Viral Metagenomics

Viral Metagenomics FAQs

• Can metagenomics detect RNA viruses?

  • Metagenomic techniques can be utilized for the detection of RNA viruses. Within the field of metagenomics, it is feasible to perform concurrent sequencing and analysis of substantial quantities of both DNA and RNA located within samples. As pertains to RNA viruses, the virus-specific RNA can be transcribed into cognate DNA prior to sequencing. This methodology permits the identification of a variety of RNA viruses present in the sample, inclusive of single-stranded and double-stranded RNA viruses, among others.

• What is the use of metagenomics to describe viral diversity?

  • Viral metagenomics can unveil the strain-based diversity intrinsic to virology. Harnessing metagenomic techniques fosters a comprehensive sequencing and incisive analysis of virus populations, shedding light on the sequence variation and differentiation between distinct viral strains. Such understanding is instrumental in deciphering the taxonomy and evolutionary relationships of viral strains, thereby augmenting accuracy within the domain of virus classification. Furthermore, metagenomics is adept at detecting an array of unknown or novel viral sequences encapsulated within samples. Analyzing these sequences paves the way for the identification of innovative virus types, invariably enhancing our understanding of viral diversity.

• What are the challenges in the analysis of viral metagenomes?

  • One of the main challenges of viral metagenomics stems from the diverse nature of viral nucleic acid types. Viruses, for example, can be categorized into double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses, etc. In addition, due to RNA viruses mutating at a faster pace than DNA viruses-coupled with the fact that DNA is easier to process from samples while RNA is unstable and requires more steps for RNA metagenomic analysis (reverse transcription)-there is an observable bias for DNA viruses in reference databases.

    Finally, the issue of sequence contamination from host organisms is significant. The source of the sample and preprocessing methodology can likely affect subsequent analysis results. Therefore, rigorous quality control measures and sample processing procedures need to be applied.

• What is the difference between 16S rRNA and metagenomics?

  • The use of metagenomics enables researchers to comprehensively study all types of microbes within a given community by providing exhaustive genomic information. Yet, despite offering high levels of precision and resolution, metagenomics involves complex data processing and analysis. Its inherent characteristics, however, make it highly efficient in meticulously identifying microbial species, and delving into their functions and metabolic potential.

    On the contrary, 16S rRNA sequencing allows for limited insights into 16S rRNA genes within the microbial community. With its comparatively reduced resolution, 16S rRNA sequencing appears simpler with regards to data treatment and analysis, making it a frequent choice for exploring phylogenetic relationships among microbes.

    More detailed descriptions of the differences are given in Microbial Species Identification Methods: 16S rRNA and Metagenomic Sequencing.

Demo

Intestinal virome in patients with alcoholic hepatitis. (Jiang et al., 2020)

Multi-omics analyses show disease, diet, and transcriptome interactions with the virome. (Mihindukulasuriya et al., 2021)

Case Study

Viral metagenomics reveals diverse viruses in the fecal samples of children with diarrhea
Journal: Virologica Sinica
Impact factor: 5.573
Published: February 2022

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Backgrounds

In less industrialized nations, particularly Southeast Asia and sub-Saharan Africa, diarrheal diseases represent the third leading cause of mortality in children under five years of age. While recent improvements in sanitation and living standards have notably reduced the incidence and mortality rate of diarrheal diseases, childhood diarrhea resultant from viral infections remains a significant public health concern in developing countries. Viruses are the primary pathogens in such cases, accounting for 75% of total incidences. Array of viruses causing diarrhea includes Human Adenovirus (HAdV), Norovirus (NoV), Sapovirus (SaV), Human Astrovirus (HAstV), Rotavirus, Enterovirus, and Picornaviruses. However, knowledge about the prevalence of these viruses in children suffering from diarrhea remains considerably limited to date.

Methods

Sample preparation:

• One hundred fresh stool samples
• Viral DNA and RNA extraction

Viral Metagenomics Sequencing:

• Reverse transcription synthesizes
• Library construction
• Miseq Illumina platform

Data analyses

• Bioinformatics analysis
• Phylogenetic analysis

Results

1. Viral metagenomic overview

In this study, analysis of 100 fecal samples from children with diarrhea revealed that among the eukaryotic viral families, Caliciviridae predominated at 78.42%, followed by Adenoviridae (8.94%), Picornaviridae (8.36%), Reoviridae (2.81%), Astroviridae (1.18%), Reoviridae (0.20%), Anelloviridae (0.04%), and Picobirnaviridae (0.03%).

Fig. 1. Taxonomic analysis of fecal virome detected in diarrheal children on the family level. A The composition of fecal virome detected in diarrheal children. The percentage of virus sequences in different virus family was shown. B Heatmap representing the reads number of each viral family in exponential form.

2. Phylogenetic analysis

In this study, nine viruses belonging to two genera of the Caliciviridae family were identified, of which eight were categorized as Norovirus (NoV), and one was part of the Sapovirus (SaV) genus. Seven viruses were under two genera of Picornaviridae, with three nearly full Enterovirus (EV) genomes and four almost complete sequences of Human parechoviruses. In addition, the presence of viruses belonging to the Mamastrovirus, Bocavirus, Mastadenovirus, and Rotavirus genera was also reported. This analysis expands the current knowledge of virodiversity in cases of paediatric diarrhoea.

Fig. 2. The phylogenetic analysis based on the complete genome sequences of NoVs and SaVs.

Fig. 3. The phylogenetic analysis of picornaviruses identified in this study.

Fig. 4. The phylogenetic analysis of human rotaviruses identified in this study.

Conclusion

In this investigation, the researchers utilized viral metagenomic sequencing to analyze the viral communities present in fecal samples from children suffering from diarrhea. They observed that a significant proportion of DNA or RNA viruses associated with diarrhea belonged to six families: Adenoviridae, Astroviridae, Caliciviridae, Picornaviridae, Reoviridae, and Picobirnaviridae. Notably, Caliciviridae exhibited the highest prevalence at 78.42%, followed by Adenoviridae (8.94%) and Picornaviridae (8.36%). In addition to known pathogens implicated in human diarrheal diseases, the researchers identified unrelated viruses such as Circoviridae and Picobirnaviridae. This study advances our comprehension of the virome in pediatric diarrhea, furnishing valuable insights for the prevention and management of viral gastroenteritis.

References

1. Braga L P P, Coutinho F H, Amgarten D E, et al. Novel virocell metabolic potential revealed in agricultural soils by virus‐enriched soil metagenome analysis. Environmental microbiology reports, 2021, 13(3): 348-354.
2. Bai G H, Lin S C, Hsu Y H, et al. The human virome: viral metagenomics, relations with human diseases, and therapeutic applications. Viruses, 2022, 14(2): 278.
3. Coutinho F H, Gregoracci G B, Walter J M, et al. Metagenomics sheds light on the ecology of marine microbes and their viruses. Trends in microbiology, 2018, 26(11): 955-965.
4. Greninger A L. A decade of RNA virus metagenomics is (not) enough. Virus research, 2018, 244: 218-229.
5. Jiang L, Lang S, Duan Y, et al. Intestinal virome in patients with alcoholic hepatitis. Hepatology, 2020, 72(6): 2182-2196.
6. Mihindukulasuriya K A, Mars R A T, Johnson A J, et al. Multi-omics analyses show disease, diet, and transcriptome interactions with the virome. Gastroenterology, 2021, 161(4): 1194-1207. e8.
7. Shixing Yang, Yumin He, Ju Zhang, et al. Viral metagenomics reveals diverse viruses in the fecal samples of children with diarrhea. VIROLOGICA SINICA, 2022, 37(1): 82-93