunch flow cytometry and high content screening microscopy for the sorting and isolation of viruses in culture. Pioneers in this field with the application of these methods to giant viruses and intracellular bacteria, we intend to relaunch the platform present in the BSL3 laboratory to apply them to the isolation of known or unknown viruses at high throughput.The objective of the unit is divided into several axes of achievement in the more or less long term.

Constitution of collections of cultivable viruses

Since most labs have abandoned cultivation, even labs equipped with cultivation capability do not have a biobank of viral strains. However, and we saw it during the COVID-19 crisis, a single reference viral strain exchanged between laboratories does not represent the broadness of viruses causing a viral epidemic or pandemic (Jaafar et al., 2021a; Pires De Souza et al., 2022). Indeed, the identification of variants has made it possible to understand the escape from antibodies, regardless they are naturally generated after infection or vaccination or monoclonal and produced by an industrial company (Le Bideau et al., 2022, Pires de Souza et al., 2021; Boschi et al. 2021; Jaafar et al., 2021a; Boschi et al., 2022). In addition, we have shown in two different articles that within the same variant/genotype there were differences in growth capacity and reactivity to antibodies. However, almost all of the studies that focus on viral physiopathology and/or specific antiviral immunity continue to use the few age-old strains available. In the former MEPHI unit, we started to generate collections whose objective was to have a hundred isolates of each viral species then to inoculate 5-10% of the samples collected over time, to expand our collection of viral isolates and in order to have representativeness over time. We had historically started with the measles virus for which we retrospectively inoculated PCR-positive samples and isolated more than 100 measles isolates, whose genomes were then sequenced to assess the genetic profile of the strains involved in the 2017-2019 epidemic (Jaafar et al., 2021b). Moreover, measles virus strains were subsequently used to carry out serological works by serum neutralization and western blot in order to understand vaccine failures (Jaafar et al., submitted).

We continued with SARS-CoV-2 for which we were pioneers in the high-throughput isolation of strains with currently a collection of 6500 isolates that cover all the variants and sub-variants having circulated in France (Boschi et al., 2021). We continue prospectively to isolate and store all new SARS-CoV-2 variants and sub-variants that newly emerge. In parallel, we are concentrating our efforts on the production of a prospective collections of cultivable respiratory viruses, among which Influenza A and B viruses, Parainfluenza viruses 1 to 4, Coronaviruses OC43 and 229AE, Rhinoviruses, and Respiratory syncytial viruses.

Improved isolation procedures for culturable viruses

Although many respiratory viruses can be cultivated, their isolation remains relatively tedious for some of them, as was the case with the SARS-CoV-2 Omicron BA.2. Indeed, either these viruses do not grow on the usual cell lines or their culture does not give a cytopathic effect. The first step will consist in the analysis of the bibliography in order to master all the stages of attachment and penetration of the targeted viruses. We will therefore first try to improve the performance of culture isolation, which will then be used for the next objective. We are going to test several existing modified cell lines, such as Vero cells over-expressing TMPRSS2, initially planned to isolate SARS-CoV-2 but that, given the low specificity of this protease, allowed us to perform the easier isolation of Parainfluenza virus 1 (preliminary personal data). The test of about twenty different cell lines is planned. Then we will test different molecules allowing the attachment of viral particles to cells. For example, vimentin has been shown to increase SARS-CoV-2 uptake by non- or low-permissive cells. Finally, for recalcitrant viruses, we will test the transformation of existing lines so that they express the necessary receptors-coreceptors, in the same spirit as Vero-Slam cells were modified to isolate measles or cells over-expressing TMPRSS2 were used for SARS-CoV-2. For the detection of growth, we need to develop methods, again at high-throughput, to screen culture wells. These methods go through the use of automated analysis of the supernatant with prior enrichment (Pires De Souza et al., 2022) or through methods based on the molecular content of the cells (Hitachi patent in co-development with IHU).

Development of simple methods for the isolation of non/poorly cultivable viruses

In this case, the strategy will be somewhat comparable to the previous case, except that viruses are badly cultivable or non-cultivable. In addition to testing a large number of cell lines transformed or not from our collection, we will try, as in the previous case, to use the modification of existing lines by adding receptors or molecules improving the viral uptake or the microenvironment as has been done for porcine enteric calicivirus (Chang et al., 2004). Another approach is co-culture with other viruses that could be facilitators, similar to the isolation of virophages that we had achieved using a reporter virus (Gaia et al., 2013). We will not start with strategies using primary cells. These are theoretically very suitable for this purpose, but given the cost and low yield we will not use them. Even if the strategy has proven being efficient through some examples (Pyrc et al., 2010), these systems are excessively expensive, the yield very modest and particularly uncertain. We checked this with SARS-CoV-2, which grew very well on many lines (Vero, Caco-2,…) and even more if these were transformed (Matsuyama et al., 2020), while the culture efficiency was very modest on Calu-3 type lung cells (Pires de Souza et al., 2021) and on a tracheo-bronchial primary cell line (personal data). Furthermore, we will continue to work on filtered samples in order to avoid the use of antibiotics which interfere with prolonged cultures, and the non-use of which represented a major advance for viral culture (La Scola et al., 2020).

In addition to the cell lines, an improvement should be obtained on the way of making the culture. Clearly the small culture flasks are poorly suited because too much volume dilutes the samples. The shell-vials that are an excellent alternative will soon no longer be marketed. During the COVID-19 crisis we developed strategies for SARS-CoV-2 isolation and worked on cells allowing to work on small volumes but also to detect viral growth by concentrating the viruses on the slides to promote observation by electron microscopy of supernatants, but also to observe the intracellular localization of viruses (Le Bideau et al., 2021; Le Bideau et al., 2022). We will continue to develop such systems based on a measurement of cellular suffering and the modification of the atomic content of the cells as assessed with the high-throughput electron microscope available at IHU, and on the detection of viral particles by automated image analysis.

Isolation of new viruses

The rest of the project will consist of high-throughput inoculation of respiratory samples for which the multiplexed syndromic approach (essentially the FTD Respiratory Pathogens and Biofire assays) has remained negative in order to isolate undetected viruses, either of genotypes that escaped PCR detection or completely new. The strategy most often used and to which we have adhered at one point is not the right one. Given the heaviness of viral cultures, until now we have favored the inoculation of samples from patients with severe infections and of deep samples, mainly bronchoalveolar fluids from patients in intensive care units with lung infections causing severe acute respiratory disease syndrome. We realized during the COVID-19 crisis that these patients ultimately excreted little or no viable virus at this stage, but rather traces of nucleic acids. Positive cultures were obtained in the community and the severity of the disease was due to the terrain and not to the virus. Our strategy will therefore rather consist of high-throughput research for respiratory viruses in people suffering from respiratory symptoms sufficiently diseased to lead to the PCR diagnosis of one or several respiratory viruses, but not necessarily severe. We thus hope to isolate (and detect for some by metagenomics) the presence of new viral genotypes of known viruses or of new viruses. The method will be the one we are currently trying to modernize, namely combo inoculation of cell lines proposed almost 30 years ago (Brumback et al., 1994), but with cells more or less transformed with addition of molecules promoting non-specific adhesion and modification of the microenvironment. For this, we plan to inoculate the bottoms of the remaining collection tubes of all patients for whom we received a respiratory sample and multiplex PCR testing will be negative. This represents approximately 7500 samples per year, which means that the high-throughput detection systems will have to be functional to start this work. After the isolation of one or more viruses, the rest will ultimately be quite simple, and will include genome sequencing of the isolated virus(es), specific PCR design and retests of nucleic acid extracts to assess the frequency of this agent. A new virus discovered over the period and the description of its disease and its epidemiology is the main and ultimate objective of this work, in particular in the cohort of immunocompromized patients monitored by V. Moal.

Status report on the viral genomics of respiratory infection viruses: approaches of our team for the constitution of genomic banks and monitoring of epidemics

surveillance to gain a better knowledge and understanding of the virological, epidemiological and clinical features of viruses of different lineages and variants (see for instance: Lv et al., 2020; Li et al., 2021; Lemey et al., 2021; Aksamentov et al., 2021; Colson et al., 2022; Burel et al., 2022; Jaafar et al., 2022; Cao et al., 2022). Viral genome analysis has indeed notably allowed deciphering the emergence and evolution of SARS-CoV-2 variants, their transmissibility and pathogenicity, and their fitness and capabilities to escape immune responses elicited by earlier infection or by vaccination. This also allowed determining the genetic variability along the genome, the nucleotide and amino acids that may be critical for viral replication, transmissibility, pathogenicity and immune escape. Such approaches are of substantial importance to try delineating antiviral strategies. Results and observations obtained for SARS-CoV-2 sharply indicate that genome characterization and genomic surveillance should also be performed for the other respiratory viruses than SARS-CoV-2. However, there is a very huge gap between genome sets available for SARS-CoV-2 and for the other respiratory viruses. Indeed, compared to the approximately 15 million genomes available from world sequence databases for SARS-CoV-2, the numbers of genomes that are available from these databases for most of these other respiratory viruses are incredibly weak: for instance, 50, 70, 370 and 48 for Parainfluenza viruses 1, 2, 3 and 4, respectively; 42, 220, 69 and 46 for Human coronaviruses (HCoV) 229, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, respectively; or 314 for bocaviruses. For influenza viruses, the number of genomes is bigger, approximately 50,000 according to the NCBI Influenza virus database (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi), but for the period from 2021 to 2022, this numbers fall below 10,000 which is about 6 times less globally that the level of our single institute for SARS-CoV-2. This represents large gaps to be filled.

Implementation of Artic-like universal PCR amplification systems for all respiratory viruses.

Considering the deficit in respiratory virus genomes at the global, country and local scales, a goal of the team will be to obtain by NGS large sets of genomes of these respiratory viruses other than SARS-CoV-2. This is an achievable goal considering that about 15,000 infections with one or more respiratory viruses apart from SARS-CoV-2 are diagnosed in our institute per year, and PCR-positive specimens are stored at -20°C or -80°C in a large biobank.

The sequencing of the genomes of respiratory viruses requires, in absence of PCR amplification prior NGS, a high viral RNA or DNA load in the clinical sample. With the experience of SARS-CoV-2, it has been observed that NGS of viral genomes most commonly failed in case of respiratory samples for which the cycle threshold value (Ct) of qPCR was above 18 (Younoussa-Abdoulaye et al., to be submitted). However, such virus RNA loads are found in less than 10-20% of the samples. This drawback to virus genome obtaining has been overcome through the so-called Artic procedure, which consists in generating by PCR short (about 400-800 nucleotide-long) amplicons that cover the whole genome by overlapping, and was initially implemented for the sequencing of Zika virus genomes (Quick et al., 2017) and was used at the global scale for SARS-CoV-2 (Artic: https://artic.network/). We will design Artic-like systems comprising two sets of multiple pairs of primers for each species of respiratory viruses (namely the four endemic coronaviruses (229E, OC43, NL63, HKU1), Influenza viruses A/H3N2, A/H1N1 and B, RSV A and B, the four Parainfluenza viruses 1-4, Metapneumovirus, Bocaviruses, Rhinoviruses, Enteroviruses, Adenoviruses). Such designs of PCR systems along the whole genome length are hampered by the substantial genetic diversity of viral species. Therefore, we will use bioinformatics tools that were previously implemented for PCR design by our former teams or by other non-IHU teams including SVARAP (Colson et al., 2006), GEMI (Sobhy and Colson, 2012), and the online Primal Scheme primer designer software (Quick et al., 2017; Primalscheme: https://primalscheme.com/), and we will perform manual curation of primer designs, then test PCR primer pairs individually and as pools to determine the most appropriate PCR conditions. This approach will allow growing our own databases of hundreds of genomes of respiratory viruses detected in the framework of routine clinical virology diagnostics.

Analyses of respiratory virus genomes.

The analysis of respiratory virus genomes will be carried out with tools available online and in the literature, some used for SARS-CoV-2 genome (for instance Nextstrain (Hadfield et al., 2018; https://nextstrain.org/) and Influenza virus genomes analysis (for instance: https://www.bv-brc.org/; https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database) after adaptation, and others set up in house by our team. Analyses will list all mutations (substitutions, indels) at the nucleotide and amino acid levels, and will perform their qualitative and quantitative assessments among which the delineation of lineages based on hallmark sets of mutations and the prediction of changes at the protein level by structural predictions. Phylogenetic analyses will be performed with various tools including FastTree (Price et al., 2009), Auspice (https://docs.nextstrain.org/projects/auspice/en/stable/), and MEGAX (Kumar et al., 2018). Intra-sample quasispecies will be analyzed using an in-house tool (QuasiS; Bader et al., submitted). Recombinations within and between respiratory viruses of same and different species (in case of coinfections) will be studied using accurate genome sequence scrutiny (Burel et al., 2022) as we previously performed for SARS-CoV-2 and by available tools such as the RDP4 (Martin et al., 2017) and Simplot (Samson et al., 2022) programs. Influenza virus genome segments’ reassortments will also be searched (Ding et al., 2021). For all bioinformatics analyses, we will be able relying on the IHU bioinformatics platform and team.

History of past epidemics and monitoring of future epidemics.

A major goal will be to analyze and depict the evolution of respiratory virus genomes among infections diagnosed in our institute not only during the cold months, but along the whole year, and along the whole study period which will approximately cover a decade considering both retrospective and prospective data. This is a real issue as the seasonality and epidemiological trends of respiratory infections are not well-known around a whole year. Furthermore, there are also scarce data about how respiratory virus genomes evolve, and how the incidence, prevalence and distribution of the lineages of these viruses change overtime around a whole year. We have detected by PCR over the past 5 years in the framework of routine clinical virology diagnostics about 45000 respiratory viruses other than SARS-CoV-2 in respiratory samples. For the past 12 month-period, 15000 such positive diagnoses were obtained, with cases along the whole period including during the hot months. Our project will allow deciphering the genotypic patterns of these respiratory viruses and the emergence, spread, outcome, and disappearance of each of the epidemics linked to a given lineage for a given species. We have already tested our strategy at a small scale with HCoV-229E and Influenza A virus. Seventy HCoV-229 genomes were obtained for the 2021-2022 winter season and phylogeny reconstruction delineated two different clusters, indicating that two distinct lineages co-circulated during this short time frame (Py et al., in preparation). Regarding Influenza A/H3N2 virus, more than 500 genomes have been obtained, which showed reassortments of genome segments 4 and 6 between co-circulating lineages (Le Targa et al., in preparation), and the importation from Comoros of viral strains during summer of year 2021 (Assoumani et al., 2021). These results strongly encourage us to continue on this path.

Combination of genomic and metagenomic data for the detection of new respiratory virus genotypes and new viruses

As for all infections, we observe through our exhaustive in-house weekly epidemiological surveillance system (Colson et al., 2016) that among the respiratory samples (mostly nasopharyngeal samples) sent to our laboratory for the diagnosis of viruses, no infectious etiology is found for approximately 50% of them; this despite the fact that the syndromic diagnostic approach by multiplex real-time PCR has been used since several years. As viruses are the infectious agents the most common in respiratory samples, we suspect that other viruses are present in the nasopharyngeal samples that remain undiagnosed because they escape to qPCR testing targeting specifically a set of viruses: SARS-CoV-2, the four endemic coronaviruses, Influenza viruses A/H3N2, A/H1N1 and B, Respiratory syncytial virus, the four Parainfluenza viruses, Metapneumovirus, Bocaviruses, Rhinoviruses, Enteroviruses, Adenoviruses. The reasons why viruses may remain undiagnosed can be multiple. These include (i) low virus abundance; (ii) mutations, insertions or deletions in PCR target regions that make viruses escaping to PCR detection; (iii) recombinations between different viral lineages or within members of a same lineage that generate nucleotide substitutions, deletions or insertion and change the PCR target sequence; (iv) genetically-divergent viruses belonging to known viral taxa, which may include viral variants and viruses belonging to new lineages that were not considered in the design of the PCR primers; or (v) unknown, new viruses. Detecting undiagnosed viruses will be very valuable to improve the rate of diagnosis of respiratory infections; to study the importance in human pathogeny of these undiagnosed viruses, and to gain a better understanding of their infections including their interactions with diagnosed viruses as well as bacteria. Therefore, to counteract these reasons that hamper improving and expanding the diagnosis, study and characterization of viruses associated with respiratory infections, we will use several strategies.

DNA/RNA metagenomics

DNA/RNA metagenomics has developed in parallel with next-generation sequencing and has proven being a powerful approach to detect and identify viral sequences among NGS reads generated from environmental or clinical samples (Mokili et al., 2012; Kawasaki et al., 2021; Mao et al., 2022; Johansen et al., 2022). However, its use involves pitfalls and obstacles that have to be overcome. Among them are the relative paucity of viral sequences in clinical samples compared to the abundance of eukaryotic sequences, and the difficulty to identify divergent and unknown sequences as by definition there are no similar sequences in databases worldwide (Borrato et al., 2020).  

To overcome the overwhelming presence of eukaryotic sequences, and at a lower level of prokaryotic sequences, due to the presence of human cells and bacteria (mostly commensal or contaminating) in significant quantities and with large genomes in clinical samples, which causes a sequencing depth bias and prevent from sequencing viral nucleic acid in sufficient amount for its identification (and beyond for the assembly of a full-length genome from NGS reads), we will perform depletion of these eukaryotic and prokaryotic sequences by different ways: filtration through 0.2 and 0.045 µm filters, ultracentrifugation, and use of nucleases before DNA/RNA extraction; use of DNAses post-extraction, and before reverse transcription for RNA metagenomics; ribodepletion; and targeting of methylated DNA (Oyola et al., 2013). Finally, random PCR amplification can be performed in order to enrich the metagenomes in viral sequences, using for instance the Genomiphi strategy (Sato et al., 2004).

Viral sequence identification in DNA/RNA metagenomes

The identification of viral sequencing is primarily by sequence similarity searches against viral sequence databases, which implicates that these databases have to comprise sequences representing as comprehensively as possible the sequence space of genetic diversity for a given viral taxon (Lu et al., 2022; Borrato et al., 2020). Therefore, the paucity of whole genome datasets for a majority of respiratory viruses limits the current approaches to identify viral sequences in metagenomic datasets (Johansen et al., 2022). Such conditions are obviously not reached for many respiratory viral species for which only dozens of genomes are publicly available. Therefore, we will enrich the genome sequence sets of known respiratory viruses, and therefore the sequence swarm of genetic diversity, by strategies described in paragraph 3.b.

Beyond, our former team has previously shown that the scrutiny of metagenomic datasets failed to identify sequences too genetically distantly-related to known viruses or for which there is no genome available (Boratto et al., 2020; Schulz et al., 2020). We will implement different strategies that will use multiple sequences as baits: (i) Use of large capsid and RNA polymerase sequences’ databases, which proved being efficient to find viral sequences in metagenomes (Yutin et al., 2015; Sharma et al., 2014); (ii) Use of the Rfam tool, which is a database of 3,444 viral families each represented by a multiple sequence alignment of known RNA sequences as well as a covariance model that can be used to seek additional family members (Kalvari et al., 2021); (iii) Use of the SCOP database of protein motifs (http://scop.mrc-lmb.cam.ac.uk/scop/) and of the software HMMER3 based on Hidden Markov models (HMM) that exploits the specific positions in protein-encoding codons (Bzhalava et al, 2018); (iv) Use of networks to try catching genetically-divergent sequences from known viruses (Watson et al., 2019; Lopez et al., 2015); this strategy can relate two sequences that are too genetically distant by creating a network with intermediate sequence allowing a transitive relationship involving these two sequences.

Importantly, we will not neglect any NGS read (with a sufficient sequencing quality), as our former team has demonstrated that seeking in the sequencing “trash” was fruitful to detect previously unidentified genetic elements (Desnues et al., 2012). Thus, NGS reads remaining unclassified (i) will be clusterized; (ii) will be compared cautiously to abovementioned sequence databases through BLAST searches; (iii) will be attempted to be assembled de novo. Finally, we will also use several metagenome binning strategies that will notably rely on viral motifs and k-mers (Kieft et al., 2022; Johansen et al., 2022). Finally, our former team implemented efficient in house tools to seek in metagenomes for giant virus sequences (Verneau et al., 2016; Pires de Souza et al., 2021), and this tool will be used with the most accurate abovementioned databases.

Besides, in addition to search for recombinant viral genomes within and between respiratory viruses of same and different species using the RDP4 (Martin et al., 2017) and Simplot (Samson et al., 2022) programs, we will perform the search of composite genes using a previously described approach that involved our former team and can deal with viral genome mosaicism by detecting composite (or mosaic) sequences (Jachiet et al., 2014).

Molecular testing of culture supernatants inoculated with clinical samples

We will perform targeted multiplex PCR assays on culture supernatants (see paragraph 2) after inoculation with clinical samples and 3 passages (re-inoculations), regardless a cytopathic effect has been observed, since some viruses can infect cells but do not induce cytopathogenicity. Concurrently, DNA/RNA metagenomics will be performed on the supernatant, after the same treatments than described in previous paragraph 4.a.

Relationships between human epidemics of respiratory viral infections and environmental monitoring

Wastewater-based epidemiology is considered as an early warning system for outbreaks (Sims et al., 2020). In the context of the COVID-19 pandemic, the detection and quantification of the SARS-CoV-2 in wastewater has been revealed being an efficient tool for tracking the spread, prevalence, and molecular epidemiology of the virus in a community. SARS-CoV-2 detection and quantification in wastewater has been widely used in several countries. It has been shown that the evolution of RNA concentrations in wastewater preceded by a few days (Melvin et al., 2021) the epidemic curve in patients with a high predictive value. This approach will be useful in the surveillance of some targeted small geographical areas (Wurtz et al., 2021), or institutions (for example, hospitals or retirement homes) by collecting wastewaters at the exit of these institutions. Its application to the surveillance of aircraft or cruise ships blackwaters would be a powerful tool to adapt and activate intense measures of lockdown to the only targeted (and infected) individuals that may import the virus into virus or variant-free areas (Ahmed et al., 2020). Thus, rapid advances are necessary for applying this surveillance tool to infectious diseases in real life and in any laboratory, especially in remote areas where broad-scale human testing is not possible. They imply optimization of storage and viral enrichment methods, development of selective and sensitive molecular tools, and pre-amplification systems by PCR.

Pre-treatment and viral enrichment

In collaboration with the Bataillon des Marins Pompiers de Marseille, we tracked since the beginning of the pandemics the concentrations of SARS-CoV-2 RNA in the Marseille wastewaters. Current methods of storage and enrichment are diverse and are not standardized. Moreover, they sometimes require specialized and expensive equipment. We recently assessed different methods of storage (that have to minimize viral loss) and enrichment in the aim to expand their use in any laboratory. We showed that a simple storage at +4°C combined with a filtration step at 0.8 µm on filtration units, and ultrafiltration with centricons, largely commercialized, were efficient for conservation and concentration (personal data). The simple equipment required for this procedure will allow enlarging collection locations to developing countries.

Molecular methods of detection and quantification of viral epidemics

It will be useful to extend this surveillance tool in wastewater particularly efficient to other respiratory viruses: Influenza A and B viruses, Respiratory Syncytial Virus (RSV) A and B, Metapneumovirus, the four Human Coronaviruses NL63, HKU1, 229E and OC43, and the four parainfluenza viruses. This project will allow to early detect the beginning of each seasonnal outbreak and, as for the SARS-CoV-2, will provide a global trend in a community. Currently, the Bataillon des Marins Pompiers de Marseille screens weekly the presence, in addition to the SARS-CoV-2, of the 4 human seasonal coronaviruses, the parainfluenza 3 and 4, the RSV and the Influenza A by using a commercialized kit that allows a semi-quantification in wastewater samples of Marseille. We aim to extend this system by developing real time PCR systems with an absolute quantification providing a result in copies/mL, as for what is currently performed for the SARS-CoV-2.

PCR amplification systems prior next-generation sequencing

It should be noted that viral quantification only in wastewater does not provide a sufficient information. The identification of new variants will provide valuable data on the evolution of the epidemics. Several teams have demonstrated that it was possible to identify by sequencing the different variants present in these wastewater samples. The surveillance of variants is currently based on full genome sequencing of clinical samples, which is only representative of symptomatic patients and/or other patients who have visited the hospital or laboratory for screening. Monitoring variants via wastewater could be a more efficient, faster, and representative method for monitoring the emergence and spread of variants.

We showed for example that the repartition of SARS-CoV-2 variants within wastewater of a district of Marseille differed from those found in the general wastewater network, in particular with the significant overrepresentation of low prevalent variants. This over-representation was correlated with sequencing data from patients living in this area (Wurtz et al., 2021). We also identified very early during the epidemic wave linked to the Omicron variant, the presence of this variant in blackwaters from the aircraft arriving in Marseille from Addis-Abbeba in December 2021 while the Omicron variant was not yet established in Marseille and while green passports indicating a negative PCR test for SARS-CoV-2 were still mandatory for each embarking passenger (Le Targa et al., 2022). The sequencing of wastewater from aircrafts or even of cruise ships may be useful in tracking variants spreading, and its implementation may be a powerful prevention tool for public health by targeting the individuals requiring a lockdown (and thus limiting restricting measures) to avoid the spreading of an emerging variant in a preserved area. Developing similar approaches for Influenza A and B viruses, Respiratory Syncytial Virus A and B, bocaviruses, Metapneumovirus, the four human coronaviruses NL63, HKU1, 229E, and OC43, and the four parainfluenza viruses 1-4 to wastewater samples, and studying the presence and epidemiological trends over time of these viruses and their lineages and variants through analyzing specific sequences and hallmark mutations is the one-year mobility project (from December 2022) of one researcher of the team (SA). This project will be carried out in collaboration with the Institute of Molecular and Cellular Pharmacology (https://www.ipmc.cnrs.fr/cgi-bin/site.cgi; Sophia Antipolis), which has tracked the diffusion of the UK variant based on the Nanopore sequencing of wastewater samples from some 20 different geographical sites in the city of Nice (Rios et al., 2021). This task will aim to expand our knowledge on the genomics of respiratory viruses and at the team scale, to develop our skills in bioinformatics and genomic data analysis. It will be a proof of concept for a potential future application to gastroenteritis viruses.

Valuation aspect of the new agents identified and ethical aspects of the research

This project will provide a more open vision of the set of viruses present in respiratory samples and will provide very interesting data on the performance of viral culture and metagenomics to detect known and unknown viruses. Detecting undiagnosed viruses will be also very valuable to study the importance in human pathogeny of these undiagnosed viruses, to gain a better understanding of their infections including their interactions with diagnosed viruses as well as bacteria. Improving the diagnosis rate of respiratory infections is of great interest in virology and in the clinics. This project may allow improving the clinical and therapeutic management of the patients.

Data generated including viral isolates and genome sequences will be stored appropriately. For the case of viral strains, they could be shared with other biobanks or with other research teams in the setting of new collaborations, or with industrials. For the case of genome sequences, they will be deposited on sequence databases that allow their free full access as for instance GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

This project will include retrospective studies conducted on patients’ respiratory samples sent to our diagnosis laboratory for the purpose of diagnosis of respiratory viruses, then stored in our diagnosis laboratory biobank. These nasopharyngeal samples will be fully anonymized. No additional sample will be collected from the samples than those sent by clinicians for diagnosis. In this framework, research will not be on humans. In the case of other studies that will involve clinical data: patient archiving and documentation system (PADS) will be deposited and a personal protection committee will be set up.

Conclusions

Overall, our team is experienced in virus discovery and characterization through culture and genomics. It aims to use culture, and NGS for the discovery of new and reemerging viruses, the identification of new viral variants, and also for the better characterization of existing viruses, for acquiring more comprehensive evidence reflecting viral evolution and differentiating viral strains, and expand genomic surveillance implemented in clinical virology laboratory for SARS-CoV-2 to other respiratory viruses.

This team encompasses members of various ages with different university backgrounds and diverse and complementary skills. Team’s members know each other very well and have worked and published together many articles.

The team’s goals are very exciting since they regard an original, interesting and important thematic in terms of research and clinical practice.

We are confident that our team will be effective in achieving these goals due to the support of technological platforms of high level available at IHU Méditerranée Infection, the contribution of past experiences of team’s members and the shared body of their skills. Altogether, for this team, the materials, technologies and people appear appropriate to reach these goals.

Publications

Last 5 years on Pubmed

References

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