Viral metagenomics

Metagenomic analysis uses whole genome shotgun sequencing to characterize microbial diversity in clinical and environmental samples. Total DNA and/or RNA are extracted from the samples and are prepared on a DNA or RNA library for sequencing.[21] These methods have been used to sequence the whole genome of Epstein-Barr virus (EBV) and HCV, however, contaminating nucleic acids can affect the sensitivity to the target viral genome with the proportion of reads related to the target sequence often being low.[22][23] Due to the uncontrollable nature of environmental DNA samples, the most abundant organisms in the environmental sample are the highest represented in the sequencing data and require large samples to achieve full coverage. That being said, shotgun sequencing ensures that these organisms that would previously go unnoticed in culture dependent methods are represented by some sequence segments.[24]

Metagenomics can be used for pathogen discovery or diagnosis with the proper bioinformatic tools and databases that can evaluate the possible pathogen. Metagenomics requires no prior knowledge of the viral genome as it does not require primer or probe design, allowing for rapid response to emerging threats.[21] 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 or virus-associated cancers.[21]

PCR amplicon enrichment enriches a portion of the viral genome prior to sequencing. This is done via PCR amplification of primers that are complementary to a known, highly conserved nucleotide sequence.[21] PCR amplicon enrichment is then followed by whole genome sequencing methods and has been used to track the Ebola virus,[25] Zika Virus,[26] and COVID-19[27] epidemics. PCR amplicon sequencing is more successful for whole genome sequencing of samples with low concentrations. However, with larger viral genomes and the heterogeneity of RNA viruses multiple overlapping primers may be required to cover the amplification of all genotypes. PCR amplicon sequencing requires knowledge of the viral genome prior to sequencing, appropriate primers, and is highly dependent on viral titers, however, PCR amplicon sequencing is a cheaper evaluation method than metagenomic sequencing when studying known viruses with relatively small genomes.[21]

Target enrichment is a culture independent method that sequences viral genomes directly from clinical sample using small RNA or DNA probes complementary to the pathogens reference sequence. The probes, which can be bound to a solid phase and capture and pull down complementary DNA sequences in the sample.[21] 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. DNA capture is followed by brief PCR cycling and shotgun sequencing. Success of this method is dependent available reference sequences to create the probes and is not suitable for characterization of novel viruses.[21] This method has been used to characterize large and small viruses such as HCV,[23] HSV-1,[28] and HCMV.[29]

Viral metagenomics methods can produce erroneous chimerical sequences.[30][31] These can include in vitro artifacts from amplification and in silico artifacts from assembly.[31] Chimeras can form between unrelated viruses, as well as between viral and eukaryotic sequences.[31] The likelihood of errors is partially mitigated by greater sequencing depth, but chimeras can still form in areas of high coverage if the reads are highly fragmented.[30]

Plant viruses pose a global threat to crop production but through metagenomic sequencing and viral database creation, modified plant viruses can be used to aid in plant immunity as well as alter physical appearance.[32] Data obtained on plant virus genomes from metagenomic sequencing can be used to create clone viruses to inoculate the plant with to study viral components and biological characterization of viral agents with increased reproducibility. Engineered mutant virus strains have been used to alter the coloration and size of various ornamental plants and promote the health of crops.[33]

Viral metagenomics contributes to viral classification without the need of culture based methodologies and has provided vast insights on viral diversity in any system. Metagenomics can be used to study viruses effects on a given ecosystem and how they effect the microbiome as well as monitoring viruses in an ecosystem for possible spillover into human populations.[1] Within the ecosystems, viruses can be studied to determine how they compete with each other as well as viral effects on functions of the host. Viral metagenomics has been used to study unculturable viral communities in marine and soil ecosystems.[3][10]

Viral metagenomics is readily used to discover novel viruses, with a major focus on those zoonotic or pathogenic to humans. Viral databases obtained from metagenomics provides quick response methods to determine viral infections as well as determine drug resistant variants in clinical samples.[21] The contributions of viral metagenomics to viral classification have aided pandemic surveillance efforts as well as made infectious disease surveillance and testing more affordable.[19] Since the majority of human pandemics are zoonotic in origin, metagenomic surveillance can provide faster identification of novel viruses and their reservoirs.[34]

Viral metagenomics has been used to test for virus related cancers and difficult to diagnose cases in clinical diagnostics.[35] This method is most often used when conventional and advanced molecular testing cannot find a causative agent for disease. Metagenomic sequencing can also be used to detect pathogenic viruses in clinical samples and provide real time data for a pathogens presence in a population.[34]