Molecular and Serological Characterization of the SARS-CoV-2 Delta Variant in Bangladesh in 2021

Novel SARS-CoV-2 variants are emerging at an alarming rate. The delta variant and other variants of concern (VoC) carry spike (S)-protein mutations, which have the potential to evade protective immunity, to trigger break-through infections after COVID-19 vaccination, and to propagate future waves of COVID-19 pandemic. To identify SARS CoV-2 variants in Bangladesh, patients who are RT-PCR-positive for COVID-19 infections in Dhaka were screened by a RT-PCR melting curve analysis for spike protein mutations. To assess the anti-SARS CoV-2 antibody responses, the levels of the anti-S -proteins IgA and IgG and the anti-N-protein IgG were measured by ELISA. Of a total of 36 RT-PCR positive samples (75%), 27 were identified as delta variants, with one carrying an additional Q677H mutation and two with single nucleotide substitutions at position 23029 (compared to Wuhan-Hu-1 reference NC 045512) in the genome sequence. Three (8.3%) were identified as beta variants, two (5.5%) were identified as alpha variants, three (8.3%) were identified as having a B.1.1.318 lineage, and one sample was identified as an eta variant (B.1.525) carrying an additional V687L mutation. The trend of higher viral load (lower Cp values) among delta variants than in the alpha and beta variants was of borderline statistical significance (p = 0.045). Prospective studies with larger Bangladeshi cohorts are warranted to confirm the emergence of S-protein mutations and their association with antibody response in natural infection and potential breakthrough in vaccinated subjects.


Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in Wuhan province, China, in December 2019, has become responsible for the Corona-virus disease 2019 (COVID-19) pandemic, causing over five million reported COVID-19 deaths worldwide, of which 25% are reported from the United States and India [1]. The global and confirmed the mutations by sequencing. The titers of anti-spike protein and antinucleocapsid protein antibodies were measured by ELISA. In this study, we detect SARS CoV-2 variants circulating in unvaccinated subjects and show that the delta variant is predominant in Dhaka city during the two-week period between 26 May and 6 June 2021. The limited sample size is not sufficient to assess the statistical significance of the antibody responses found among patients infected with SARS CoV-2 variants carrying rare amino acid mutations in the spike protein sequence. The findings underscore the importance of monitoring the emergence of SARS-CoV-2 variants during natural SARS-CoV-2 infection in a low-income country with 3 percent of its population receiving COVID-19 vaccines.

Study Design and Sample Collections
This study is a cross-sectional single-center study on thirty-six Bangladeshi adults testing RT-PCR SARS-CoV-2 positive with symptomatic COVID-19 between 26 May 2021 and 6 June 2021 at Dhaka Medical College. All cases were randomly selected for the survey of SARS CoV-2 variants and had not received any COVID-19 vaccination. Nasopharyngeal specimens were collected and stored at −80 • C in a virus collection and preservation medium (Khang Jian Medical Apparatus Ltd., Taizhou, China) and transported on dry ice to Germany. Serum samples matched to the date of swab collection, n = 36, and at follow-up on day 13 or day 14, n = 28, or on day 17, n = 1, post onset of COVID-19 symptoms (POCS) were kept at −20 • C storage in screw-tight cryopreservation vials (Greiner-bio one).

Quantification of Viral RNA
The viral RNA of nasopharyngeal specimens was extracted with a Magnapure 96 instrument (Roche, Mannheim, Germany) using the DNA and Viral nucleic acid kit (Roche, Penzberg, Germany). The RNA concentration in each sample was determined by reverse transcriptase polymerase chain reaction (RT-PCR) using the LightMix ® Modular SARS-CE assay (50-0776-96, TIB MolBiol, Berlin, Germany) and programming on 480II light cycler (Roche, Penzberg, Germany). The point at which the amplification curve crossed the vertical threshold line in the RT PCR cycle (Cp) was reported as positive if the sample Cp was less than 40.

Bioinformatics
Sequence and phylogenetic analyses were conducted using software program MEGA version X. The derived sequences were compared with the data from Global Initiative on Sharing All Influenza Data (GISAID) database and analyzed according to the phylogenetic assignment of named global outbreak (PANGO) lineages [27].

Assessment of Anti-SARS CoV-2 Antibodies
The anti-SARS-CoV-2 spike (S) protein IgA and the anti-SARS-CoV-2 S-protein IgG Quanti vac ELISA (EUROIMMUN Medizinische Labordiagnostika AG, Germany) were measured according to the manufacturer's instructions. The positive IgA reactivity was reported as OD450nm − OD620nm > 0.22 and the positive anti-S-protein IgG > 11 RU/mL IgG. The positive anti-nucleocapsid (N)-protein IgG reactivity was according to the recommended cut-off value of NovaTec units (NTU) > 10 for positive results (Novatec Diagnostics, Dietzenbach, Germany); NTU = X × 10/QC, where X = OD450 nm − OD620 nm of the test sample and QC = OD450 nm − OD620 nm of the quality control equivocal serum sample. E > 10 NTU. All ELISA measurements were performed on a Multiskan 96 well reader (Lot 357−706872, Thermofischer Scientific Life Technologies, Darmstadt, Germany).

Statistical Analysis
The Cp values, the levels of the anti-SARS CoV-2 S-proteins IgA and IgG, and the levels of the anti-N-protein IgG are presented as the mean with a standard deviation and as medians with ranges. Comparisons of the values between patient groups were assessed by a nonparametric Mann-Whitney sum rank test or Chi-square analysis. A p value of ≤ 0.05 was considered statistically significant with differences between independent groups. Correlation analyses were calculated with the Spearman's rank correlation coefficients. The correlation coefficients r > 0.4 or r < −0.4 with significance at p < 0.05 were considered strongly positive or strong negative associations, respectively. Statistical analysis was performed with MedCalc version 14 for Windows (MedCalc Software, Mariakerke, Belgium).

SARS CoV-2 Variants
Of the 36 (14%) RT-PCR-positive cases, 5 belonged to known variants: two alpha (H69V70del and N501Y) and three beta (K417N, E484K, and N501Y), designated as group 1 (Table 1). Of a total 36 RT-PCR-positive Dhaka COVID-19 cases, 27 (75%) were identified as delta variants. Of the 27, 24 had amino acid substitutions G142D, T478K, and P681R, matching the known delta variants in India, and were assigned to group 2. The other 3 of the 27 delta variants had aberrant melting curves in the VirSNiP SARS-CoV-2 typing assay and were assigned after sequencing to the group 3 "delta plus" ( Table 1). The sequencing of spike gene fragments from these three delta plus variants showed one carrying an additional Q677H mutation and the other two car-rying single nucleotide substitutions C23045T compared with the reference wild-type Hunan SARS-CoV-2 strain NC-045512 in the genome sequence ( Figure 1 and Table 2). The remaining 4 of the 36 (11%) RT-PCR-positive cases in group 4 were other variants: three were matched to the PANGO lineage B.1.1.318, of which two carried nucleotide substitution T23287C without amino acid change ( Table 2). The variant of interest (VOI), the eta variant belonging to the B.1.525 lineage, revealed a novel amino acid substitution V687L in addition to A67V and Q677H (Table 2).

Bangladeshi COVID-19 Patient Characteristics
All thirty six of the enrolled SARS-CoV-2 RT-PCR patients were unvaccinated adults with symptomatic COVID-19 in the Dhaka district. The group 1 alpha or beta SARS-CoV-2 variants presented clinical symptoms at similar frequency to the clinical symptoms of group 2 delta SARS-CoV-2 variants, with the exception of loss of smell or taste, which was reported at significantly higher frequencies among group 1 than group 2.

SARS CoV-2 Variant Viral Loads
The median Cp 24 of delta variants in group 2 is significantly lower than the median Cp 31 of alpha or beta variants in group 1 (Table 1 and Figure 2). The variants in group Viruses 2021, 13, 2310 5 of 12 3 and group 4 show a trend for lower Cp values than that of group 1, but the number of cases is insufficient to assess if the differences in viral loads are of statistical significance.    Viruses 2021, 13, x FOR PEER REVIEW 5 of 12 Table 1. Patient characteristics. The patient characteristics in four groups of SARS-CoV-2 variants were compared. The pvalues are either the rank sum of median differences between group 1 versus group 2 in the Mann-Whitney independent test or the difference in proportions between group 1 versus group 2 in the Chi-square analysis (b). Time points (A) = day 2 or 3 and (B) = day 13 or 14 post onset of COVID-19 symptoms (POCS). NR= not relevant.    Table 2.  Table 2.

Bangladeshi COVID-19 Patient Characteristics
All thirty six of the enrolled SARS-CoV-2 RT-PCR patients were unvaccinated adults with symptomatic COVID-19 in the Dhaka district. The group 1 alpha or beta SARS-CoV-2 variants presented clinical symptoms at similar frequency to the clinical symptoms of group 2 delta SARS-CoV-2 variants, with the exception of loss of smell or taste, which was reported at significantly higher frequencies among group 1 than group 2.

SARS CoV-2 Variant Viral Loads
The median Cp 24 of delta variants in group 2 is significantly lower than the median Cp 31 of alpha or beta variants in group 1 (Table 1 and Figure 2). The variants in group 3 and group 4 show a trend for lower Cp values than that of group 1, but the number of cases is insufficient to assess if the differences in viral loads are of statistical significance.

Antibody Responses in SARS-CoV-2 Variants
Antibodies were detected against both the spike (S) protein ( Figure 3) and the nucleocapsid (N) protein (Figure 4). Of the 24 (8%) delta variant cases, 2 gave the negative anti-S-protein IgG. Only 1 of the 24 delta variant cases tested negative for both anti-S-protein and anti-N-protein antibodies. The one case of eta variant B.1.525, although positive for anti-S-protein IgG, gave no detectable anti-N-protein reactivity (Figure 4). One of the three B.1.1.318 lineage cases were below the anti-S-protein cut-off on day 2 POCS (Figure 3D). Anti-S-protein antibody seroconversion (negative reactivity on day 2 and positive reactivity on days 16 or 17 POCS) was found in three cases: one beta variant ( Figure 3A) and two Viruses 2021, 13, 2310 7 of 12 delta variants ( Figure 3B). The anti-S-protein IgA in the cases of alpha or beta variants of group 1 show a trend for higher levels than anti-S-protein IgA levels of the delta variants in group 2 (Table 1).
S-protein IgG. Only 1 of the 24 delta variant cases tested negative for both anti-Sand anti-N-protein antibodies. The one case of eta variant B.1.525, although posi anti-S-protein IgG, gave no detectable anti-N-protein reactivity (Figure 4). One of th B.1.1.318 lineage cases were below the anti-S-protein cut-off on day 2 POCS (Figu Anti-S-protein antibody seroconversion (negative reactivity on day 2 and positive r ity on days 16 or 17 POCS) was found in three cases: one beta variant ( Figure 3A) a delta variants ( Figure 3B). The anti-S-protein IgA in the cases of alpha or beta var group 1 show a trend for higher levels than anti-S-protein IgA levels of the delta v in group 2 (Table 1).   (Figure 4). One of th B.1.1.318 lineage cases were below the anti-S-protein cut-off on day 2 POCS (Figu Anti-S-protein antibody seroconversion (negative reactivity on day 2 and positive ity on days 16 or 17 POCS) was found in three cases: one beta variant ( Figure 3A) a delta variants ( Figure 3B). The anti-S-protein IgA in the cases of alpha or beta var group 1 show a trend for higher levels than anti-S-protein IgA levels of the delta v in group 2 (Table 1).  To assess the associations between the levels of anti-N-protein versus anti-S-protein antibodies, regression analyses were made on day 2 or day 3 and on day 16 or day 17 POCS. The serum anti-N-protein IgG was positively correlated with the serum anti-S-protein IgG at both time points ( Figure 5). Curiously, the anti-S1-protein IgG by Euroimmun Quant ELISA consistently gave higher activities above the cut-off than the activity of the anti-Nprotein IgG by Novalisa, both on day 2 or day 3 and on day 14 post onset of COVID-19 symptoms (POCS).
Viruses 2021, 13, x FOR PEER REVIEW 8 of 12 Figure 4. Anti-N-protein antibody levels of SARS-CoV-2 variants. The anti-N-protein IgG reactivity for patients infected with COVID-19 variants described in Figure 2; a = day 2 or day 3 POCS, and b = day 16 or day 17 PCOS. The horizontal dotted line indicates the cut-off for Novalisa positivity at > 10 NTU.
To assess the associations between the levels of anti-N-protein versus anti-S-protein antibodies, regression analyses were made on day 2 or day 3 and on day 16 or day 17 POCS. The serum anti-N-protein IgG was positively correlated with the serum anti-S-protein IgG at both time points ( Figure 5). Curiously, the anti-S1-protein IgG by Euroimmun Quant ELISA consistently gave higher activities above the cut-off than the activity of the anti-N-protein IgG by Novalisa, both on day 2 or day 3 and on day 14 post onset of COVID-19 symptoms (POCS).

Discussion
The extensive genomic surveillance of SARS CoV-2 viral isolates in Bangladesh throughout the pandemic is motivated by the urgency to identify the next strains of SARS CoV-2 carrying S-protein mutations or other genetic changes [25][26][27][28][29][30][31][32][33][34]. In the first and second pandemic waves in Bangladesh, the alpha and beta variants of the B.1.1.7 and B.1.351 lineages were associated with an Ro of approximately 1.2 [27]. In the event that viral transmissibility and COVID-19 severity were to increase and threaten the collapse of the already fragile health care in the country, the government needs to rapidly implement lockdowns and to possibly develop vaccines derived from the novel SARS CoV-2 sequences [28].
The delta variant is estimated to have emerged in May 2021 in Bangladesh [26,33]. Our findings of 75% delta variant among COVID-19 patients testing SARS CoV-2 RT-PCR positive between 26 May 2021 and 6 June 2021 strongly indicate that the delta variant is well-established in Dhaka city at the time of this study and likely to be the predominant variant in Bangladesh. There are currently 1.4 million delta variants deposited in the GSAID database, of which 6818 carry the Q677H amino acid substitution; 291 of the Q677H delta variants are reported from Asia and one entry was from Bangladesh. In this respect, the Q677H delta variant in Dhaka city is a rare mutation. The Q677H is positioned adjacent to the S1/S2 cleavage site. Its effect on cell tropism and transmissibility is predicted by molecular modeling [34,35]. So far, there is no evidence that Q677H is dangerous in terms of conferring increased SARS-CoV-2 viral pathogenicity or COVID-19 disease severity [36,37]. The two delta variants carrying the C23029T single nucleotide substitution is of no relevance for the virus pathology. This synonymous substitution caused irregular melting temperatures of the E484K RT-PCR typing probe but the S-protein sequence is identical to the known delta variant.

Discussion
The extensive genomic surveillance of SARS CoV-2 viral isolates in Bangladesh throughout the pandemic is motivated by the urgency to identify the next strains of SARS CoV-2 carrying S-protein mutations or other genetic changes [25][26][27][28][29][30][31][32][33][34]. In the first and second pandemic waves in Bangladesh, the alpha and beta variants of the B.1.1.7 and B.1.351 lineages were associated with an Ro of approximately 1.2 [27]. In the event that viral transmissibility and COVID-19 severity were to increase and threaten the collapse of the already fragile health care in the country, the government needs to rapidly implement lockdowns and to possibly develop vaccines derived from the novel SARS CoV-2 sequences [28].
The delta variant is estimated to have emerged in May 2021 in Bangladesh [26,33]. Our findings of 75% delta variant among COVID-19 patients testing SARS CoV-2 RT-PCR positive between 26 May 2021 and 6 June 2021 strongly indicate that the delta variant is well-established in Dhaka city at the time of this study and likely to be the predominant variant in Bangladesh. There are currently 1.4 million delta variants deposited in the GSAID database, of which 6818 carry the Q677H amino acid substitution; 291 of the Q677H delta variants are reported from Asia and one entry was from Bangladesh. In this respect, the Q677H delta variant in Dhaka city is a rare mutation. The Q677H is positioned adjacent to the S1/S2 cleavage site. Its effect on cell tropism and transmissibility is predicted by molecular modeling [34,35]. So far, there is no evidence that Q677H is dangerous in terms of conferring increased SARS-CoV-2 viral pathogenicity or COVID-19 disease severity [36,37]. The two delta variants carrying the C23029T single nucleotide substitution is of no relevance for the virus pathology. This synonymous substitution caused irregular melting temperatures of the E484K RT-PCR typing probe but the S-protein sequence is identical to the known delta variant.
The amino acid mutations T95I, Y144del, E484K, D614G, and P681H of the B. The major limitation of our study is the low number of RT-PCR-positive COVID-19 cases investigated during the brief two-week sampling period. The positive anti-S protein IgG and anti-N protein IgG on the day of RT-PCR positive nasopharyngeal swab and on day 16 or day 17 post onset of COVID-19 symptoms in 33 out of 36 COVID-19 patients indicate prior SARS CoV-2 infection. Our finding is supported by reports of high anti-S protein IgG on the day of positive swab among unvaccinated COVID-19 patients in the United Kingdom having confirmed SARS-CoV-2 re-infection [39]. On this basis, we conclude that the patients with the delta variant likely have pre-existing antibodies from either the first Bangladesh pandemic wave in April to June 2020 [29][30][31][32] or the second Bangladesh pandemic wave in March 2021, which were predominantly of the SARS CoV-2 alpha or beta variants [25][26][27][28]40].
The Euroimmun Quant ELISA detects anti-S1 protein IgG that has been calibrated to the antibody neutralizing activity assessed by the suppression of cytopathic effect on Vero cells infected with the German SARS CoV-2 strain01/2020, lineage B.3. [41]. Thus neutralizing antibodies were not directly assessed in our study on unvaccinated Bangladeshi adults with natural SARS CoV-2 infection. Although we assume the neutralizing antibodies are in part represented by the anti-S1 protein IgG binding activity in the Euroimmun ELISA, we cannot distinguish between anti-S-protein IgG directed against the delta variant versus other variants.
Our finding of anti-N-protein IgG positively correlated with anti-S-protein IgG confirms the positive correlation previously reported for unvaccinated natural SARS CoV-2 infection [42] and are consistent with the anti-N-protein IgG levels in Novalisa previously reported among Bangladeshi COVID-19 patients [43]. During the first 21 days POCS, anti-N protein IgG levels are typically higher than the anti-S-protein IgG levels [42]. Anti-S protein IgG reach peak levels at 21 days and remain positive beyond 180 days POCS [39]. This trajectory of antibody response may not necessarily apply for asymptomatic and mild cases of COVID-19 which do not develop nucleocapsid antibodies or for populations having SARS CoV-2 reinfection [39,44]. The S1-protein IgG Euroimmun ELISA reported higher rates of sample positivity compared with the S protein IgG Novalisa or the S-protein IgG automated immunoassays of Roche, Diasorin and Abbot [45]. In this respect, we are cautious about interpreting the S-protein IgG levels and acknowledge the higher sensitivity of Euroimmun ELISA for anti-S-protein IgG detection compared with the ELISA or the automated immunoassays of other manufacturers.

Conclusions
The emergence of SARS CoV-2 variants in unvaccinated populations of low-income countries is a complex process dependent on the coronvavirus-2 molecular epidemiology and specific socioeconomic conditions. In many Bangladeshi households, the extended families of grandparents, parents, and children often live together, which likely raises the risk of viral transmission. Our findings on the Bangladeshi SARS CoV-2 delta variants underscore the potential risk of under-vaccinated and under-monitored populations of low-income countries regarding local public health and the global cooperative response against pandemics.

Institutional Review Board Statement:
The study was carried out with the approval of the local Institutional Review Board of the National Institutes of Laboratory Medicine Referral Centre No. NILMRC/IRB/2020-06 Dhaka, Bangladesh. In addition, our present study was conducted in compliance with the principles of the Declaration of Helsinki, Good Clinical Practice guidelines, and local regulatory requirements.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. All patient information and data on the analyses of blood and swab specimens were kept anonymized.
Data Availability Statement: All of the sequencing data and information for this study are available in the GISAID. The accession numbers are provided.