Rotaviral Diseases and Their Implications

*Kirti Nirmal and Seema Gangar*

#### **Abstract**

Rotaviruses (Latin rota, "wheel"), the name derived from the wheel-like appearance of the virions when viewed by negative-contrast electron microscopy Rotavirus, are one of the foremost causes of rigorous peadiatric diarrhea globally. According to WHO, it is the primary cause of severe diarrhea among young children, leading to 4.5 million hospitalizations and more than 700,000 deaths of children aged 5 and under annually. The viruses are present in the stool of an infected person and can remain viable for a long time on contaminated surfaces, including people's hands. They are transmitted by fecal-oral route. Fecal contamination of food and water are common reservoirs and fingers, flies and fomites play vehicular role in transmission of rotaviruses. Both symptomatic and asymptomatic infections can lead to viral transmission due to shedding of viruses, often observed in close contacts, day care centers or via infected food handlers or healthcare workers. The disease manifested the symptoms of rotavirus infection, which may last up to 8 days and comprises fever, nausea, vomiting, abdominal cramps, and frequent, watery diarrhea. Two types of the rotavirus vaccine RotaTeq (RV5) and Rotarix (RV1) are available. Both vaccines are administrated orally, not as a shot. This chapter focuses on new information related to the clinical presentation and pathogenesis of rotavirus infection and its implications.

**Keywords:** rotavirus, disease, virus, systematic review

#### **1. Introduction**

Rotaviruses are recognized as a major cause of viral gastroenteritis among children since 1973 when Ruth F. Bishop and his colleagues observed the virus particles in the duodenal epithelial cells of gastroenteritis patients by electron microscopy [1]. As per WHO, Rotaviruses are responsible for approximately 453,000 deaths/year among children aged <5 years, globally, and over 2 million children are hospitalized each year with pronounced dehydration from rotavirus infection [2].

#### **2. Taxonomy**

The rotavirus (RV) genome is comprised of eleven segments of double-stranded RNA (dsRNA) and is contained within a non-enveloped, icosahedral particle.

During assembly, a highly-coordinated selective packaging mechanism ensures that progeny RV virions contain one of each genome segment. It has two subfamilies: Sedoreovirinae and Spinareovirinae [3].

#### **3. Morphology**

The name Rotaviruses (Latin *rota*, "wheel") is derived from the wheel-like appearance of the virions when viewed by negative-contrast electron microscopy (**Figure 1**). The mature virion has an approximate diameter of about 100 nm and possesses icosahedral symmetry. It is made up of three concentric protein layers and thus also known as a "triple-layered particle" or TLP. The mature virion lacks a lipid envelope (**Figure 2**) [4]. However, there is the acquisition of a transient lipid envelope during the budding of immature particles into the endoplasmic reticulum (ER). The triple-layered capsid encloses 11 discrete segments (seg 1–11) of linear dsRNA, which codes for six structural viral proteins, (VP1, VP2, VP3, VP4, VP6, and VP7) and six non-structural viral proteins (NSP1-NSP6). Each segment of dsRNA codes one protein except for segment 11, which codes two proteins (**Figure 3**) [5].

The outermost layer of the capsid is an icosahedral-shaped lattice made up of 780 copies of VP7 (38 kDa), a glycoprotein, and 120 copies of the spike protein VP4 (88 kDa) [6]. As per cryo-EM studies, VP4 has a large globular domain that is buried inside the inner layer, and it forms the spikes that emanate through the outer layer, which helps in cell attachment, penetration, hemagglutination, neutralization, and virulence of Rotaviruses [7]. The VP4 or the spike protein has a trypsin cleavage site along its length. The infectivity of virus particles gets enhanced by several folds on proteolytic cleavage of VP4 (88 kDa) into VP8<sup>∗</sup> (28 kDa) and VP5<sup>∗</sup> (60 kDa). It induces conformational changes to stabilize the spike protein. VP8<sup>∗</sup> and VP5<sup>∗</sup> remain

#### **Figure 1.**

*Transmission electron micrograph of rotavirus virions (viral particles). (https://www.cdc.gov/rotavirus/about/ photos.html) (taken permission from original resources).*

*Rotaviral Diseases and Their Implications DOI: http://dx.doi.org/10.5772/intechopen.109466*

#### **Figure 2.**

*3D graphical representation of rotavirus virions (viral particles). Rotavirus is known and named for the wheel-like appearance visible under an electron microscope (taken permission from original resources).*

**Figure 3.** *Structure of rotavirus.*

associated with the virion and facilitate virus entry into cells [8]. The intermediate layer of the virion is formed by 260 VP6 trimers in an icosahedral shape. The outer two layers consist of 132 aqueous channels of three types (types I–III), which play an important role in the transcription and translocation of mRNA. The virus particles that lack the outer layer are non-infectious and known as double-layered particles (DLPs), and particles that lack the outer two layers are known as single-layered particles (SLPs) [9]. The VP2 is the most abundant protein; it interacts with genomic RNA on the inside and the VP6 layer on its outer side. The rest of the core proteins are present in small quantities and provide the enzymatic functions required for producing the capped mRNA transcripts [10]. The components of outermost layer (VP7 and VP4) carry epitopes for eliciting neutralizing antibodies independently. Based on this, a dual classification system was established decades ago to define Rotavirus G (VP7 is a **G**lycoprotein) and P (VP4 is **P**rotease sensitive) genotypes. So far, 36 G genotypes and 51 P serotypes have been identified [11].

#### **4. Nucleic acid**

The genome of rotaviruses consists of 11 discrete segments of linear dsRNA of 18,555 bp size. The size of the segments ranges from 667 bp to 3302 bp. The genome is A + U rich (58–67%). **Table 1** describes the size, protein products encoded by segments of dsRNA, and functions of proteins of the Rotaviruses group [5, 11]. The minor proteins VP1 and VP3 form a heterodimer, which is anchored to the inner surface (N-terminal residues) of the VP2 layer and is known as the transcription enzyme complex. The VP2 layer in addition to proper positioning of the transcription enzyme complex helps in the regulation of endogenous transcription of the genome [5, 12]. The transcription of the genome is a dynamic process, the dsRNA segments move around the anchored transcription enzyme complex (**Figure 3**). The similar assembly of the structures has been observed in members of ortho-reoviruses; however, their capping enzyme is outside the innermost layer (**Table 1**).

#### **5. Replication**

Rotaviruses enter the body via the fecal-oral route and infect the epithelial cells of the villi in the small intestine. The virus multiplies in the cytoplasm of the epithelial cells [12]. The rotaviruses replicate well in continuous cell cultures derived from monkey kidneys. The replication cycle is approximately completed in 12–15 h at 37°C. The replication cycle can be divided into the following steps (**Figure 4**) [8]. The following steps need to be followed for replication of the virus. Attachment (mediated by VP4 and VP7), Penetration and uncoating, Synthesis of the plus strand of ssRNA (which also acts as mRNA), Formation of viroplasm, RNA packaging, synthesis of minus-strand and DLP formation, and Virus particle maturation (TLP formation) and release.

#### **5.1 Attachment**

The VP4 or the spike protein is the viral attachment protein of the TLPs. The VP8\* subunit of VP4 (at the tip of the spikes) interacts with the sialoglycans (such as gangliosides GM1 and GD1a) and non-sialoglycans receptors such as histoblood group antigens (HBGAs), receptors present on the host cell [13, 14]. Several integrins (α2β 1, ανβ3, αxβ2, and α4β1) and heat shock protein 70 (hsc70) serve as co-receptors, which interact with VP5\* or VP7 [15]. The VP8\* subunit also mediates hemagglutination in some strains of rotaviruses [10, 16]. The VP4 spike undergoes conformational change the moment it interacts with the host cell receptors leading

*Rotaviral Diseases and Their Implications DOI: http://dx.doi.org/10.5772/intechopen.109466*


#### **Table 1.**

*The number of and size of each gene segment and the functions of their encoded proteins.*

to the exposure of the VP5\* subunit, which is normally hidden under VP8\*. The trypsin facilitates this virus's entry into cells by inducing conformational changes and stabilizing the spike protein.

#### **Figure 4.**

*The rotavirus replication cycle. The rotavirus triple-layered particles (TLPs) first attach to sialo-glycans on the host cell surface, followed by interactions with other cellular receptors (integrins and Hsc70). Virus is then internalized by receptor-mediated endocytosis. Removal of the outer layer, triggered by the low calcium of the endosome, results in the release of transcriptionally active double-layered particles (DLPs) into the cytoplasm. The DLPs start rounds of mRNA transcription, and these mRNAs are used to translate viral proteins. Once enough viral proteins are made, the RNA genome is replicated and packaged into newly made core in specialized structures called viroplasms. Addition of VP6 protein to the core forms the DLPs. The newly made DLPs bind to NSP4, which serves as an endoplasmic reticulum (ER) receptor, and bud into the ER. NSP4 also acts as a viroporin to release Ca2+ from intracellular stores. Transiently enveloped particles are seen in the ER. The transient membranes are removed as the outer capsid proteins VP4 and VP7 assemble, resulting in the maturation of the TLPs. The progeny virions are released through cell lysis.*

#### **5.2 Penetration and uncoating**

After binding, the virusparticles (TLPs) penetrate the host cell either by receptormediated endocytosis or direct membrane penetration. Due to low Ca2+ concentrations in endosomes, the outer capsid proteins (VP4 and VP7) solubilize and release the transcriptionally active DLP form in the cytoplasm of the host cell [17].

#### **5.3 Synthesis of the plus strand of ssRNA and viroplasm formation**

The transcription complexes (RdRp/VP1/Capping enzymeVP3) in the core of the DLPs are complex with the dedicated segment of viral dsRNA [17, 18]. The negative strand of the genomic RNA acts as a template, and the transcription complex synthesizes a capped, non-polyadenylated plus (+) ssRNA, which is released in the cytoplasm via type I aqueous channels [18]. These plus (+) ssRNA transcripts serve two functions. First, they are used for the translation of virus-encoded proteins in the cytoplasm, thus also called mRNA, and later on in the replication cycle, they act as templates for negative-sense strand synthesis and producing dsRNA genome segments [16–18].

#### **5.4 Formation of the viroplasm**

Two non-structural proteins NSP2 and NSP5 mediate the synthesis of virion assembly in cytoplasmic inclusions termed viroplasms. NSP2 octamer forms a complex with VP1, VP2, and tubulin to form viroplasms. The NSP2 octamer has a binding site for which NSP5 and (+)ssRNA compete, thus it regulates the balance between translation and replication [19].

#### **5.5 RNA packaging, synthesis of minus-strand RNA, and DLP formation**

The transcription complex interacts with VP2 decamer, NSP5, and NSP2 and forms the core particle The 11 different (+) ssRNA segments interact with the transcription complex and VP2 and are then packaged [20]. This interaction leads to the synthesis of a negative-sense strand and results in the formation of cores containing a complete set of 11 dsRNA segments. Once formed, VP6 is added to the core leading to the synthesis of DLPs [20].

#### **5.6 Virus particle maturation (TLP formation) and release**

The NSP4 is a transmembrane glycoprotein, located in the endoplasmic reticulum (ER). It interacts with the VP6 protein and acts as an intracellular receptor for DLPs [21]. The NSP4 acts as viroporin as it increases intracellular Ca2+ levels by releasing Ca2+ from intracellular stores. The elevated levels of intracellular Ca2+ are needed to stabilize the outer layer of TLPs, and it activates a kinase-dependent pathway, which leads to autophagy [22]. NSP4 also acts as viral enterotoxin and interacts with noninfected intestinal cells [21, 22]. Interaction of NSP4 and DLP is crucial in the maturation process. The DLPs are recruited to the ER. Budding of DLPs through the ER results in the transient acquisition of a lipid envelope followed by the acquisition of the outermost layers by the addition of VP4 and VP7 to the DLPs. The newly formed TLPs are then released by either cell lysis or exocytosis [8].

#### **5.7 Physical properties**

The outermost layer (VP7 and VP4) of the infectious triple-layer particles (TLPs) can be destabilized with Ca2+-chelating agents, such as EDTA, leading to the loss of the outermost layer and formation of double-layered particles (DLPs). The DPLs are non-infectious. The virion became non-infectious by treating it with 95% ethanol, 0.1% sodium dodecyl sulfate, beta-propiolactone, chlorine, formalin, and phenols. The hemagglutinin activity of VP4 is lost at 45°C or by freezing and thawing [4, 5].

#### **6. Pathogenesis**

Rotaviruses are ubiquitous, and TLPs are relatively stable in the environment. They are transmitted by the fecal-oral route. Fecal contamination of food and water are common reservoirs and fingers, flies and fomites play a vehicular role in the transmission of rotaviruses. Both symptomatic and asymptomatic infections can lead to viral transmission due to the shedding of viruses, often observed in close contact, with day care centers, or via infected food handlers or healthcare workers [23].

The rotaviruses mainly infect the villi of small intestinal mucosa leading to loss of microvilli, villous atrophy, and necrosis of the gut epithelium. The virions extensively replicate in the cytoplasm of enterocytes and damage their transport mechanism resulting in electrolytes and fluid malabsorption leading to increased osmotic pressure in the gut lumen and subsequently onset of diarrhea [8, 24].

The NSP4 protein, encoded by rotaviruses, is a viral enterotoxin that induces calcium ion-dependent chloride secretion into the intestinal lumen via activating signal transduction pathways through phospholipase C. The NSP4 inactivates the Sodium-Glucose-Lactose-Transporter proteins system (SGLT1) that mediates the reabsorption of water, sugar, and body electrolytes. The NSP4 also stimulates the enteric nervous system by inducing the secretion of serotonin (5-HT: 5-hydroxytryptamine) from enteroendocrine cells due to an increase in intracellular calcium concentration, thus increasing gut motility [24, 25].

#### **7. Clinical manifestations**

Rotaviruses are the major cause of diarrheal illness in infants and children worldwide. The infectious dose is low (<100 TLPs), and the incubation period is of 18–36 h followed by acute onset of symptoms. The symptoms vary from mild to severe watery diarrhea, vomiting, abdominal pain, fever, and dehydration. Diarrhea may last for


**Table 2.** *Summary of rotavirus.* *Rotaviral Diseases and Their Implications DOI: http://dx.doi.org/10.5772/intechopen.109466*

5–7 days. The majority of patients usually recover completely. Infected adults are usually asymptomatic or rarely exhibit symptoms. However, infants and children may suffer because of severe loss of fluid and electrolytes leading to dehydration or death if untreated. The excretion of viruses in the stool may persist up to 50 days after the onset of diarrhea (**Table 2**).

#### **8. Epidemiology**

As per WHO, more than 25 million outpatient visits and more than 2 million hospitalizations each year globally are due to severe, dehydrating diarrhea by rotavirus infections in children aged less than 5 years. Generally, the mortality rate is higher in low and middle-income countries as compared with high-income countries. In low and middle-income countries, approximately 75% of children acquire their first episode of rotavirus infection within 12 months of age, whereas children of 2–5 years of age are predominantly infected in high-income countries. The group A rotaviruses (RVA) is the major group that accounts for more than 90% of rotavirus infections in humans worldwide [26]. Rotavirus gastroenteritis is predominantly seen during cool, dry seasons thus also known as "winter diarrhea" and in tropical areas, it can be seen all the year around [27]. Nosocomial outbreaks of rotaviruses are common in pediatric wards, elderly, and immunocompromised patients.

#### **9. Lab diagnosis**

A fresh stool sample should be collected in well-labeled, clean wide-mouth container for the presence of the virus, virus-specific antigen, or RNA by direct or indirect detection methods. Rectal swabs can also be taken, but they are not ideal samples [27].

#### **9.1 Direct method**

The demonstration of the viral particle in stool sample by electron microscopy shows a typical wheel-shaped virion. The sensitivity and specificity can be improved by immune-electron microscopy by adding specific antiviral antibodies to the specimen or by utilizing magnetic microparticles functionalized with monoclonal antibodies to enhance the ability to capture, concentrate, separate, and detect infectiously [28]. The rotavirus can be cultured on cell lines such as monkey kidney cell line to confirm viral viability and also improves the molecular detection of the virus in clinical samples where low viral load is present, although the method is highly sensitive, it is time-consuming, expensive, highly prone to contamination, and is usually available at research-based laboratories [20].

#### **9.2 Indirect method**

Indirect detection of viruses by a variety of molecular assays to detect antigens, antibodies, or viral RNA is more popular, cost-effective, and less time-consuming as compared with direct methods. Commercially available antigen detection kits based on various principles such as immunochromatography or latex agglutination are widely used point-of-care tests for rotavirus diagnosis in resource-limited nations.

The ELISA-based antigen detection techniques are the most widely recognized screening platform with high sensitivity and specificity. A large sample volume can be tested on a 96-well plate [28, 29]. Other immunoassays such as radioimmunoassay, counter-Immuno-electrophoresis, and fluorescent antibody staining are also used.

#### **9.3 Molecular method**

The detection of the rotaviral genome through polymerase chain reaction–based methods (e.g., RT-PCR, RT-qPCR, real-time PCR, and multiplex RT-PCR) are revolutionary as a research tool as well as for diagnosis. Newer PCR-based techniques such as RT-qPCR assays are more sensitive and specific than antigen detection and are suitable for genotyping and sequencing with faster turnaround time as compared with conventional methods [29].

#### **10. Treatment**

The treatment is mainly supportive and consists of rehydration by the oral or intravenous route to restore the loss of water and electrolytes. Commercial preparations of oral rehydration solution (ORS) are available over the counter. To restore the damaged mucosal epithelial lining, the ORS is supplemented with zinc tablets [30].

There is currently no antiviral drug approved for the treatment of rotavirus infections.

There have been studies that demonstrate the anti-rotavirus activity of some drugs such as Nitazoxanide (100 mg in 12–47 months and 200 mg in 4 years old, twice daily for consecutive 3 days) an oralsynthetic anti-parasitic agent inhibits viro plasma formation; gemcitabine, an anticancer drug, with pyrimidine nucleotide inhibitor activity inhibits rotavirus as well. Resveratrol (Inhibitor of viral protein synthesis), Ziyuglycoside II (Inhibitor of TLR4/NF-kB pathway), Brequinar (pyrimidine biosynthesis inhibitor), 20 -*C*-methyl nucleosides (viral polymerase inhibitor), Racecadotril (Intestinal enkephalinase inhibitor, which suppresses the secretion of water and electrolytes into the gut), ML-60218 (RNA polymerase III inhibitors), and Genipin (Entry inhibitor) are potent antiviral drugs [31, 32].

#### **11. Prevention**

General preventive measures:

The rotavirus infection can be prevented by good hygiene measures such as personal hygiene mainly hand and sanitary. Maintenance of food safety standards, safe water supply, and environmental hygiene are essential in preventing outbreaks at food outlets. The WHO recommends continued breastfeeding as maternal antibodies have a protective role and help in reducing the duration and severity of diarrhea [33].

#### **11.1 Rotavirus vaccine**

The rotavirus vaccines that have been licensed by WHO are RotaTeq, Rotarix, Rotavac, and ROTASIIL for the prevention and control of rotavirus gastroenteritis *Rotaviral Diseases and Their Implications DOI: http://dx.doi.org/10.5772/intechopen.109466*

#### *11.1.1 RotaTeq (RV5)*

RotaTeq (RV5) is an oral live attenuated pentavalent vaccine developed by Merck and Co. Inc., USA, and approved by WHO in 2008. RotaTeq is given in three doses at ages 2 months, 4 months, and 6 months. It is a human-bovine reassorted live attenuated vaccine containing four human VP7(G) types (G1, G2, G3, and G4) and one human VP4(P) type. The efficacy of the vaccine in developed nations such as in European countries is higher at 98%; however, the efficacy reduces in low socioeconomic countries such as Asia at 51% and African countries at 64%. Multiple factors such as malnutrition, enteric co-infections, poor maternal health, prevalent strain diversity, and poor maintenance of cold chain storage lead to reduce effectiveness and efficacy of the vaccine in low socioeconomic countries [34, 35].

#### *11.1.2 Rotarix (RV1)*

Rotarix (RV1) is an oral live attenuated monovalent vaccine developed by GlaxoSmithKline Biologicals and approved by WHO in 2009. It is human rotavirus G1P8 genotype months for the prevention of rotavirus gastroenteritis caused by G1 and non-G1 types (G3, G4, and G9). The vaccine is available in lyophilized form, which can be stored at 2°–8°C. It should be used within 24 h of reconstitution in provided diluent. The vaccine is given in two doses (1 ml each) 4 weeks apart. The first dose should be administered before 15 weeks of age, and the two dose series should be completed before 8 months. Similar to RotaTeq, the efficacy reduces to 62–85% in developing countries as compared with developed countries (96%) [36].

#### *11.1.3 Live attenuated, oral vaccine by Bharat Biotech, India, in 2018*

Recently, WHO prequalified another live attenuated, oral vaccine by Bharat Biotech, India, in 2018. It is a monovalent liquid frozen vaccine containing wild-type reassortant G9P11 (116E) strain of rotavirus, prepared in Vero cells. The vaccine is administered as a 3-dose regimen, 4 weeks apart, beginning at 6 weeks of age. The vaccine should not be administered to children older than 8 months of age. The vaccine can be stored at 2–8°C for 7 for months and −20°C for long-term (5 years). The vaccine efficacy is 56.4% (37–70%) in the first year of life in developed countries [37, 38].

#### *11.1.4 Rotasiil*

Rotasiil, developed by the Serum Institute of India, is a pentavalent vaccine containing a lyophilized preparation of live attenuated human-bovine rotavirus reassortant G1, G2, G3, G4, and G9 strains. The vaccine is available in Lyophilized, Thermostable lyophilized and Liquid formulations and thus can be kept at <40°C for up to 18 months and <25°C for up to 30 months. The three doses are given at 6 weeks, 10 weeks, and 14 weeks [38].

#### *11.1.5 Rotavin-M1 (POLYVAC, ThànhphË Hà NÎi, Vietnam) and Lanzhou lamb (Lanzhou Institute of biological product, China)*

Recently, two vaccines have been licensed in Vietnam and India are Rotavin-M1 (POLYVAC, ThànhphË Hà NÎi, Vietnam) and Lanzhou lamb (Lanzhou Institute of biological product, China) respectively though yet to be licensed by WHO, and their coverage is limited [38].

#### *11.1.6 Rotavin-M1*

Rotavin-M1 is a live attenuated human rotavirus strain G1P8 available in the liquid frozen formulation, which can be stored 2–8°C for 2 months and 20°C for 24 months. Two doses are required minimum at 6 weeks of age, for 4 weeks apart. On the other hand, Lanzhou lamb is a live attenuated lamb G10P15 rotavirus strain available in the liquid formulation, which can be stored at 2–8°C for 12 months [37, 38]. The vaccine is given as a single dose followed by annual boosters for children aged between 2 months and 3 years [35]. Rotavirus causes acute gastroenteritis among children less than 5 years of age. Early detection and treatment are crucial to prevent mortality. Improvement in vaccination coverage, safe water supply, personal and environmental hygiene, continued surveillance, and knowledge of circulating genotypes of the virus in the environment are needed.

Rotavac and Rotasiil are manufactured in India, while others are manufactured outside India.

#### **12. Conclusion**

Rotavirus remains the leading cause of severe acute dehydrating diarrhea among infants and young children for four decades with a substantial effect on morbidity and mortality. This infection is primarily localized to the intestinal enterocytes leading to necrosis of mucosal cells by various mechanisms and subsequently secretorydriven watery diarrhea. It is to be expected that together with rotavirus-mediated gastroenteritis such extra-intestinal manifestations will become less prevalent as a consequence of the introduction of anti-rotavirus vaccination. Clinicians, particularly pediatricians caring for rotavirus-infected children, should be aware of the possible extra-intestinal complications of rotavirus infection. The currently available vaccines have significantly reduced mortality in the developed world, but vaccine efficacy, safety, and cost still pose a significant challenge to developing countries. Considering that the attack rate and mortality are relatively higher in developing countries, the control of rotavirus-attributable gastroenteritis mainly relies on vaccine coverage along with good hygienic measures. The introduction and expansion of the use of next-generation RV vaccines with better efficacy, safety, and low cost are necessary along with a multifaceted health approach to address the infections by rotavirus. A new generation of rotavirus vaccines will soon be licensed in many countries and available for more widespread use. Identifying the full value of these vaccines to prevent mortality from rotavirus in developing countries is still several years away and each of the vaccines must first show its effectiveness in poor populations in Africa and Asia. Although many hurdles remain to ascertain the effectiveness of these vaccines in key target populations and their affordability and to remove lingering concerns about safety from intussusception, the presence of two candidate vaccines provides an important new instrument to decrease the morbidity and mortality associated with rotavirus diarrhea.

*Rotaviral Diseases and Their Implications DOI: http://dx.doi.org/10.5772/intechopen.109466*

#### **Author details**

Kirti Nirmal\* and Seema Gangar Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India

\*Address all correspondence to: doctorkirtinirmal@gmail.com

© 2023 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.

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#### **Chapter 3**
