Viral metagenomics

Viral metagenomics is the study of viral genetic material sourced directly from the environment rather than from a host or natural reservoir. The goal is to ascertain the viral diversity in the environment that is often missed in studies targeting specific potential reservoirs. It reveals important information on virus evolution and the genetic diversity of the viral community without the need for isolating viral species and cultivating them in the laboratory. With the new techniques available that exploit next-generation sequencing (NGS), it is possible to study the virome of some ecosystems, even if the analysis still has some issues, in particular the lack of universal markers. Some of the first metagenomic studies of viruses were done with ocean samples, and revealed that most of the sequences of DNA and RNA viruses had no matches in databases. Subsequently, some studies about the soil virome were performed with a particular interest on bacteriophages, and it was discovered that there are almost the same number of viruses and bacteria. This approach has created improvements in molecular epidemiology and accelerated the discovery of novel viruses. Viral metagenomics is the study of viral genetic material sourced directly from the environment rather than from a host or natural reservoir. The goal is to ascertain the viral diversity in the environment that is often missed in studies targeting specific potential reservoirs. It reveals important information on virus evolution and the genetic diversity of the viral community without the need for isolating viral species and cultivating them in the laboratory. With the new techniques available that exploit next-generation sequencing (NGS), it is possible to study the virome of some ecosystems, even if the analysis still has some issues, in particular the lack of universal markers. Some of the first metagenomic studies of viruses were done with ocean samples, and revealed that most of the sequences of DNA and RNA viruses had no matches in databases. Subsequently, some studies about the soil virome were performed with a particular interest on bacteriophages, and it was discovered that there are almost the same number of viruses and bacteria. This approach has created improvements in molecular epidemiology and accelerated the discovery of novel viruses. Acknowledging the importance of viral metagenomics, the International Committee on Taxonomy of Viruses (ICTV) recognizes that genomes assembled from metagenomic data represent actual viruses and encourages their official classification following the same procedures as those used for viruses isolated and characterized using classical virology approaches. The IMG/VR system and the IMG/VR v.2.0 – the largest interactive public virus database with over 760,000 metagenomic viral sequences and isolate viruses – serves as a starting point for the sequence analysis of viral fragments derived from metagenomic samples. The virus detection method and host assignment approach in IMG/VR is described in a paper discussing Earth's virome and is fully presented as a protocol. The aim of the Global Virome Project is to expand the viral discovery to reduce the risk of harm for new viral large-scale outbreak. It is centered on the massive collection and sequencing of the majority of the planet’s unknown viruses. In fact, it was estimated that between 631,000 to 827,000 yet-to-be-discovered viral species in animal reservoirs (as mammal and bird host) are estimated to have a zoonotic potential. Total transparency of the data acquired, and the correlated possible development of some medical products (vaccine) are two main benefits of this project. The main limit of this project is the cost. To analyse the majority of viruses with a zoonotic potential, the total cost has been estimated around $1.2 billion. Another GVP’s aim is also to improve the possibility to detect with low cost sequencing viruses also in developing countries in order to avoid possible outbreaks. For a worldwide sample collection some network between different agencies and nations must be created. For example, the USAID (agency for international development) EPT (Emerging Pandemic Threats) PREDICT project is included in this plan and it is focused on the study of the biology of some dangerous viruses, such as Ebola, Lassa fever, Rift Valley fever and avian influenza. PREDICT project was founded also to discover new viral species in the animal reservoir host and individuate the main characteristics that can cause the viral transmission to human, to avoid the viral outbreak in the population. Next-generation sequencing can help the massive sequencing of this viral genome samples collected, allowing the increase of speed and efficiency and moreover reducing the cost of sequencing. To validate the possibility for these new-discovered viruses to be transmitted from animal to human, new approaches must be developed for the study of their pathogenicity. The Global Virome Project could help the current pandemic surveillance, diagnosis techniques and prevention strategies, as far as helping for pre-emptive production of vaccine and other countermeasures for candidate high-risk viruses. Another benefit of this research could be a deeper comprehension of the viral biology. Its discovers can be applied not only for medical need, but also in other field as the agricultural and food one, for example to enhance biosecurity of food. It is used to sequence all the microbial genomes in a sample by using the Shotgun approach. Its aim is to identify the nucleic acid diversity present in the sample (either DNA, RNA or both, depending on the sequencing method), in order to provide information about features of the viruses within the samples such as drug resistance, viral genotypes and virus epidemiology. The sensitivity of this method is affected by the presence of contaminating nucleic acids from the host and other microorganisms. This method has been used for the sequencing of viruses like Epstein-Barr virus (EBV) and HCV. It may be used also to provide information about cancer evolution and integrated virus genomes in cases of virus-associated cancers. This method requires a low number of PCR cycles, so the consequent risk of contamination is decreased. Although no primers or probes are required, the cost to obtain enough data is high. Because this method is agnostic to expected viral content of a sample, it can be used to identify new virus species or divergent members of known species. It therefore has a role in clinical diagnostics, such as identification of pathogens causing encephalitis. Its aim is to identify the organism and in order to do that, it enriches a portion of the genome of the virus before sequencing. For the amplification it uses specific primers for a highly-conserved target sequence. This method has been used to track Ebola virus and Zika virus during their outbreaks or to sequence the whole genome of HCMV. Another possible application is the monitoring of mutations associated to drug-resistance in order to administer the more efficient drug to the patient. Although this method is cheaper than the metagenomic approach and has a great specificity and sensitivity, it has some limits: it requires many PCR cycles so it can introduce mutations and contaminants and the primers may be subjected to mismatches. Clinical samples may lack sufficient nucleic acid to enable many PCR reactions; this makes PCR amplicon sequencing of viruses more appropriate if the viral genome is small (eg influenza, norovirus or HIV), or if the virus has been cultured to increase the available genomic material. It is an overlapping PCR method. It doesn’t require a culture step because it sequences the whole viral genome directly from the clinical sample. Small oligonucleotides, complementary to the target, are used as probes for a hybridization reaction. The probes can be bound to a solid phase or to magnetic beads in liquid phase. Capture is followed by a small number of PCR cycles and Shotgun sequencing. This method can be used to compare the genome of healthy cells and of tumoral cells in cases of virus-associated cancer. It has been used to characterize HCV, HSV-1, HCMV and other viruses. The presence of overlapping probes increases the tolerance for primer mismatches but their design requires high cost and time so a rapid response is limited.