Ocular Infection of HCMV: Immunology, Pathogenesis, and Interventions

*Yan Yan and Renfang Chen*

#### **Abstract**

Human cytomegalovirus (HCMV) retinitis accounts for 70% of herpesvirus-infected ocular diseases. Recent advances in knowledge of innate immune responses to viral infections have elucidated a complex network of the interplay between the invading virus, the target cells, and the host immune responses. Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization. Viruses have various strategies to evade or delay the cytokine response, and buy time to replicate in the host. Some signaling proteins impact the virologic, immunologic, and pathological processes of herpesvirus infection with particular emphasis on retinitis caused by HCMV. The accumulated data suggest that signaling proteins can differentially affect the severity of viral diseases in a highly cell-type-specific manner, reflecting the diversity and complexity of herpesvirus infection and the ocular compartment. By summarizing the immunological characteristics and pathogenesis of HCMV ocular infection, it will provide important information on the development of antiviral therapy, immunotherapy, and antidrug resistance.

**Keywords:** human cytomegalovirus (HCMV), retinitis, immunology, pathogenesis, resistance

#### **1. Introduction**

Human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family, which tends to establish asymptomatic and lifelong latent infection [1]. Opportunistic HCMV reactivation is a common cause of increasing morbidity and mortality in newborns, the aged population, solid organ transplant patients, hematologic malignancy, or immunodeficient patients [2, 3].

HCMV was first reported to induce HCMV retinitis in 1957 [4], which is known to predominantly target retinal vascular endothelial cells, glial cells, and retinal pigment epithelial cells in the eye [5]. HCMV keratitis or retinitis is the most common opportunistic complication of infection in immunocompromised patients [2, 6, 7], including HIV-1-infected individuals. In general, HIV-1-infected individuals who have viral retinitis tend to be severe, long-lasting, and resistant to conventional treatment with a high rate of complications and significant visual morbidity [7].

Despite the widespread use of highly active antiretroviral therapy (HAART), up to 50–85% of AIDS patients develop ocular manifestations [8–10]. It has been well known that the HCMV can infect the immune-privileged retina site, lead to severe visual loss, and affect the quality of life in HIV-1-positive individuals [5]. Opportunistic infections develop when there is a deterioration of the immune status of the individual, which can be measured with the help of CD4+ T-cell counts [5]. The proportion of HCMV retinitis manifestations was also correlated with the CD4<sup>+</sup> T-cell counts in patients [8]. Retinitis symptom has been classified into two categories, namely, infectious and noninfectious with the vast majority of manifestations occurring in the former. The infectious group mainly consists of the herpetic group of viral infections. Bacterial causes may be due to Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, alpha-hemolytic Streptococcus, *Micrococcus*, and *Bacillus*. Fungal keratitis in HIV-1-infected individuals depend on the geographic locations from which the patient comes. *Microsporidia* and *Acanthamoeba* are common protozoal pathogens. Noninfective inflammatory causes include peripheral ulcerative keratitis, keratoconjunctivitis sicca, and squamous cell carcinoma of the conjunctiva. Posterior segment lesions caused by HCMV show severe visual disorders [7]. A severity that is abnormally severe or minimally reactive makes clinicians suspect immunosuppression. In the HAART era, the incidence, visual morbidity, and mortality of HCMV-related retinitis, and other HIV-1-related retinopathies showed a decline [8, 10]. In this chapter, we will focus on the immunological mechanisms and pathological processes of HCMV infection and strive to highlight those clinical manifestations that should alert the clinicians to suspect underlining HIV-1 infection and provide a basis for intervention.

#### **2. Immunology of HCMV ocular diseases**

#### **2.1 Immunology**

HCMV has evolved a variety of mechanisms to evade host immune surveillance and to establish latent infection with the ability to reactivate when the immune surveillance is compromised [11]. The host immune responses to HCMV involve both innate and adaptive immune systems, which play an important role in resolving both primaries, reactivating, and superinfections [12]. Under the innate and adaptive immune responses, a low viral load and latent state are established in the host after HCMV infection, but under the stimulation of the irregular and intermittent viral antigen reactivation, the functions of T-cells are exhausted [13]. At the same time, the HCMV virus can also encode a large number of gene products that interfere with the immune clearance responses to evade immune surveillance [11, 14, 15]. Considering that immunological clearance and evasion are associated with clinical outcomes, we summarized the immunological mechanisms of HCMV infection.

#### *2.1.1 Innate immune responses*

The natural killer (NK) cell is an important member of the innate lymphoid cell family for defense against HCMV during the early stages of infection and before the development of adaptive immune response due to its strong ability to kill infected or transformed cells [15]. The activities of NK-cells or NKT-cells (a subset of T-cells that co-express T- and NK-cell receptors) depend on the balance between activating and inhibitory signals transduced by its receptors [15]. They are also protected by releasing anti-viral cytokine interferon (IFN)-γ or by direct lysis, or autophagy of infected cells [15]. This will determine the disease progression of HCMV infection with ocular target cells.

#### *2.1.2 Adaptive immune responses*

Accumulating studies have shown that NK-cells take part in adaptive immune responses, such as clonal expansion and immune memory, during cytomegalovirus infection [16]. Clonal expansion not only serves to amplify the number of specific lymphocytes and mount robust protective responses against the pathogen but also results in the selection and differentiation of the responding lymphocytes [16]. In both innate and adaptive lymphocytes, clonal expansion is a critical process for host defenses. It has been shown that antigen (Ag)-specific T-cell expansion was estimated up to 400,000-fold [17]. The intensity of the adaptive immune responses suppresses the acute inflammatory responses caused by HCMV, causing less ocular tissue damage and sequelae.

In primary HCMV infections, CD4<sup>+</sup> T-cells play a vital role in controlling symptomatic disease in healthy and immunocompromised patients. It is important to note that HCMV-infected cells can induce impairment of HCMV**-**specific effector CD4+ T-cell responses [18]. A subpopulation of HCMV-specific CD4+ T-cells has been shown to express Foxp3 and to perform functions similar to regulatory T-cells, such as the production of IL-10 [18, 19]. Latent infection is associated with secretory expression of CCL8, IL-10, and TGF-β [13, 18]. The presence of viral genes and viral IL-10 lead to down-regulate human leukocyte antigen (HLA) class II molecules and limit antigen presentation to CD4+ T-cells in antiviral immunity [12, 18]. At the peak of HCMV infection, HCMV-specific CD4+ T-cells are CD45RA+ CD45RO+ and express CD27+ , CD28+ , CD38+ , and CD40L<sup>+</sup> . During the latent infection period, the HCMVspecific CD4<sup>+</sup> T-cells are rich in CD27<sup>−</sup> CD28− CD4+ T-cells (5–10%) [13]. It has been known that HCMV-specific CD4<sup>+</sup> T-cells are required for the maintenance of HCMVspecific CD8+ T- and B-cell responses in adoptive T-cell immunotherapy in transplant patients [13]. CD8+ T-cells undergo extensive expansion before differentiating into cytotoxic T-cells capable of producing high levels of cytokines, including IL-2, IFN-γ, TNF-α, perforin, and granzyme B [13]. For therapeutics, CD8+ T-cells with long-term survival rates and the potential to respond to challenges are very useful in adoptive transfer strategies for treating HCMV infection. Therefore, HCMV-specific CD8<sup>+</sup> T-cell responses, including the maintenance, distribution, effector function, and metabolic requirements of these cells, have been highly interesting from a vaccine perspective.

Activated HCMV is typically controlled by CD4<sup>+</sup> and CD8<sup>+</sup> T-cell responses, while the virus replicates under the immunosuppressive condition and spreads rapidly to nearby tissues, resulting in worsening of retinitis, such as the patients accompanied with HIV-1 infection and chemotherapy for cancers. In addition to eliminating or perturbing surface immune recognition molecules (HLA I or HLA II molecules) from the antigen-presenting cells (B lymphocytes, dendritic cells, monocytes, or macrophages), HCMV immune evasion mechanisms have evolved to escape recognition and immune clearance of infected cells by effector cells through innate immunity, mimicked the inhibitory ligands or downregulate the activating ligands of NK-cells [11].

#### **2.2 Pathogenesis of HCMV ocular diseases**

Although HCMV infection can occur in healthy individuals, it is uncommon to observe symptomatic infection in individuals without immune suppression [3, 20]. HCMV retinitis is a clinical syndrome characterized by full-thickness necrotizing retinitis, which can result in profound vision loss, retinal detachment, and permanent vision loss [21]. The possible transmission paths of HCMV include blood, prenatal intrauterine infection, perinatal infection through breast milk or genital secretions, saliva, and sperm [22, 23].

In immunocompromised patients, primary HCMV infection causes severe complications, including pyrexia, viremic-septicemia, pneumonitis, and immunosuppression [24, 25]. In total, 60% of patients have been infected with HCMV prior to the onset of critical illness, and are commonly infected before adulthood [26]. It has been shown that patients who suffered HCMV reactivation during critical illness have ~2-flod the mortality rate of those who have not reactivated [27]. The clinical data suggest that the clinical outcome is not only the cause of HCMV replication but also the degree of virus-associated immune responses [3, 25]. In addition to HCMV and HIV-1 co-infected patients with lower CD4<sup>+</sup> T-cell counts have a higher risk of mortality, HCMV has sophisticated strategies to circumvent immunocyte recognition, such as changing the signals of immunomodulatory molecules and subverting T-cell and NK-cell function, and allowing it to establish lifelong infection in blood and bone marrow [16]. In these disorders, HCMV-infected individuals could be accompanied by hemophagocytic lymphohistiocytosis-associated genetic defects. The active and latent infection induce sustained systemic inflammatory responses and predisposes patients to produce autoantibodies, which increased the autoimmune disease progression [28]. It has been demonstrated that individuals infected with one HCMV strain may not necessarily be able to resist other HCMV strains [29]. HCMV also causes immunosuppression associated with T-cell exhaustion, which contributes to the persistence of infection [13].

HCMV retinitis is caused by lytic infection, the conclusion supported by clinical resolution with antiviral therapy. Healing is through fibrosis, which predisposes patients to future retinal detachment and is also the cause of severe vision loss [30]. When antiretroviral therapy is introduced, some individuals with HIV-1 develop immune-restorative uveitis, which is an inflammatory response to the presence of HCMV antigens in the eye by activating viral immediate-early gent product-2 and increasing FasL secretion [31]. This condition may cause more visual impairment in patients than potential retinitis [30]. One possible explanation is that the damage of HIV-1 infection to the blood-retinal barrier may contribute to the preferential entry of HCMV into the oculus [30].

#### **2.3 Interventions of HCMV ocular diseases**

Acute retinal necrosis was first described in Japan as acute unilateral panuveitis, retinal periarteritis, and necrotizing retinitis progressing to retinal detachment [32]. The symptoms of acute retinal necrosis include redness, ocular pain, photophobia, floaters, and blurred vision [32]. These studies suggest that the challenges in diagnosis and therapeutic challenges primarily in the absence of guidelines or evidence-based literature to follow [21].

Ganciclovir was licensed in 1989 and remains the only licensed drug sufficient to treat active HCMV infection. Although the oral prodrug valganciclovir

#### *Ocular Infection of HCMV: Immunology, Pathogenesis, and Interventions DOI: http://dx.doi.org/10.5772/intechopen.105971*

was licensed in 2001, it delivers the same active ingredient. Ganciclovir-resistant HCMV disease has become a serious clinical problem in transplanted populations. Mutations in viral kinase (UL97) or polymerase (UL54) have been shown to mediate resistance to ganciclovir and valganciclovir [33]. For strains of HCMV-resistant to ganciclovir, foscarnet is used off-label. Thus, this field would benefit from more licensed drugs that are both safe and effective anti-HCMV [30]. This becomes particularly important for clinical trials seeking to test the anti-HCMV activity of novel compounds.

In large randomized controlled trials of HIV-1-associated HCMV retinitis in the era before combination anti-HIV treatment, in which the primary endpoint was an objective progression of CMV retinitis, an intra-ocular ganciclovir implant (15% of patients progressed after 100 days of treatment) was superior to intravenous ganciclovir (65% of patients progressed). The limitation of the intra-ocular ganciclovir implant was its failure to prevent CMV disease in the contralateral eye. In a subsequent randomized controlled trial of HIV-associated CMV retinitis, treatment with oral valganciclovir (38% of patients progressed after 100 days of treatment) was similarly effective to initial intravenous ganciclovir for 4 weeks followed by oral valganciclovir (45% of patients progressed). During the latter trial, most patients were also taking a combination anti-HIV treatment. As the ocular penetration of systemically administered anti-CMV drugs is limited, current clinical guidelines include consideration of intraocular injection of anti-HCMV drugs for patients who have sight-threatening HCMV retinitis


*Abbreviations: IV, intravenous; PO, oral; BD, twice daily; FBC, full blood count; UEC, urea electrolytes creatinine; LFT, liver function test*

#### **Table 1.**

*Antiviral treatment for HCMV retinitis [32].*


**Table 2.**

*Biologic immunosuppression and HCMV retinitis [32].*

(**Tables 1** and **2**). In addition to ganciclovir, given that the retina shows acute necrosis of one eye, corticosteroid or methylprednisolone is very important because of its effects in relieving intense inflammatory responses [21].

The HCMV persistent or progressive retinitis may be resolved by systemic administration of virus-specific cytotoxic T-cells (CTLs) [34]. HCMV-specific CTL therapy may become a novel monotherapy or adjunctive therapy, or both, for retinitis, especially in eyes that are resistant, refractory, or intolerant of antiviral therapies [34]. In addition, it has been demonstrated that HCMV strain-specific antibodies play an important role in preventing viral recrudescence after transplantation [29]. Antibodies, natural killer cells, and macrophages theoretically contribute to protective immune responses and are expected to interact and cooperate with T-cells to control HCMV replication. It was also recommended that studies of active immunization should proceed concurrently with passive immunotherapy using monoclonal antibodies with defined reactivity against specific proteins of HCMV against the resistance [34]. Recently, letermovir has been used in ganciclovir-resistant patients at doses of 720–960 mg, while intravitreal therapy with formic acid or ganciclovir was also used, and by monitoring continuous hematologic, renal, and hepatic function, some patients experienced an improvement in symptoms [32].

#### **3. Conclusions**

The interaction between HCMV and the host immune system is complex. NK-cells play an important role in the virus infecting the ocular target cells and take part in the processes of innate immune response and adaptive immune response. After the acute infection in adolescent accompanied by acute symptoms, the virus easily establishes latent infection and reactivate in immunodeficient HIV-1-infected and transplanted individuals. T-cell function is important in controlling HCMV recurrence. Immunodeficient individuals are susceptible to developing HCMV retinitis, which can be treated with systemic and intraocular topical medications, but are also prone to developing drug resistance. Therefore, understanding the immunology and pathogenic mechanisms of HCMV will help us further develop effective antiviral drugs for the treatment or mitigation of HCMV ocular disease.

#### **Acknowledgements**

This work was supported by the Wuxi Key Medical Talents Program (ZDRC024) and the Top Talent Support Program for Young and Middle-Aged People of the Wuxi Health Committee (BJ2020094).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Yan Yan1,2 and Renfang Chen2,3\*

1 Laboratory for Infection and Immunity, The Fifth People's Hospital of Wuxi, Affiliated Hospital of Jiangnan University, Wuxi, China

2 Hepatology Institute of Wuxi, The Fifth People's Hospital of Wuxi, Affiliated Hospital of Jiangnan University, Wuxi, China

3 Department of Infectious Diseases, The Fifth People's Hospital of Wuxi, Affiliated to Jiangnan University, Wuxi, China

\*Address all correspondence to: 1094825330@qq.com

© 2022 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 4**

## Middle East Respiratory Syndrome Coronavirus Outbreaks

*Abdulkarim F. Alhetheel and Faisal A. Alhetheel*

#### **Abstract**

Middle East respiratory syndrome coronavirus (MERS-CoV) is a single-stranded RNA-enveloped virus that belongs to the Coronaviridae family. Initially reported in 2012 in Saudi Arabia, MERS-CoV is a zoonotic virus originating from bats and transmitted from camels to humans and among humans by contact. It causes both upper and lower respiratory tract infections and in some instances can lead to renal failure or death. This chapter provides an overview of the virologic aspects, outbreaks and risk factors, clinical symptoms, diagnostic methods, as well as prevention and management of MERS-CoV infection.

**Keywords:** MERS-CoV, outbreak, clinical symptoms, diagnosis, prevention

#### **1. Introduction**

The first case of the Middle East respiratory syndrome coronavirus (MERS-CoV) infection was reported in June 2012 in Saudi Arabia. MERS-CoV then spread to several neighboring countries, mainly Jordan and Qatar, and has since been reported in Asia, Africa, Europe, and America [1]. By October 16, 2018, 2260 confirmed cases and 803 deaths from MERS-CoV infection had been documented in 27 countries by the World Health Organization. The vast majority of cases (73%) were reported in Saudi Arabia, with only one widespread outbreak observed outside of the Arabian Peninsula in South Korea in 2015 [2]. Due to the high fatality rate (36%) [1], a lot of effort has been made to understand the origin and pathophysiology of this novel coronavirus strain to prevent it from becoming endemic in humans [3].

#### **2. Middle East respiratory syndrome coronavirus**

The first reported case of human MERS-CoV infection was in a 60-year-old man who was admitted to a private hospital in Jeddah, Saudi Arabia, on June 13, 2012. He presented with a 7-day history of fever, cough, expectoration, and dyspnea. He was a non-smoker, had no prior history of cardiopulmonary or renal disease, and was not maintained on any long-term medication. Vital sign examination showed a blood pressure of 140/80 mmHg, a pulse rate of 117 beats/minute, a temperature of 38.3°C, a respiration rate of 20 breaths/minute, and a body mass index of 35.1. Chest X-ray revealed low lung volume, bilateral enhanced pulmonary hilar vascular shadows more prominent on the left side, and accentuated bronchovascular lung markings. Multiple segmental, patchy, and veiling opacities were present in the middle and lower lung fields, and the costophrenic angles were mostly blunted. The cardiac silhouette was not enlarged, and the aorta was dilated and unfolded. Chest X-ray was repeated after 4 days, which showed that the opacities became denser and more confluent. Computed tomography performed 4 days after admission revealed few subcentimetric mediastinal and hilar lymph nodes, bilateral dependent airspace opacities with air bronchograms, scattered areas of ground-glass opacity, interstitial septal thickening, and nodularity in the upper lobes, with minimal bilateral pleural effusion. Collectively, these findings are suggestive of an infection. On the day of admission, oseltamivir, levofloxacin, piperacillin-tazobactam, and micafungin were started. Three days later, meropenem treatment was initiated, since meropenem-sensitive *Klebsiella pneumoniae* was identified in tracheal lavage sample collected on day 2. *Staphylococcus aureus* was detected in a sputum sample performed on admission. *Acinetobacter* was identified in tracheal aspirate collected on the day of death. No other pathogens were detected in respiratory specimens, and no bacterial growth was detected in blood samples.

The patient was transferred to the intensive care unit (ICU) for mechanical ventilation on the second day. Laboratory findings obtained on admission showed normal white blood cell counts except for a relatively high percentage of neutrophils (92.5%) and a low percentage of lymphocytes (4.3%). Liver enzymes, blood urea nitrogen, and creatinine levels were within the normal range. A small increase in the liver enzymes was noted from day 7 onward, with alanine aminotransferase levels of 20, 78, and 47 international unit (IU)/liter (l) on days 1, 7, and 8, respectively, and aspartate aminotransferase levels of 33 and 96 IU/l on days 1 and 8, respectively. The patient tested negative for the human immunodeficiency virus; however, testing for pneumocystis pneumonia was not performed. On the third day following admission, blood urea nitrogen and creatinine levels significantly elevated, and on the eighth day, white cell count began to rise and reached a peak of 23,800 cells per cubic millimeter by day 10, with neutrophilia, persistent lymphopenia, and progressive thrombocytopenia. Arterial oxygen saturation ranged from 78% to 98%. On day 11 (June 24, 2012), the patient died of progressive respiratory and renal failure [4].

#### **2.1 The source of MERS infections**

In 2012, a new coronavirus strain was detected in patients from the Arabian Peninsula with severe respiratory symptoms known as MERS-CoV. Camels were identified as the source of the infections; however, the role of these animals in transmitting the infection is not well understood. Approximately 300 isolated MERS-CoV genomes had been sequenced from humans and camels during the epidemic. Previous attempts to understand the MERS-CoV epidemic relied on these data or reports of case numbers; however, this led to conflicting results at odds with other sources of evidence. Nevertheless, Dudas et al. [5] determined the relationship among MERS-CoV strains and reconstructed their family tree by analyzing their sequenced genomes.

#### **2.2 Genome structure and function**

MERS-CoV, a lineage C betacoronavirus (BCoV), has a positive-sense singlestranded RNA (ssRNA) genome of approximately 30 kb (**Figure 1A** and **B**) [6, 7]. As of 2016, phylogenetic analysis of MERS-CoV had been performed on 182 full-length

genomes and multiple concatenated genome fragments, including 94 from humans and 88 from dromedary camels [9, 10]. The MERS-CoV genomes share more than 99% sequence identity, indicating low mutation and variance rates. The MERS-CoV genome is divided into two clades: clade A, which contains only a few strains, and clade B, to which most strains belong [10].

Similar to other coronaviruses, approximately two-third of the 5′ end of the MERS-CoV genome consists of the replicase complexes open reading frame (ORF1a) and (ORF1b). The remaining one-third encodes the structural protein spike (S), envelope (E), membrane (M), and nucleocapsid (N) as well as five accessory nonreplicating proteins (ORF3, ORF4a, ORF4b, ORF5, and ORF8b) likely involved in viral pathogenesis (**Figure 1B**) [6, 11–15]. Typical of coronaviruses, the MERS-CoV accessory proteins are not homologous with any known host or viral proteins other than those closely related to lineage C BCoV [10]. MERS-CoV structural and accessory protein-coding plasmids transiently transferred into cells showed that ORF4b is localized mostly in the nucleus, whereas all other proteins are localized in the cytoplasm (S, E, M, N, ORF3, ORF4a, and ORF5) [16]. In addition, MERS-CoV deletion mutations of ORFs 3–5 attenuate replication in human airway-derived (Calu-3) cells [17], while deletion mutations of ORFs 4a and 4b attenuate replication in hepatic carcinoma-derived (Huh-7) cells [14, 18]. This highlights the importance of MERS-CoV accessory proteins in viral replication *in vitro* [19].

#### **Figure 1.**

*MERS-CoV genome and schematic structure of viral proteins. (A) Schematic structure of major MERS-CoV structure proteins. (B) The MERS-CoV genome consists of two partially overlapping replicase open reading frames (ORF1a and 1b) and several ORFs that encode viral functional structural proteins and other proteins with unknown functions [8]. Abbreviation: MERS-CoV, Middle East respiratory syndrome coronavirus.*

In response to viral infection, mammalian cells activate the type I interferon (IFN)-mediated innate immune response by producing type I IFNs (IFN-α and IFNβ). In contrast, evasion of host innate immunity through IFN antagonism, mediated by virus-encoded IFN antagonist proteins, is critical to viral pathogenesis. Each protein blocks key signaling proteins in the IFN and nuclear factor kappa B (NF-κB) pathways to enhance viral replication and pathogenesis [20–23]. Coronaviruses have evolved similar mechanisms to impede or bypass the innate immunity of their host at various levels, which ultimately contribute to viral virulence. Moreover, various coronavirus proteins disrupt signal transduction events required for the IFN response [24], often by interfering with host type I IFN induction.

MERS-CoV weakly induces type I IFN late during infection. In addition, MERS-CoV M, ORF4a, ORF4b, and ORF5 proteins are strong INF antagonists [16]. Studies using transient overexpression of the MERS-CoV accessory proteins ORF4a, ORFb, and ORF5 showed that they inhibit both IFN induction [16, 25, 26] and NF-κB signaling pathways [26]. MERS-CoV ORF4a, a double-stranded RNA (dsRNA) binding protein [25], potentially antagonizes antiviral IFN activity by inhibiting interferon production (IFN-beta promoter activity, IRF-3/7, and NF-kB activation) and the ISRE promoter element signaling pathway [16]. On the contrary, MERS-CoV ORF4b belongs to the 2H-phosphoestras (2H-PE) family and possesses phosphodiesterase (PDE) activity. Although MERS-CoV ORF4b is detected primarily in the nucleus of both infected and transfected cells [16, 25, 26], cytoplasmic expression levels are sufficient to inhibit activation of RNase L, a potent interferon-induced antiviral protein [16, 26]. MERS-CoV ORF4b was the first identified RNase L antagonist expressed by human or bat coronaviruses. It inhibits type I IFN NF-κB signaling pathways, providing a mechanism through which MERS-CoV can evade innate immunity [14, 26]. In addition, the MERS-CoV replicase nonstructural proteins (nsp1, nsp3, and nsp14) have been shown to interfere with innate immune signaling pathways through differing mechanisms [19, 27, 28]. In short, MERS-CoV has developed various mechanisms to evade the host immune system [29].

#### **3. MERS-CoV infections and outbreaks**

Between September 2014 and January 2015, a MERS-CoV outbreak resulting in 38 cases and 21 deaths was reported in Taif, Saudi Arabia. Clinical and public health records showed that 13 patients were healthcare personnel (HCP) and 15 patients, including 4 HCP, were associated with 1 dialysis unit. Serological studies done on three additional HCP in the same dialysis unit showed a positive report for MERS-CoV infection. Viral RNA was then measured from serum specimens of 15 patients in the acute phase, and full spike gene-coding sequencing was obtained from 10 patients, forming an unrelated cluster where sequences from 9 patients were closely related. Contrastingly, similar gene sequences among patients not linked by time or location suggest unidentified route of viral transmission. In short, circulation persists in multiple healthcare settings over an extended period, underscoring the importance of strengthening MERS-CoV surveillance and infection control practices [30].

Between May and July 2015, a large outbreak of MERS-CoV infection occurred in South Korea, which resulted from a traveler returning from the Middle East. This outbreak led to 186 confirmed cases in the country due to a primary case [31]. Patient 1 was diagnosed at Samsung Medical Center after transmitting the virus to several healthcare facilities. Patient 14 was exposed to Patient 1 outside the hospital and sought medical attention at the institution without knowing his infection status. Therefore, the experience gained from South Korea's first MERS-CoV case and a case following single-patient exposure in an emergency room showed the importance of investigating the epidemiology of MERS-CoV infection in a crowded areas such as an emergency room for the potential presence of super-spreaders [2].

#### **4. MERS-CoV clinical features**

MERS-CoV affects both upper and lower respiratory tracts in humans and may lead to complications ranging from renal failure to death. The symptoms in a patient with MERS-CoV are fever, sore throat, runny nose, and muscle ache. Some of the cases have developed to severe diseases by progression to acute respiratory distress syndrome. In severely ill patients, X-rays and other scans showed multilobar airspace disease [32].

Extra-pulmonary manifestations are common in severe cases; 30% of critical cases had gastrointestinal symptoms like nausea, vomiting, and diarrhea. Kidney disease has been reported for about 50% of critical MERS-CoV cases. Laboratory results showed leukopenia, lymphopenia, anima, and thrombocytopenia. Also, partial to moderate increase in amino transferase level is usual in MERS-CoV infection [32].

Herein we present the cases of two immunocompromised patients with MERS-CoV. In April 2013, two MERS-CoV cases were reported following nosocomial transmission from one patient to the other in a French hospital. Patient 1 visited Dubai, while patient 2 lived in France and had not traveled abroad. Both patients presented with fever, chills, and myalgia; however, patient 1 also complained of diarrhea. Respiratory status deteriorated, leading to acute respiratory failure requiring mechanical ventilation and extracorporeal membrane oxygenation (ECMO), and both patients experienced acute renal failure. MERS-CoV RNA was detected in lower tract specimens from both patients using reverse transcriptase polymerase chain reaction (RT-PCR) (e.g., cycle threshold [CT] values of 22.9 for upE and 24 for Orf1a from patient 1; CT values of 22.5 for upE and 23.9 for Orf1a from patient 2), whereas nasopharyngeal swab specimens were weakly positive or indeterminate. The patients shared a room for 3 days, and the incubation period was estimated to be 9–12 days for the second case. Patient 1 died on May 28 due to refractory multiple organ failure [33].

Another MERS-CoV case was presented in an old man with multiple myeloma. On March 8, a 73-year-old patient from Abu Dhabi developed flu-like symptoms with fever and a non-productive cough. He was admitted to the Mafraq Hospital in Abu Dhabi and was diagnosed with pneumonia. He was then intubated on day 9 due to progressive hypoxia and acute respiratory distress syndrome (fraction of inspired oxygen, 60%; positive end-expiratory pressure, 10 cm H2O). The patient received intensive antimicrobial treatment with meropenem, levofloxacin, vancomycin, caspofungin, acyclovir, and oseltamivir during his stay in the ICU without major improvement of his pulmonary function. The patient was then transferred to the Klinikum Schwabing on March 19, 2013. Of note, relatives reported that the patients owned camels. He was diagnosed with multiple myeloma in 2008 and received several lines of treatment in the past few years, including high-dose chemotherapy with autologous stem cell transplantation in 2009. In November 2012, the patient had a relapse of multiple myeloma and was treated with lenalidomide and dexamethasone. During his stay in Munich, thrombocytopenia was observed. Interestingly, thrombocytopenia was also reported in early cases of MERS-CoV infection [4] including two of the four patients from a family cluster in Saudi Arabia [34] and two cases reported

in France [33]. The patient then developed renal insufficiency on day 14 requiring dialysis. Despite continuous invasive ventilation and antibiotic treatment, the health status of the patient worsened, and he died on day 18 due to septic shock with signs of hemolysis and acute coagulation disorder [35].

On September 14, 2012, the United Kingdom Health Protection Agency (HPA) Imported Fever Service was notified of a case of unexplained severe respiratory illness in an ICU in London. The patient was a 49-year-old man who had recently been transferred from Qatar and had a travel history to Saudi Arabia. He developed mild undiagnosed respiratory illness while visiting Saudi Arabia in August 2012, which was fully resolved. On September 3, he presented to a physician in Qatar with cough, myalgia, and arthralgia and was prescribed oral antibiotics. Five days later, he was admitted to Qatar Hospital with a fever of 38.4°C and hypoxia (saturation of 91% in room air). Chest X-ray revealed bilateral lower-zone consolidation, and the patient required intubation and ventilation and was then transferred to London via air ambulance. The patient was clinically unstable and required manual ventilation during the transfer. On admission to the ICU in London, he remained severely hypoxic with arterial oxygen partial pressure of 6.5 kPA on 100% oxygen with optimized pressure ventilation. He required low-dose norepinephrine to maintain blood pressure. C-reactive protein was high (350 mg/L), and creatinine was high (353 μmol/L), with normal liver function and coagulation. The patient was treated with corticosteroids and broad-spectrum antibiotics, including meropenem, clarithromycin, and teicoplanin. Colistin and liposomal amphotericin B were later added. The patient's condition deteriorated with progressive hypoxia between September 11 and 20. His C-reactive protein level peaked at 440 mg/L and procalcitonin level at 68 ng/ml. His renal function also worsened, and hemofiltration was initiated on September 14. He was then transferred to a specialist ICU, and ECMO was initiated on September 20 (day 17 of illness). On October 2, he remained stable but was fully dependent on ECMO after 13 days (day 30 of illness) [36].

#### **5. Diagnostic tests for MERS-CoV**

MERS-CoV identification by diagnostic testing is crucial for tracking down cases of MERS-CoV, selecting appropriate treatment modalities to improve patient health, and lowering MERS-CoV symptoms and mortality rate. To date, RT-PCR is the mainstay test to diagnose MERS-CoV. However, like other tests, it has some limitations, including a long turnaround time and a lack of common measurements and correlations with viral load (VL). Most laboratories determine only CT values—which are inversely related to VL—to predict the viral concentration and disease progression as well as serve as a cut-off marker for diagnosis. However, few studies have evaluated the relationship between CT values and clinical severity [37]. Nevertheless, screening for MERS-CoV by RT-PCR upstream of the envelope gene (upE) is recommended, followed by confirming the presence of one of the following genes: open reading frame 1A, 1B genes, or nucleocapsid (N) [38]. Serology testing is another method to diagnose MERS-CoV.

Similar to other viruses, detecting antibodies and antigens by molecular methods may sometimes lag behind detecting the viral genome. To date, kinetics of antigen production in nasopharyngeal samples have not been studied. Moreover, viral antibodies usually appear 10 days after illness onset and are further delayed in severely ill patients requiring mechanical ventilation [39].

#### *Middle East Respiratory Syndrome Coronavirus Outbreaks DOI: http://dx.doi.org/10.5772/intechopen.108574*

An enzyme-linked immunosorbent assay (ELISA) capture assay that can detect NP antigens of MERS-CoV virus in nasopharyngeal samples has been recently developed [40]. The assay is highly sensitive (detecting MERS-CoV-NP of less than 1 ng/mL) and specific (specificity of 100%) for MERS-CoV and can also be used in animals. Song et al. developed a rapid immunochromatographic assay to detect MERS-CoV nucleocapsid protein from camel nasal swabs, with a sensitivity of 93.9% and specificity of 100%. This assay is promising and worthy of replication in both camels and humans; however, antigen detection assays are not widely available. Nevertheless, this type of assay is valuable for ruling infections in or out.

Perera et al. [41] produced and optimized a microneutralization test to detect specific antibodies for MERS-CoV. Serial dilutions of serum sample were incubated with the Vero cells/MERS-CoV virus, and after 3-day incubation at 37°C, the antibody titers were scored based on virus cytopathic effect (CPE). Also, they developed a MERS-CoV spike pseudoparticle neutralization test [41], in which HIV/MERS spike pseudoparticles were used to infect Vero E6 cells. After 2 days, infected cells were lysed and antibodies that resulted in 90% luciferase reduction were reported as the ppNA antibody titer. As opposed to virus neutralization test, the pseudoparticle neutralization assay does not require biosafety level-3 (BSL3) containment.

An indirect immunofluorescent antibody assay to detect MERS-CoV antibodies was carried out using either whole virus in Vero cells [42, 43] or Vero cells transfected with MERS-CoV spike or nucleocapsid proteins [42]. ELISA utilizing S1 protein was also used to investigate the epidemiology of viral exposure [44]. To date, no studies have compared ELISA to either immunofluorescences assay (IFA) or neutralization assays.

Western blotting has been previously used to confirm antibody specificity to other viruses, such as SARS-CoV [45]. In addition, western blotting assays are needed to confirm antibody specificity in MERS-CoV, which can be in the form of genetically engineered specific MERS-CoV antigens blotted on the membrane.

Overall, MERS-CoV diagnostic testing and molecular techniques are the first-line methods used to confirm MERS-CoV infections. RT-PCR or sequencing of lower respiratory samples (tracheal aspirates and bronchoalveolar lavage samples) are recommended for viral detection. Thus, serological testing is a valuable tool to confirm suspected MERS-CoV cases; however, the virus cannot be detected in respiratory samples [46].

#### **6. Prevention and treatment of MERS-CoV**

Documenting the source of infection is key to preventing viral spread of MERS-CoV. Outbreaks are caused by viral transmission within healthcare settings facilitated by overcrowding, poor compliance with basic infection control measures, unrecognized infections, super-spreaders, and poor triage. However, actual contributing factors leading to MERS-CoV infection have not yet been systematically studied, but viral, host, and environmental factors are suggested to play major roles.

MERS vaccines can induce humoral and cellular immune responses. Specifically, a suitable MERS vaccine must induce a strong humoral immune response and, depending on the immunization route, activate B cells to produce systemic IgG and secretory IgA antibodies that bind to the virus and mediate systemic and mucosal responses [47–49], respectively. Serum IgA is also induced upon vaccination, particularly through the mucosal or intranasal routes [48]. The antibodies then neutralize MERS-CoV infection by blocking viral binding of the cell via the cellular receptor dipeptidyl-peptidase 4 (DPP4) and thus inhibiting cell entry [50, 51]. B cells can become

antigen-specific memory B cells that can further boost immunization and induce rapid recall antibody responses [52]. However, this outcome has not been extensively studied in MERS-CoV vaccines.

Non-human primate (NHP) models were initially established as effective vehicles for MERS-CoV infection and vaccine evaluation; however, no vaccine against MERS-CoV is currently available for human use. Nevertheless, progress has been made since the emergence of the MERS-CoV in 2012. Unlike the SARS vaccines, which are developed based on attenuated or inactivated SARS-CoV and can potentially recover virulence factors [53–57], recombinant MERS-CoV vaccines can be developed based on recombinant viral particles using reverse genetics. For instance, a recombinant MERS-CoV with specific mutations is produced using a panel of contiguous cDNAs covering the whole viral genome and propagated to high titers in different tissue types. Additionally, an engineered mutant MERS-CoV that lacks the structural protein E was rescued and replicated in cells expressing the viral E protein [17, 18]. Using reverse genetics, developing replication-competent and propagation-defective MERS-CoV candidate vaccines that can provide a platform for designing live-attenuated MERS-CoV vaccines becomes possible. However, as recombinant MERS viruses contain major viral components and virulence factors, safety concerns need to be addressed, and their efficacy requires further assessment in appropriate animal models.

#### **6.1 Viral-vector-based MERS vaccines**

MERS vaccines can also be developed using viral vectors that express main MERS-CoV proteins, including the S proteins. As such, several MERS vaccine candidates have been produced and evaluated for immunogenicity in hDPP4-expressing mouse models and camels [47, 58–60].

Ad5 or Ad41 vectors expressing full-length S or S1 protein of MERS-CoV induce S-specific antibody and/or T-cell response in a mouse model via the intramuscular (IM) or intragastric route, effectively neutralizing MERS-CoV infection in vitro [58, 61]. In addition, IM or subcutaneous vaccination of mice with an MVA-based full-length S vaccine elicited the MERS-CoV challenge. Intranasally or intramuscularly administered MVA-S vaccine also induced mucosal immunity in camels, causing a significant decrease of excreted infectious viral RNA transcripts after MERS-CoV challenge. Similarly, a recombinant MV-based MERS vaccine expressing full-length or truncated S protein of MERS-CoV induced significant MERS-CoV, neutralizing antibodies and T-cell response, protecting mouse transducers with hDPP4 from the MERS-CoV challenge [62]. Although viral-vector-based vaccines can produce strong immune responses and/or protection, they may have unwanted safety and potency limitations.

#### **6.2 Nanoparticle-based MERS vaccines**

Nanoparticles can be used as delivery vehicles for MERS vaccines. The MERS-CoV full-length S protein can be prepared and purified from pellets of infected baculovirus insect cells. In the absence of adjuvants, nanoparticles induce a low level of MERS-CoV neutralizing antibodies in mice. However, by adding adjuvants such as aluminum hydroxide (Alum) or matrix M1, neutralizing antibodies become significantly increased and maintained. In addition, matrix M1 promotes increased production of neutralizing antibodies compared to alum [63]. Thus, adjuvants are required for MERS nanoparticle vaccines to promote immunogenicity. However, the efficacy and protection of this vaccine type have not yet been evaluated in MERS-CoV challenge models.

#### **6.3 DNA prime/protein-boosted MERS vaccines**

DNA priming followed by protein boosting could be used to develop MERS vaccines and subsequently expand DNA immunogenicity and efficacy. In this combined vaccination plan, DNA was constructed to encode the full-length MERS-CoV S protein, while the protein was expressed as the viral S1 subunit [64]. Studies have demonstrated that IM/electroporation priming of full-length S DNA and IM boosting of S1 protein of MERS-CoV with Ribi or alum (aluminum phosphate, AlPO4) adjuvant in mice and rhesus macaques induced robust neutralizing antibodies against MERS-CoV infection, conferring the protection of NHPs against MERS-CoV-induced radiographic pneumonia. However, the potential for vaccine-induced immune pathology needs to be investigated further.

#### **6.4 Subunit MERS vaccines**

Protein-based subunit vaccines against MERS-CoV have also been developed [49, 50, 65, 66]. While some subunit vaccines are designed based on the full-length S1 protein [64], most are based on viral RBD [49, 50, 65–67]. RBD-based vaccines have been evaluated for immunogenicity and protection in several MERS-CoV animal models, including hDPP4-transduced and hDPP4-Tg mice, as well as in NHPs [65–70]. The antigenicity and functionality of RBD proteins have also been extensively investigated.

Subunit vaccines do not induce the immune system as strongly as the other previously mentioned vaccines. However, the immunogenicity of subunit vaccines can be significantly enhanced by adding an ideal adjuvant via the appropriate route [48, 69]. In addition, maintaining a suitable conformation of the protein antigens in the vaccine, such as MERS-CoV RBD proteins [49, 50], is essential.

Subunit vaccines are the safest vaccine type since they do not contain viral genetic material. They are composed of antigens essential for developing protective immune responses, thus excluding the possibility of recovering virulence or inducing adverse reactions [71–73]. In contrast to vaccines based on the full-length S or S1 protein, RBD-based MERS subunit vaccines contain major neutralizing epitopes and lack nonneutralizing immunodominant domains; thus, they possess minimal risk of inducing non-neutralizing antibodies that can potentially lead to harmful immune responses or enhancement of virus infection [49, 74, 75]. This review aimed to provide guidelines for the development of effective and safe MERS vaccines.

#### **7. Conclusion**

With every passing years, our knowledge of MERS-CoV virus is improving; fewer cases of MERS-CoV have been reported as more studies improve our understanding of the virus. Appropriate diagnostic testing such as RT-PCR, documenting causes of viral outbreaks, and developing infection control units in every hospital have played key roles in hindering viral spread and preventing MERS-CoV from becoming endemic in humans, also lowering the risk of human infection by controlling animalto-human transmission of the virus by vaccinating animals to prevent any transmission. There are studies that support developing potential therapies and vaccines to prevent infections [32].

#### **Author details**

Abdulkarim F. Alhetheel1,2\* and Faisal A. Alhetheel3

1 Department of Laboratory Medicine, King Saud Medical City, Riyadh, Saudi Arabia

2 Department of Pathology, College of Medicine, King Saud University, Riyadh, Saudi Arabia

3 College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

\*Address all correspondence to: aalhetheel@ksu.edu.sa

© 2022 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 5**

## A Review on Viral Outbreak in India with Special Reference to COVID-19

*Aishwarya Khamari, Monika Khamari, Akshya Kumar Mishra, Jijnasa Panda, Debashish Gardia and Ratikanta Rath*

#### **Abstract**

COVID-19, Middle East respiratory syndrome (MERS), and SARS are three severe pandemics linked to novel coronaviruses that have so far impacted people in the twenty first century. These acute respiratory tract infections (ARTIs) are brought on by viruses that are all exceedingly contagious and/or have caused large mortality. On January 7, 2020, a patient in Wuhan, China, with pneumonia-like symptoms had a novel coronavirus found in lung fluid. In 1980, the smallpox disease was formally deemed extinct worldwide. The cause of smallpox is unknown. The discovery of smallpox-like lesions on Egyptian mummies indicates that the illness has existed for at least 3000 years. The Ebola virus, a member of the filovirus family that affects both humans and other primates, causes the severe illness known as Ebola virus disease (EVD). The idea that swine influenza was a sickness related to human flu was originally put forth when pigs were ill during the 1918 flu pandemic at the same time as humans. Because viruses vary in their structural, anatomical, and molecular makeup, distinct viral diseases can be detected or tested using different methodologies, procedures, or diagnostic tools. Viral vaccines come in a wide variety of varieties in the pharmaceutical industry. From a medical perspective, several treatments are used for various viral illnesses.

**Keywords:** COVID-19, flu, testing, outbreak, treatment, Indian context, pandemics, Ebola virus disease

#### **1. Introduction**

COVID-19, Middle East respiratory syndrome (MERS), and SARS are three severe pandemics linked to novel coronaviruses that have so far impacted people in the twenty first century. These acute respiratory tract infections (ARTIs) are brought on by viruses that are all exceedingly contagious and/or have caused large mortality. Another zoonotic novel coronavirus with the name severe acute respiratory syndrome coronavirus 2 is the cause of the recently identified COVID-19 sickness, a highly contagious viral infection (SARS-CoV-2). Similar to the other two coronaviruses like SARS-CoV-1 and MERS-CoV, SARS-CoV-2 is most likely to have originated from bats, which have long served as established reservoirs for a range of lethal coronaviruses [1]. In December 2019, there were several reports of individuals in the province of Hubei who were admitted to hospitals with a brand-new illness characterised by pneumonia and respiratory failure and brought on by a novel coronavirus (SARS-CoV-2) (China). On February 11, 2020, the World Health Organization (WHO) identified this agent as the COVID-19 causal agent. 2019 (Coronavirus Disease). Despite the use of significant containment measures, the disease later spread to other Asian countries, the Middle East, and Europe. On March 11, Tedros Adhanom Ghebreyesus, the director general of the WHO, said that COVID-19 was a pandemic [2, 3].

Numerous studies show that after the coronavirus infection (COVID-19) outbreak, anxiety around it has significantly increased. To measure COVID-19 fear, a number of questionnaires have been developed concurrently. The several questions could cover a wide range of subjects, and COVID-19 dread is not necessarily a widely accepted idea. We conducted structural equation modelling and network analysis on four scales in an online convenience sample to examine the underlying structure of COVID-19 fear [4].

It is more crucial to comprehend the organisation and structure of conspiracy theories and misleading information about the COVID-19 epidemic in order to counteract the harm that these dubious claims pose as the pandemic spreads. We found distinct belief clusters when surveying Americans on their views on 11 of these ideas. These belief clusters correlated with various individual-level traits (like support for Trump and mistrust of scientists) and behavioural intentions (like taking a vaccine or participating in social activities) [5].

The rapid development of diagnostics for the novel virus was made possible by the genome assembly and release of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in January 2020. Due to this, the largest global testing programme in history was launched and has since tested hundreds of millions of people. The massive amount of testing has stimulated innovation in the techniques, instruments, and theories that direct public health testing [6].

Physical isolation has been recommended as one of the most effective techniques to inhibit the transmission of COVID-19 before a vaccine or efficient therapy is created. How far people can be physically apart depending on both population density and behavioural characteristics. Most models developed to predict the spread of COVID-19 in the US do not explicitly take population density into account [7].

The Centres for Illness Control and Prevention developed and conducted the initial test as a result of the novel coronavirus severe acute respiratory syndrome coronavirus 2 producing coronavirus disease 2019 cases in the United States. The Centres for Disease Control and Prevention had to use the Emergency Utilization Authorization to allow both university and commercial labs to develop assays for determining the virus's existence as the number of cases increased and the necessity for testing increased. Several nucleic acid assays were developed on the basis of RT-PCR, each with its own techniques, specifications, and turnaround times. The pandemic-like spread of the illnesses made testing even more crucial. Prioritisation was required in accordance with instructions because the test supply ran out before it could satisfy demand [8].

Due to the breakdown of global cooperation and a lack of international solidarity, several low- and medium-income countries have been refused access to clinical tools in the COVID-19 pandemic response. Despite the availability and scalability of fast immunodiagnostic testing, knowledge of the dynamics of the immune response associated with infection is lacking [9].

*A Review on Viral Outbreak in India with Special Reference to COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.108575*

The US has given ongoing emphasis to the value of testing in decreasing and suppressing the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to problems with test validation at the Centres for Illness Control and Prevention, testing was put off after the first case of coronavirus disease 2019 (COVID-19) was identified in the US in mid-January 2020 [10, 11].

The coronavirus disease (COVID-19) pandemic has shifted the focus of the global discussion about how to end the epidemic to the clinical lab and SARS-CoV-2 tests. Clinical laboratories have developed, approved, and used a variety of molecular and serologic assays to look for SARS-CoV-2 infection as a result. This has been essential for identifying cases, directing isolation decisions, and controlling the transmission of disease [12].

#### **2. History**

#### **2.1 History of COVID-19**

On January 7, 2020, a patient in Wuhan, China, with pneumonia-like symptoms had a novel coronavirus found in lung fluid. On January 10, 2020, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assembled reference genome was revealed, and the first diagnostic tests to detect the virus were made available 2 weeks later. Since hundreds of millions of people have been tested for SARS-CoV-2, there has been a great deal of interest in and debate regarding diagnostic theories and procedures. In Wuhan, China, the first SARS-CoV-2 infections were found. It is still uncertain how the virus initially infected humans and if it turned into a pathogen before to or following the spillover incidence. The fifth known pandemic since the 1918 flu pandemic was caused by the uncommon human coronavirus disease of 2019 (COVID-19), which was originally discovered in Wuhan, China, in 2019. More than 200 million confirmed cases and more than 4.6 million fatalities had been reported as of September 2021, around 2°years after COVID-19 was first discovered. In this article, we present a thorough examination of the development of COVID-19, from the first case ever reported to the most recent efforts to stop the disease's global spread through vaccine campaigns. The World Health Organization (WHO) in Wuhan, China, received reports of pneumonia episodes on December 31, 2019, and as a result, the first COVID-19 cases were found. On January 7, the Chinese government determined that these instances were brought on by the 2019-nCoV, a brand-new coronavirus. A few weeks later, on January 30, 2020, the WHO deemed the fast expanding COVID-19 epidemic a Public Health Emergency of International Concern. The new coronavirus wasn't officially given a name until February 11th, when COVID-19 was assigned. The first instances of COVID-19 were discovered after reports of pneumonia episodes were received by the World Health Organization (WHO) in Wuhan, China, on December 31, 2019. The rapidly spreading COVID-19 epidemic was classified as a Public Health Emergency of International Concern by the WHO a few weeks later, on January 30, 2020. On February 11th, COVID-19 was given as the novel coronavirus's official name [1, 13–17].

#### **2.2 History of smallpox**

The cause of smallpox is unknown. The discovery of smallpox-like lesions on Egyptian mummies indicates that the illness has existed for at least 3000 years. The first written account of a disease akin to smallpox was produced in China during the fourth century CE (Common Era). India saw one of the worst smallpox epidemics

of the twentieth century in 1974, 3 years before smallpox was completely eradicated. More than 15,000 people contracted smallpox and died as a result between January and May 1974. West Bengal, Bihar, and Odisha are three Indian states where the majority of the fatalities occurred. There were many thousands of people who were still alive but were either blind or deformed. India reported 61,482 smallpox cases to the World Health Organization (WHO) during these 5 months. In 1974, India was home to over 86% of all smallpox cases in the globe, primarily as a result of this pandemic. On May 24, 1975, a smallpox patient was found in India, and by January 1975, an operation known as "Target Zero" had been started in an effort to eradicate all remaining cases. In 1980, the smallpox disease was formally deemed extinct worldwide. Despite the fact that this programme was first introduced in 1958, it did not move swiftly because of disagreements between the WHO and the Indian government on logistics. Progress was only truly accomplished after the WHO was reorganised in the middle of the 1960s in India. Donald Henderson, a U.S. Public Health Services Officer in New Delhi, said that "If this attention and concern can last for the foreseeable future, smallpox will be eliminated. Everything seems to be in working order, though we don't think we're being overconfident. By June 1975, we hope to have eradicated smallpox in Asia" [18–23].

#### **2.3 History of Ebola**

History of the disease. The Ebola virus, a member of the filovirus family that affects both humans and other primates, causes the severe illness known as Ebola virus disease (EVD). The illness almost simultaneously spread to the Democratic Republic of the Congo (DRC) and Sudan in 1976 (now South Sudan). An EVD outbreak was reported in the Beni Health Zone in North Kivu Province on October 8, the DRC's Ministry of Health reported. Three suspected cases were later discovered in September 2021, and other cases in the same health zone were eventually confirmed. Sequencing results revealed a connection to the outbreak that struck the same area in 2018–2020, demonstrating that an EVD survivor's chronic infection was most likely the root cause of this outbreak. On December 16, 2021, 42 days after the final confirmed patient was removed from care, the 13th EVD outbreak in the DRC was ruled to be over legally. The Democratic Republic of the Congo's Ministry of Health (MOH) revealed on February 7, 2021 that an Ebola virus disease (EVD) case had been identified in North Kivu Province's Biena Health Zone. Later incidents were verified. EVD was present in North Kivu prior to the largest Ebola outbreak in the DRC's history, which occurred from 2018 to 2020 and was declared over on June 25, 2020. According to sample sequencing, cases from the 2018 to 2020 outbreak are connected to patients in this pandemic. It is likely that these cases resulted from sexual transmission of the virus or from a survivor who relapsed with a chronic infection. On May 3, 2021, the outbreak was determined to be over. On June 1, 2020, the DRC government announced a fresh Ebola outbreak in Mbandaka, Equateur Province of western DRC. The DRC government received technical support from international partners including the CDC to aid in response operations. This outbreak, the eleventh to hit the DRC, started as the tenth was still rapidly expanding over the east of the country. The DRC government announced the 10th Ebola epidemic on August 1 in the nation's eastern North Kivu province. Instances were also reported in the provinces of South Kivu and Uganda. In order to coordinate efforts and offer technical advice regarding laboratory testing, contact tracing, infection control, border health screening, data management,

#### *A Review on Viral Outbreak in India with Special Reference to COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.108575*

risk communication and health education, vaccination, and logistics, the CDC worked with the DRC government, neighbouring nations, local and international partners. A number of probable Ebola virus disease (EVD) cases were reported in the Likati health zone in the province of Bas Uele on May 11 by the Democratic Republic of the Congo's Ministry of Public Health, which also alerted other international public health organisations to the situation. Eight suspected instances, including two fatalities, were listed in the original report. On May 12, there was word of a third fatality. Two samples proved positive for Ebola Zaire during testing by the Institute National de Recherche Biomédicale (INRB) in Kinshasa. Health's epidemiologic, diagnostic, clinical, and communication efforts to contain the outbreak were supported by teams from international organisations like the CDC, WHO, MSF (Doctors without Borders), and others. The outbreak solely impacted the western province of Equator, even though it spread to several villages close to the town of Boende. The Ebola virus strain that caused it, meanwhile, was quite similar to the one that was responsible for the outbreak in Kikwit in 1995. This outbreak had nothing to do with the significant outbreak that was happening concurrently in West Africa. The probable death of an EVD patient was reported by the Uganda Ministry of Health on May 6, 2011. The Uganda Virus Research Institute's newly created CDC Viral Haemorrhagic Fever lab quickly identified the Ebola virus in a blood sample (UVRI). This outbreak was contained in part by the ability to quickly confirm the presence of the Ebola virus through laboratory testing carried out in-country, the clinical staff's early, strong suspicion of hemorrhagic fever, the appropriate use of personal protective equipment and barrier methods to safeguard hospital staff, and the ability to quickly stop the spread of the virus [24–28].

#### **2.4 History of swine flu**

The idea that swine influenza was a sickness related to human flu was originally put forth when pigs were ill during the 1918 flu pandemic at the same time as humans. The first influenza virus was found to be the cause of illness in pigs around 10°years later, in 1930. The World Health Organization (WHO) categorised the 2009 swine flu pandemic, which was brought on by the H1N1 influenza virus and lasted from June 2009 to August 2010, as the third recent pandemic caused by the H1N1 virus (the first being the 1918–1920 Spanish flu pandemic and the second being the 1977 Russian flu). According to two separate US investigations, the first two occurrences were discovered in April 2009. A prior triple reassortment of human, swine, and avian flu viruses combined with an additional Eurasian pig flu virus to produce what initially looked to be a novel strain of the H1N1 virus, giving rise to the term "swine flu" [29–32].

#### **3. Structure**

#### **3.1 Corona virus composition**

The lengthy RNA polymers that are tightly packed into the centre of coronavirus particles are encased in a protective capsid, which is a lattice of repeating protein molecules called the coat or capsid proteins. In coronaviruses, these proteins are referred to as nucleocapsids (N) [33, 34].

#### **3.2 The smallpox virus's structure**

The variola virus, a large double-stranded DNA pathogen with a shape akin to a brick, is serologically reactive with other members of the poxvirus family, including camel pox, vaccinia, cowpox, and ectromelia. Unlike other DNA viruses, the variola virus replicates in the cytoplasm of parasitized host cells [35, 36].

#### **3.3 Ebola virus's structure**

The Ebola virus (EBOV), a member of the family Filoviridae and genus Ebolavirus, has seven genes in its non-segmented, single-stranded RNA: (a) nucleoprotein (NP), (b) viral protein 35 (VP35), (c) VP40, (d) glycoprotein (GP), (e) VP30, (f) VP24, and (g) RNA polymerase (L) [37, 38].

#### **3.4 The swine flu virus's structure (H1N1)**

The RNA genome of the H1N1 influenza virus is around 13.5 kb in size, and its virions range in size from 80 to 120 nm. Hemagglutinin (HA) and neuraminidase, two envelope proteins (NA), are the 11 different proteins that are encoded by each of the eight segments that make up the swine influenza genome [39–41].

#### **4. Spreading**

#### **4.1 How is the Corona virus transmitted?**

When an infected person coughs, sneezes, or speaks, droplets or microscopic particles known as aerosols are emitted from their mouth or nose, dispersing the virus into the atmosphere. Anyone within 6°feet of that individual can breathe it into their lungs. Communication by air. The virus can hang about in the air for up to 3°hours, according to study [42–44].

#### **4.2 The smallpox virus: How does it spread?**

Smallpox spreads via contact with infected individuals. Smallpox is frequently spread from person to person by prolonged, direct face-to-face contact. Smallpox can also spread by contact with contaminated objects, such as contaminated bedding, clothing, or human fluids [20, 45, 46].

#### **4.3 How does the Ebola virus circulate?**

The only method to get Ebola is by direct contact with blood or other bodily fluids (such vomit, diarrhoea, urine, breast milk, sweat, or semen) from an infected person who is displaying Ebola symptoms or has recently passed away from Ebola [47, 48].

#### **4.4 H1N1 spreads in what way?**

The H1N1 virus spreads similarly to seasonal flu, according to the CDC. Droplets from an infected person's cough or sneeze, as well as touching something they recently touched and then contacting your eyes, mouth, or nose can all spread the flu [49, 50].

*A Review on Viral Outbreak in India with Special Reference to COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.108575*

#### **5. Testing**

#### **5.1 The COVID-19 test**

If you are currently infected with SARS-CoV-2, the virus that causes COVID-19, a viral test will look at samples taken from your mouth or nose. The two main types of viral tests are nucleic acid amplification tests (NAATs) and antigen testing. Depending on the circumstance, one test type may be recommended over another. All tests should adhere to the FDA's regulations. A laboratory setting is used for the majority of NAATs, including PCR-based testing. They are frequently the most reliable tests, regardless of whether a person has symptoms or not. These tests identify virus genetic material, which may stay in your body for up to 90 days after a positive test result. As a result, you should not utilise an NAAT if you had a positive test within the past 90 days. Antigen test results are available in 15–30 minutes. They are less reliable than NAATs, especially for people who do not show symptoms. A single, negative antigen test result cannot exclude an infection. For the best probability of identifying infection after a negative antigen test, the test should be repeated at least 48 hours later (known as serial testing). On rare occasions, a second NAAT may be suggested to confirm the outcomes of an antigen test [6, 51, 52].

#### **5.2 How is the small pox identified?**

Smallpox can be identified based on the patient's clinical signs and symptoms. The condition can be positively identified by extracting the virus from lesions or blood and by checking for viral-specific antibodies in the blood [9, 53].

#### **5.3 Virus testing for Ebola**

After symptoms manifest, blood can be tested for the Ebola virus. Up to 3 days after the initial signs and symptoms arise, the virus may not be visible. Polymerase chain reaction (PCR) is one of the most often used diagnostic procedures because it can detect extremely low amounts of the Ebola virus [54–56].

#### **5.4 H1N1 swine flu testing**

Polymerase chain reaction (PCR) testing is becoming more common in many hospitals and labs. This test could be administered to you while you are in the hospital or at the doctor's office. PCR testing, which is more sensitive than other techniques, can be used to identify the flu strain [57–59].

#### **6. Treatment**

#### **6.1 Treatment for COVID-19**

Turn off the patient in a well-ventilated space. Utilise a triple-layered medical mask, and after 8 hours, discard it (or sooner if it becomes moist or obviously dirty). If a caregiver enters the room, the patient and the caregiver could consider donning N 95 masks. The mask must first be sterilised with 1% sodium hypochlorite before being discarded. Take a rest and drink enough of drinks to maintain proper hydration. Always use appropriate breathing strategies. Use an alcohol-based product to disinfect your hands after regularly washing them for at least 40 seconds with soap and water. Give no access to your personal goods to family members. Ensure that a 1% hypochlorite solution is used to clean the area's commonly touched surfaces, such as tabletops, doorknobs, and handles. Check the temperature every day. To check oxygen saturation, a pulse oximeter should be used every day. Contact your medical physician right away if you notice any worsening of your symptoms [60, 61].

#### **6.2 Treatment for smallpox**

To stop an outbreak of smallpox, health officials would use vaccines. There is currently no known cure for smallpox in humans, despite the fact that some antiviral drugs may help with treatment [62, 63].

#### **6.3 Therapy for Ebola**

Delivering fluids and electrolytes (body salts) intravenously or orally (intravenously). taking medication to control fever, reduce nausea and vomiting, stabilise blood pressure, and relieve pain. Treating any further infections that may develop [64, 65].

#### **6.4 Therapy for swine flu**

Some of the antiviral drugs used to treat seasonal flu can also be used to treat H1N1 swine flu. The three antivirals zanamivir (Relenza), peramivir (Rapivab), and oseltamivir (Tamiflu) tend to be the most effective ones; nevertheless, oseltamivir is ineffective against some swine flu strains. These drugs might help you recover more quickly [32, 66].

#### **6.5 COVID-19 vaccines vaccination**

To avert this pandemic, a large segment of the population must be immune to the virus. The safest method to do this is through immunisation. In the past, vaccines have been a common method employed by humans to lessen the prevalence of infectious diseases that are lethal. A number of research teams stepped up to the plate and developed SARS-CoV-2 vaccines when the pandemic began less than a year ago. The aim now is to make these vaccines available to people everywhere. It will be vital that everyone receives the appropriate protection, not only those in wealthy countries. A COVID-19 vaccination, especially a booster, effectively protects recipients from developing severe illness, necessitating hospitalisation, and even dying. The COVID-19 vaccine is safe—much safer than getting COVID-19 from a person. People who have received the COVID-19 vaccine may benefit from additional protection from the vaccine, such as protection from having to stay in the hospital for a future infection. Similar to vaccines for other diseases, people are most protected when they receive the recommended number of doses plus boosters [67–69].

The U.S. Food and Drug Administration (FDA) has approved the smallpox vaccine ACAM2000®, (Smallpox [Vaccinia] Vaccine, Live), a replication-competent vaccine, for use in those who have been identified as having a high risk of getting smallpox. India had smallpox vaccination in 1904–1907 [70, 71].

#### **6.6 Ebola virus illness vaccine**

The Ebola Zaire Vaccine, Live, also known as V920, rVSV-G-ZEBOV-GP, or rVSV-ZEBOV, has been licenced by the U.S. Food and Drug Administration (FDA) for use in preventing Zaire ebolavirus disease in adults 18 years of age and older as a single dose administration [72–75].

#### **6.7 Swine flu vaccine**

The use of one dose of the 2009 H1N1 influenza vaccine has been authorised by the U.S. Food and Drug Administration (FDA) for people 10 years of age and older. It is recommended that children between the ages of 6 months and 9 years receive two doses of the immunisation. These two dosages should be separated by 4°weeks. The swine flu vaccine is reliable and secure. Nevertheless, a large number of people who were not at risk of contracting the virus had health problems as a result of the 1976 vaccine campaign. In contrast, the effective 2009 vaccination campaign helped to stop the H1N1 influenza pandemic in 2010 [32, 76].

#### **7. Indian context**

The primary causes of morbidity and mortality in both humans and animals continue to be infectious diseases, which has a significant financial impact on India's healthcare system. The country has had a number of epidemics and outbreaks of infectious diseases. Major epidemic diseases including cholera, leprosy, malaria, and plague have all traditionally been successfully controlled. Due to the country's varied geography, extreme geoclimatic fluctuations, and unequal population distribution, viral disease dispersion patterns are uniquely displayed. The dynamic interconnections of biological, social, and ecological variables as well as unanticipated features of the interaction between people and animals present additional challenges with regard to the origins of infectious diseases. Understanding the impact of the conditions required for the emergence and developing strengthened surveillance systems that can lessen human suffering and mortality are just two of the significant problems faced in the control and prevention of emerging and re-emerging infectious diseases. The important emerging and re-emerging viral infections of public health significance that have previously been incorporated into the Integrated Disease Surveillance Programme have been reviewed in this article.

The cholera epidemic had a significant impact on British colonial India on numerous occasions in the nineteenth century, including in the years 1817, 1829, 1852, 1863, 1881, and 1899, according to studies. Slum dwellers and the poor in rural areas, primarily in Northern Indian provinces like Punjab, Delhi, and United Provinces, made up the majority of the pandemic's casualties (current Uttar Pradesh and Uttarakhand). It gradually spread to nearby provinces, with the Madras presidency in 1877 suffering the most. Instances were noted in 1899 in Calcutta, Madras, and Bombay, three important provinces. The virus expanded to a number of countries following each epidemic, including the US, China, Arabia, Persia, and Russia. In 1992, there was a major cholera outbreak on India's southern peninsula. A cholera outbreak that followed the Orissa floods of 2001 claimed the lives of 33 individuals while infecting 34,111 others [77–79].

#### **7.1 Smallpox (1974)**

A smallpox outbreak struck West Bengal, Bihar, and Orissa in 1974. About 85% of all incidents that were reported globally were in India. In the worst smallpox pandemic of the twentieth century, around 15,000 individuals perished. Thousands of survivors suffered from blindness and deformities. The WHO launched the fight to eradicate smallpox. In 1980, the WHO deemed it extinct [80–82].

#### **7.2 Influenza (1918–1920)**

The H1N1 influenza virus caused the deadly pandemic known as the Spanish Flu or Spanish "Influenza," which claimed the lives of 20–50 million people worldwide. The flu first came in 1918, and the following year, in the fall, a second, more severe wave of the illness reappeared and swept the globe. The second wave originated in Bombay, India, and afterwards spread to Sri Lanka and the rest of the world. With an estimated death toll of 10–20 million, India served as the pandemic's mortality epicentre. One of the reasons the outbreak subsided later was the weather in India. In humid settings, the influenza virus cannot survive and cannot spread [83–85].

#### **7.3 Polio (1970–1990)**

India was affected by the polio epidemic between 1970 and 1990. India was the developing country that was most badly damaged till the late 1990s. Post-polio paralysis was widespread in children. Both urban and rural regions were severely impacted. India was the source of 40% of all polio cases that have been reported worldwide. Despite the fact that oral vaccinations were initially given there in the 1960s, India was declared polio-free in January 2011 [86].

#### **7.4 Plague outbreaks (1994, 2002, 2004)**

1994 saw a plague outbreak in Surat, Gujarat, however it was over in less than 2°weeks. The amazing panic it caused and the repercussions it had on the entire planet, nevertheless, made it noteworthy. There were only 1000 reported incidents, involving 53 fatalities. Panic and quarantine concern caused a population evacuation and internal migration [87, 88].

#### **7.5 Encephalitis in Japan (2005)**

Japanese encephalitis is a flavivirus illness that injures the brain and causes swelling that is transmitted by mosquitoes. The virus that is causing the sickness has genes in common with viruses that cause dengue and yellow fever. 2005 saw 90 occurrences in Bihar and 1145 cases from 14 districts in Uttar Pradesh. About 296 persons, or one-fourth of all those impacted, passed away. Annual reports of encephalitis cases are still common, mostly in the north (Uttar Pradesh) [89–91].

#### **7.6 Chikungunya (2006)**

In 2006, Chikungunya broke epidemic in India. Nationwide, there were almost 15 lakh recorded cases. The southern states of Gujarat, Madhya Pradesh, Maharashtra, and the Andaman and Nicobar Islands reported the majority of the cases. It was found *A Review on Viral Outbreak in India with Special Reference to COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.108575*

that Aedes mosquitoes carried the illness. Chikungunya-related deaths were underreported for a number of reasons. The outbreak was contained in part by eradicating mosquito breeding grounds, implementing additional vector control measures, promoting awareness, etc. A dengue outbreak occurred that same year, resulting in 10,344 cases and 162 fatalities [92, 93].

#### **7.7 H1N1 flu (2010 and 2015) (2010 and 2015)**

About 18,500 people died from H1N1 flu, also referred to as swine flu, in 2010. Over 27,000 confirmed cases, including 981 fatalities, were reported in India. With 30,000 cases nationally and 1731 fatalities, the flu made a comeback in 2015. The worst affected states were Gujarat, Maharashtra, and Rajasthan [94, 95].

#### **7.8 COVID-19**

India has tallied more than 18,000 confirmed cases, 600 of which have been linked to COVID-19-related fatalities. As of April 18, 2020, the COVID-19 death rate in India was 3.3%, according to the Ministry of Health. More vulnerable individuals include those who are older and/or have co-morbid disorders [96, 97].

#### **8. Conclusions**

Global social and economic conditions have been considerably disrupted by the pandemic, leading to the worst recession since the Great Depression. Supply chain instability led to widespread shortages of items, particularly food supplies. The resulting practically universal lockdowns resulted in a record-breaking decrease in emissions. The primary causes of morbidity and mortality in both humans and animals continue to be infectious diseases, which has a significant financial impact on India's healthcare system. The country has had a number of epidemics and outbreaks of infectious diseases. Major epidemic diseases including cholera, leprosy, malaria, and plague have all traditionally been successfully controlled. Due to the country's varied geography, extreme geoclimatic fluctuations, and unequal population distribution, viral disease dispersion patterns are uniquely displayed. The dynamic interconnections of biological, social, and ecological variables as well as unanticipated features of the interaction between people and animals present additional challenges with regard to the origins of infectious diseases. Two of the major challenges in the control and prevention of emerging and re-emerging infectious diseases are understanding the effects of the conditions necessary for their emergence and creating strengthened surveillance systems that can reduce human misery and mortality. This article reviews the significant emerging and re-emerging viral illnesses of public health importance that have previously been included in the Integrated Disease Surveillance Programme. India is always at danger from newly emerging and re-emerging viral infections that are important for public health because of its great geoclimatic variety. With an emphasis on epidemiology and disease burden, illness surveillance needs to be strengthened across the country. In-depth knowledge of disease biology, particularly that of disease vectors and the effects of the environment on disease, is also urgently needed. It is also necessary to increase emergency preparedness for these diseases and response by focusing on the "one health" idea. India had a gradual rise in the number of cases after the first case was identified on January 30, 2020. However, given that

testing approach and skills have progressively improved, India's meagre testing efforts may reflect an underestimate of COVID-19 circumstances. Additionally, the clear selective policy of only screening symptomatic individuals contributed to the underrepresentation of the genuine case counts. This brought to light the fact that there are incidences in India that go unreported. It is essential to develop a universal testing method for all symptomatic, asymptomatic, pre-symptomatic, and post-symptomatic cases in order to successfully stop the spread of COVID-19, which is on the rise. Given its vast population and high danger of community transmission, this is particularly true in India. India uses the corona virus spike proteins to represent different COVID-19 defence systems (**Figure 1**). To combat COVID-19, the Indian government has undertaken a number of activities, including testing, vaccination, mask and sanitizer use, genome sequencing, government and public awareness campaigns, research, and the improvement of health infrastructure. With the aid of a large number of tests, including RT-PCR and rapid antigen testing kits, India's indigenous COVID-19 vaccine COVAXIN, developed by Bharat Biotech in collaboration with the Indian Council of Medical Research (ICMR) - National Institute of Virology (NIV), and the vaccine Covishield manufactured and large-scale production by the Serum Institute of India, played a significant role in inhibiting the rapid spread of the pandemic (UK). There was a severe shortage of masks and sanitizers during the COVID-19's initial phase, but internal production, large-scale production, and distribution severely damaged the chain. Genome sequencing in India occasionally helped to detect the different altered Corona virus strains. In India, public awareness campaigns and government regulations were key in preventing the COVID-19 virus from spreading. For the COVID-19, which included research labs, institutes, and universities all over India, researchers

*Different strategies for COVID-19 in India represented as spike proteins of Corona virus.*

*A Review on Viral Outbreak in India with Special Reference to COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.108575*

worked tirelessly to oversee the Research and Development units. Instead of India's underdeveloped healthcare infrastructure, ongoing work is being done to create appropriate facilities with proper management of healthcare infrastructure, such as converting regular hospitals into COVID-19 hospitals that are specially outfitted and redesigned to meet the needs of patients infected with the virus.

#### **Author details**

Aishwarya Khamari1 \*, Monika Khamari<sup>2</sup> , Akshya Kumar Mishra<sup>3</sup> , Jijnasa Panda1 , Debashish Gardia4 and Ratikanta Rath<sup>5</sup>

1 School of Life Sciences, Sambalpur University, Sambalpur, India

2 Department of Biotechnology and Bioinformatics, Sambalpur University, Sambalpur, India

3 Department of Microbiology, Batakrushna College of Pharmacy, Nuapada, India

4 Department of Pharmacology, Batakrushna College of Pharmacy, Nuapada, India

5 Department of Zoology, Government Women's College Sambalpur, Sambalpur, India

\*Address all correspondence to: khamariaishwarya@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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Section 3
