**Metagenomic Applications**

**Chapter 2**

**Provisional chapter**

**Potential Applications and Challenges of**

**Potential Applications and Challenges of** 

DOI: 10.5772/intechopen.75023

Complex association of human host and pathogenic viruses makes a necessity to understand the overall host and virus interaction network. Identification of virus population and its systematic classification will help in understanding the viral association with the disease outcome. Metagenomics is a recently developing approach for the detection of pathogens in the samples with precise interpretation in a short period of time. Metagenomic approaches have been employed for studying the predominance or spread of the virus within a particular locality and nature of virus during infection. Metagenomics is basically a collective approach of lab-based techniques and in-silico methods for identification of pathogenic viruses without culturing them in specific aseptic conditions. Lack of unique conserved genes in viruses has made metagenomics study difficult in this juncture. Other challenges in the field of metagenomics are like cellular DNA contamination, free environmental DNA contamination and continuous evolution of viruses. Recent studies have shed light on the advancement of this field in virus identification and characterization however still needs further investigations to overcome the challenges. Current chapter focuses on the application and challenges faced in metage-

**Keywords:** metagenomics, viral metagenomics, gastrointestinal infections, applications

In Latin, the term virus means toxin, virus are obligate intracellular parasites with RNA or DNA as a genetic material. They vary in size from ~20 nm to ~1.5 μm and simple machinery. Viruses cant able to replicate themselves as they are intracellular parasites and require susceptible host

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Metagenomics in Human Viral Infections**

**Metagenomics in Human Viral Infections**

Prudhvi Lal Bhukya and Renuka Nawadkar

Prudhvi Lal Bhukya and Renuka Nawadkar

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

nomic analysis of human viral infections.

of metagenomics

**1. Viruses**

http://dx.doi.org/10.5772/intechopen.75023

**Abstract**

#### **Chapter 2 Provisional chapter**

#### **Potential Applications and Challenges of Metagenomics in Human Viral Infections Potential Applications and Challenges of Metagenomics in Human Viral Infections**

DOI: 10.5772/intechopen.75023

Prudhvi Lal Bhukya and Renuka Nawadkar Prudhvi Lal Bhukya and Renuka Nawadkar

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75023

#### **Abstract**

Complex association of human host and pathogenic viruses makes a necessity to understand the overall host and virus interaction network. Identification of virus population and its systematic classification will help in understanding the viral association with the disease outcome. Metagenomics is a recently developing approach for the detection of pathogens in the samples with precise interpretation in a short period of time. Metagenomic approaches have been employed for studying the predominance or spread of the virus within a particular locality and nature of virus during infection. Metagenomics is basically a collective approach of lab-based techniques and in-silico methods for identification of pathogenic viruses without culturing them in specific aseptic conditions. Lack of unique conserved genes in viruses has made metagenomics study difficult in this juncture. Other challenges in the field of metagenomics are like cellular DNA contamination, free environmental DNA contamination and continuous evolution of viruses. Recent studies have shed light on the advancement of this field in virus identification and characterization however still needs further investigations to overcome the challenges. Current chapter focuses on the application and challenges faced in metagenomic analysis of human viral infections.

**Keywords:** metagenomics, viral metagenomics, gastrointestinal infections, applications of metagenomics

#### **1. Viruses**

In Latin, the term virus means toxin, virus are obligate intracellular parasites with RNA or DNA as a genetic material. They vary in size from ~20 nm to ~1.5 μm and simple machinery. Viruses cant able to replicate themselves as they are intracellular parasites and require susceptible host

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

for their propagation. Extracellular viral particles are noninfectious in nature. They can infect a wide range of hosts including plants, bacteria, fungi, algae, protozoa, vertebrate or non-vertebrate animals. In nature, around 1 × 1031 number of different viruses are present. The number itself suggests the diversity of viruses in nature. They play a very important role such as an increase in diversity via horizontal gene transfer in hosts, and nutrient recycling [1]. Report from Hooda et al. showed the abundance of viruses in nature is around 1000 times more than observed via cell culture dependent technique [1, 2]. This suggests the large pool of viruses is still unknown, only around 219 pathogenic viruses have been yet identified [2, 3].

of embryonated eggs or laboratory animals. Discovery of tissue culture technique in the 1900s provides an indispensable tool for in vitro virus culture. Tissue culture technique has been then recognized as a "gold standards" for virus discovery. Major advantages of using tissue culture technique for virus identification are an amplification of viruses, characterization of the virus, functional studies, drug targeting, and genome extraction. Due to authentic results and sensitivity of the technique, tissue culture-based techniques are still in use for virus discovery, as well as immune responses study, altered gene expression and characterization of viruses. Successful use of tissue culture technique in virus identification depends on crucial steps involved such as collection of a sample from high titer area of the body, immediate transport of sample, sample processing and selection of appropriate cell line [10]. The major defects of traditional method for virus identification are difficulties in identification of susceptible cell line, time-consuming and laborious in nature [10]. Moreover, culture-based virus identification is further succeeded with the evolution of new scientific techniques and modification in existing techniques. Shell vials with centrifugation, PRE-CFE stain technique, immune-based techniques, e.g., ELISA, agglutination, precipitation, flocculation, microscopy-based techniques, reduced the time of virus identification but

Potential Applications and Challenges of Metagenomics in Human Viral Infections

http://dx.doi.org/10.5772/intechopen.75023

21

Gradually field of virology shifted their particles toward molecular biology methods. Together, traditional culture-based methods and molecular biology techniques are used hand in hand for studying virus associated samples [11]. Broadly molecular biology methods are of two types: sequence dependent and sequence independent. Both the methods have proven its

**1. Sequence-dependent method**: These techniques are most sensitive molecular biology techniques; it can amplify selective DNA from mixed samples [12]. Since the time of discovery of PCR, it has opened the door for many other variations of PCR for multiple gene modulations. The basic backbone of molecular biology PCR is, it has been used in several approaches such as for sequencing of known viruses depending on similarly in sequence in DNA or consensus sequence of previously known viruses, RFLP and diagnostic purposes [13–16]. Another technique, microarray introduced in 1995, it is used mainly for gene expression studies, used in gene profiling, usually in infected samples. Two methods have been used for discovery of new viruses, taxa, gammaretroviruses and xenotropic murine leukemia virus, SARS-CoV are few best examples [17]. The subsequent studies were unable

**2. Sequence-independent method:** This approach is independent of prior knowledge of virus genome sequence. Sequence subtractive hybridization and representational difference analysis were methods used for detection of gene expression studies and comparison of genome sequence repetitively [18]. Use of these methods was helpful for detection of human herpes simplex virus type 8 (HHV-8) [9, 19], GBV-A, GBV-B virus [20, 21], Tonovirus

usefulness; many viruses have been identified using these techniques.

compromising sensitivity.

**4.2. Molecular methods**

to reproduce the earlier results [6, 7].

and norovirus [22].
