Clinical, Virological, Immunological, and Genomic Characterization of Asymptomatic and Symptomatic Cases With SARS-CoV-2 Infection in India

Abstract

Purpose: The current global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the investigation with clinical, biochemical, immunological, and genomic characterization from patients to understand the pathophysiology of viral infection.

Methods: Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2-confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, and treatment regimen were collected from a hospital; viral load was determined by RT-PCR; and the levels of cytokines and circulating antibodies in plasma were assessed by Bio-Plex and isotyping, respectively. In addition, whole-genome sequencing of viral strains and mutational analysis were carried out.

Results: Analysis of the biochemical parameters highlighted the increased levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT, and ferritin in symptomatic patients. Symptomatic patients were mostly with one or more comorbidities, especially type 2 diabetes (66.6%). The virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. On the other hand, viral load was higher in plasma and serum samples of symptomatic patients, and they develop sufficient amounts of antibodies (IgG, IgM, and IgA). The levels of seven cytokines (IL-6, IL-1α, IP-10, IL-8, IL-10, IFN-α2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO, and MDC) were remarkably higher in asymptomatic patients. The whole-genome sequence analysis revealed that the current isolates were clustered with 19B, 20A, and 20B clades; however, 11 additional changes in Orf1ab, spike, Orf3a, Orf8, and nucleocapsid proteins were acquired. The D614G mutation in spike protein is linked with higher virus replication efficiency and severe SARS-CoV-2 infection as three patients had higher viral load, and among them, two patients with this mutation passed away.

Conclusions: This is the first comprehensive study of SARS-CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and thereby advance the implementation of effective disease control strategies.

Keywords: COVID-19; D614G mutation; SARS-CoV-2; asymptomatic and symptomatic patients; cytokines; disease severity.

Figure 1

Viral dynamics in patients with asymptomatic and symptomatic disease. (A) Bar diagram depicting the ΔΔCT of OP samples collected from patients with asymptomatic and symptomatic COVID-19 at different days of disease onset. (B, C) Graph showing ΔΔCT of individual plasma and serum samples collected from asymptomatic and symptomatic COVID-19 patients. (D) Bar diagram representing the viral copy number of OP samples collected from patients with asymptomatic and symptomatic COVID-19 at different days of disease onset. (E, F) Graph depicting viral copy number of individual plasma and serum samples collected from asymptomatic and symptomatic COVID-19 patients. The Mann–Whitney (non-parametric, two-tailed) test was performed. All error bars were SD. p value ≤ 0.05 was considered statistically significant (*) and p value ≤ 0.01 was considered to be very significant (**).

Figure 2

Analysis of SARS-CoV-2-specific (IgG+IgM+IgA) antibodies in asymptomatic and symptomatic patients. Bar graph representing antibody units (IgG+IgM+IgA) of plasma samples from a COVID-19 patient. Line depicting the cutoff value of positive samples which was 59.3. Negative (NC) and positive (PC) controls delivered by detection kit were included to warrant test validity.

Figure 3

Evaluation of the cytokine responses between asymptomatic and symptomatic COVID-19 patients. The bar diagram depicting the expression levels of cytokines in pg/ml. Cytokines were measured after disease onset between asymptomatic and symptomatic patients. The Mann–Whitney (non-parametric, two-tailed) test was performed. p < 0.05 was considered statistically significant (*), and p < 0.005 was considered to be very significant (**). ns, not significant. All error bars were SD.

Figure 4

Analysis of the circulating antibodies in asymptomatic and symptomatic COVID-19 patients. The bar diagram representing circulating antibody isotypes (IgA, IgM, IgG1, IgG2, IgG3, IgG4, and IgE) which were evaluated from COVID-19-positive plasma samples. The Mann–Whitney (non-parametric, two-tailed) test was performed. p < 0.05 was considered statistically significant (*), and p < 0.005 was considered to be very significant (**). ns, not significant. All error bars were SD.

Figure 5

The phylogenetic analysis of the whole-genome sequences of four SARS-CoV-2 strains from human oropharyngeal swab samples is depicted. The phylogenetic tree was generated using the maximum likelihood method with 1,000 bootstrap value by the IQTREE2 tool. The tree was constructed using WH-01 (NC_045512.2) as a reference strain along with four strains from each of the countries, namely, USA, Brazil, Canada, Australia, South Africa, United Kingdom, and South Korea. The sequences for reference strains were retrieved from the GISAID database. The viral isolates are depicted by hCoV-19/country/strain ID/year of isolation/accession number. The bootstrap values are mentioned at major branch points of the tree.

Figure 6

Mutation profiles of SARS-CoV-2 strains. Dot plot summarizing the single nucleotide variants in four studied cases. The x-axis represents the nucleotide change with position and region, and the y-axis represents cases. The dots represent the presence or absence of a mutation (small dot absent and large dot presence), and different colors represent different types of mutations. High-quality mutations (depth > 20) are represented by round dots and triangles represent low-quality mutations. PS: low-quality site only represented in spike 23403: A>G change.