Pathogenesis, immune response and laboratory diagnosis of severe acute respiratory syndrome associated Coronavirus 2

The CoVID-19 caused by a novel strain of coronavirus, known as Severe Acute Respiratory Syndrome Associated Coronavirus 2 (SARS-CoV-2), has spread worldwide with about 15 million confi rmed cases and more than half a million deaths as of now according to Center for Disease Control and Prevention (CDC). The fi rst incidence of the disease as clustered outbreak, reported in the Wuhan city of China during early December 2019 originated in the Huanan Seafood Market. The SARS-CoV-2 genome sequenced in January 2020 revealed the novel coronavirus have greater similarity to (96%) bat coronavirus compared with (79%) Severe Acute Respiratory Syndrome Associated Coronavirus (SARS-CoV) and (50%) Middle Eastern Respiratory Syndrome coronavirus (MERSCoV) that had caused an earlier outbreaks in China (2002) and Middle East (2012) respectively [1]. The intermediate host for SARS-CoV and MERS-CoV was implicated to be civet cats and camel respectively [2]. The SARS-CoV-2 debuted into human host causing severe respiratory disorders and the intermediate host is unclear, probably bat [3] snake or pangolin [2]. However SARS CoV-2 is more contagious than the SARS-CoV and MERSCoV [4].

of whales and birds) and deltacoronavirus (virus isolated from pigs and birds) [7]. The alphacoronavirus consists of many animal virus and human virus (HCoV-229E and HCoV-NL63). The betacoronavirus genus includes prototypes of mouse Hepatitis Virus (HMV) and the human viruses such as the HCoV-OC43, SARS-HCoV, HCoV-HKU1, MERS-CoV, SARS-CoV including the SARS-CoV-2 capable of causing mild to severe respiratory infections. The Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV) proposes to use the convention SARS-CoV-2/host/location/ isolate/date to name a particular isolate (for example SARS-CoV-2/human/Wuhan/X1/2019) with various characteristics like pathogenic potential in human and other hosts. In publications it would be further extended with a sequence database ID linking it to the specifi c genome sequence of the virus in the public database such as GenBank enabling further epidemiological and other studies [5].

Genome and proteins
The virus belonging to coronavirus subfamily, have the largest RNA genome of 28 to 32 kb. The enveloped single stranded positive RNA virus contain the replicase locus encoded at its 5' end and various structural genes arranged in the order Heamaggluttin esterase or HE (not seen in SARS-CoV 2), spike or S, small membrane or envelope or E, membrane or M and nucleocapsid or N at its 3'end [8]. Inside the envelope, the helical capsid structure is the RNA complexed with N whereas the S trimer, embedded in envelop, gives the virus a crown like morphology and hence giving the name 'corona" [9].
The Spike (S) glycoprotein of the SARS-CoV-2 is cleaved by a trypsin-like host serine protease (TMPRSS) into S1 and S2 domains to mediate viral entry into the host cell through the angiotensin-converting enzyme 2 (ACE2) receptors. The S2 domain is conserved in coronavirus forms the highly coiled stalk portion while the divergent S1 domain forms the bulb of the spike. The binding of S1 domain to the ACE2 receptors results in conformational changes in S2 domain leading to the fusion of virion and host cell membrane [6]. The interaction between S and corresponding receptors on the host cell determines the species range and tissue tropism of the virus. However S protein in SARS-CoV 19 have a peculiar furin-like cleavage site missing in the MERS-CoV and SASR-CoV [10]. Further, the virus is internalized by receptor mediated endocytosis [11].
The Membrane (M) glycoprotein, a membrane protein, contributing to virion shape is the most abundant constituent of the virus [6]. The glycosylated ectodomain of M molecule, is the least conserved part and is highly sensitive to proteolysis. The endodomain of M molecule, closely connected to the membrane surface, is compact and somewhat resistant to proteolysis. The transmembrane domain of M, which aids in anchoring and inserting the protein in its native orientation in the membrane is the central organizer of the viral assembly process [12].
The small membrane or envelope (E) protein, a minor constituent of virion has a short hydrophilic and long hydrophobic segment. The palindromic hairpin configuration of the transmembrane segment in the E protein is unique to SARS CoV [13]. The E protein have signifi cant role in viral assembly, budding and spread. The N (Nucleocapsid) protein bind to the genomic RNA like beads on strings and interacts with M protein for viral assembly [14].
The coronavirus family has the largest genome among all RNA viruses enabling expansive coding capacity and gene expression strategies. The extremely large genome of coronaviruses are similar to eukaryotic mRNA with a 5'caps and 3' poly (A) tails at either ends. The gene coding for replicase is towards the 5' end whereas those coding for the structural genes like S, M and E are clustered at 3'end [15]. Another

Translation and replication
After viral entry into host cell the nucleocapsid and viral genome are disassembled. The ORFs 1a and 1b at the 5′ end of the genome RNA are translated into pp1a and pp1ab via a frameshift mechanism and co-translational proteolytic processing results in production of 16 nonstructural proteins (designated as nsp 1 to nsp 16) including various accessory proteins [17]. The ORF1a encodes proteases (nsp1 to nsp 11) which function to process pp1a and pp1ab into the mature replicase proteins, assembling machinery for RNA synthesis and prime the host cell for infection whereas the ORF1b encodes (nsp 1 to nsp 10 and nsp11 to nsp 12) multiple enzymes promoting the replicase complex in viral metabolism associated with catalyzing RNA replication, transcription and also interfere with host cell process [11].  [18]. Clinical manifestations range from fever, unproductive cough, fatigue, diarrhea, myalgia, sore throat, lymphopenia to severe pneumonia [19]. The third stage progresses to pulmonary destruction with hypoxia and acute respiratory distress syndrome [18]. The infi ltrated lung and damaged alveoli shows multinucleated giant cells containing macrophages and cells of epithelial origin along with numerous characteristic syncytium like formation [20].
There is marked lymphopenia, hemophagocytosis and in some cases diarrhea, hepatic involvement, atrophy of white pulp in speen, lymphadenopathy suggesting wide systemic spread dissemination of the virus along with copious viral shedding in respiratory and excretory secretions. Some develop cardiovascular complications, with tachyarrhythmia and thromboembolic events characterized by troponin rise [21].
Clinical investigations showed fevers, lymphopenia, highly elevated C-reactive protein, proinfl ammatory cytokines, serum ferritin, and D-Dimers. One possible explanation of these proinfl ammatory cytokine expression is the virus induced endothelial damage, apoptosis, and necrosis during viral replication and dissemination, leading to immune cell recruitment and activation, which in turn exhibited strong uncontrolled hyper-infl ammation. Lethal cases of Covid 19 showed increased number of neutrophils and activated monocyte/macrophage in the airways [19].

Host immune response
The Furthermore ACE2 receptors are lesser in children compared to adults for viral entry into the cell important for the mediation of infection [29]. Women also showed less viral load, ACE2 expression and infl ammation and higher level of CD4+ T cells and antibody response, TLR8, CD40L and CXCR3 and high antiviral IF-Gama production when compared to men.
The reasons for this is the hormonal and genetic difference that lower incidence of comorbidities affecting lungs such as smoking, cardiovascular diseases and X chromosomes that produce associated cytokines [30]. Patient over 60 showed more than 50% mortality. This extreme increase or decrease in age is correlated with an increase in severity and mortality in the disease [21].

Radiological fi nding: Typical CT scans of Covid 19 patients
show ground glass opacities mostly involving multiple lobes with a peripheral and subpleural distribution in majority of cases and a "white lung appearance" in severe cases. CT can be recommended for long term evaluation of fi brosis in lungs [31]. Chest X Rays, though not as sensitive as CT, can also be helpful in monitoring the rapid progress of infected patients in emergency cases. However the imaging reports are variable, nonspecifi c, have signifi cant overlap with pneumonia from other etiology and so the standard reference relies mainly on laboratory diagnosis for confi rmation of the disease [32].

Specimen collection, biosafety and transport
Specimen collected are nasopharyngeal (OP) and/or oropharyngeal (ON) swabs using dracon or polyester swabs from the Upper Respiratory Tract (URT), sputum or Bronchoalveolar Lavage (BAL) or endotracheal aspirate collected in sterile containers from Lower Respiratory Tract (LRT). During initial stages RNA of SARS Cov-2 is detected more from NP swabs (63%) than in OP swabs (32%) [33]. World Health Organization recommends collecting OP and NP swabs in same the tube to increase the detection. URT can be easily collected compared to sputum and BAL, which increases the exposure and biosafety risk to health workers [34]. Viral RNA peaks in URT during 7 to 10 days after disease onset and declines after that whereas in LRT the viral detection rate are high after 3 rd week of illness onset and maintains thus throughout [35]. Other specimens including rectal swabs, stool, urine and blood in appropriate to disease severity and to assess cross reactivity leading cross protection and long term immunity from an individual level [34]. The viral antigens mostly used in serological assays are the N protein, S protein and RBD of the S protein. Sensitivity wound be higher if N and S proteins are used together [38].  [38]. Other molecular methods such as loop-mediated isothermal amplifi cation, multiplex isothermal amplifi cation followed by microarray detection, and CRISPR (clustered regularly interspaced short palindromic repeats)-based assays are also being evaluated [34].