Detection of SARS-CoV-2 N501Y mutation by RT-PCR to identify the UK and the South African strains in the population of South Indian state of Telangana

ObjectiveTo detect N501Y mutation of the SARS-CoV-2 spike protein by RT-PCR to distinguish (B.1.1.7) UK and (501Y.V2) South African strains from others in the population of Telangana and to determine its clinical implications.MethodsA primer-probe mix that specifically detects the mutated N501Y strain by real time RT-PCR was designed. 93 samples that were reported positive for COVID-19 by our laboratory in the month of February 2021 were tested using our own primer-probe mix for the presence of N501Y by RT-PCR. The results of RT-PCR were validated by Sanger sequencing in representative samples. Sanger sequencing of other defining spike mutations of B.1.1.7 (del 69-70, del 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H) and 501Y.V2 (K417N, E484K, N501Y and D614G) was also investigated.FindingsOut of 93 COVID-19 positive samples, 12 samples are detected positive for N501Y by RT-PCR. Sanger sequencing of these 12 samples further confirmed the presence of N501Y and other mutations that are characteristic of UK strain (B.1.1.7). The South African strain (501Y.V2) is not detected in any of our samples in this study. But, the E484K mutation that is characteristic of 501Y.V2 is detected in one N501Y negative sample.ConclusionStrain-specific RT-PCR for N501Y was developed and validated with Sanger sequencing. Such strategy facilitates quick surveillance for more transmissible and more vaccine resistant strains.

L18F substrain of SARS-CoV-2 VOC-202012/01 is rapidly spreading in England

The Variant of Concern (VOC)-202012/01 (also known as B.1.1.7) is a rapidly growing lineage of SARS-CoV-2. In January 2021, VOC-202012/01 constituted about 80% of SARS-CoV-2 genomes sequenced in England and was present in 27 out of 29 countries that reported at least 50 viral genomes. As this strain will likely spread globally towards fixation, it is important to monitor its molecular evolution. Based on GISAID data we systematically estimated growth rates of mutations acquired by the VOC lineage to find that L18F substitution in viral spike protein has initiated a substrain characterized by replicative advantage of 1.70 [95% CI: 1.56–1.96] in relation to the remaining VOC-202012/01 substrains. The L18F mutation is of significance because when recently analyzed in the context of the South African strain 501Y.V2 it has been found to compromise binding of neutralizing antibodies. We additionally indicate three mutations that were acquired by VOC-202012/01 in the receptor binding motif of spike, specifically E484K, F490S, and S494P, that may also give rise to escape mutants. Such mutants may hinder efficiency of existing vaccines and expand in response to the increasing after-infection or vaccine-induced seroprevalence.

In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2–Spike RBD Interface

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike–ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure–function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.

In silico investigation of the new UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 variants with a focus at the ACE2-Spike RBD interface

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor and several in silico methods to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both UK and South African strains) should be favorable for the interaction with ACE2 while the K417N and E484K substitutions (South African) would seem unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the predicted less favorable (regarding binding) K417N and E484K Spike replacements. Our finding suggests that, if indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions outside the direct Spike-ACE2 interface.HihglightsTransmission of the UK and possibly South African SARS-CoV-2 strains appears substantially increased compared to other variantsThis could be due, in part, to increased affinity between the variant Spike proteins and ACE2We investigated in silico the 3D structure of the Spike-ACE2 complex with a focus on Spike K417N, E484K and N501YThe N501Y substitution is predicted to increase the affinity toward ACE2 (UK strain) with subsequent enhanced transmissibility and possibly pathogenicityAdditional substitutions at positions 417 and 484 (South African strain) may pertub the interaction with ACE2 raising questions about transmissibility and pathogenicity

Measles-based Zika vaccine induces long-term immunity and requires NS1 antibodies to protect the female reproductive tract.

Zika virus (ZIKV) can cause devastating effects in the unborn fetus of pregnant women. To develop a candidate vaccine that can protect human fetuses, we generated a panel of live measles vaccine (MV) vectors expressing ZIKV-E and -NS1. Our MV-based ZIKV-E vaccine, MV-E2, protected mice from the non-lethal Zika Asian strain (PRVABC59) and the lethal African strain (MR766) challenge. Despite 100% survival of the MV-E2 mice, however, complete viral clearance was not achieved in the brain and reproductive tract of the lethally challenged mice. We then tested a combination of two MV-based vaccines, the MV-E2 and a vaccine expressing NS1 (MV-NS1[2]), and we observed durable plasma cell responses, complete clearance of ZIKV from the female reproductive tract, and complete fetal protection in the lethal African challenge model. Our findings suggest that NS1 antibodies are required to enhance the protection achieved by ZIKV-E antibodies in the female reproductive tract.

Measles-based Zika vaccine induces long-term immunity and requires NS1 antibodies to protect the female reproductive tract in the hCD46 IFNα/β receptor knockout mice

Zika virus (ZIKV) can cause devastating effects in the unborn fetus of pregnant women. To develop a candidate vaccine that can protect human fetuses, we generated a panel of live measles vaccine (MV) vectors expressing ZIKV-E and -NS1. Our MV-based ZIKV-E vaccine, MV-E2, protected mice from the non-lethal Zika Asian strain (PRVABC59) and the lethal African strain (MR766) challenge. Despite 100% survival of the MV-E2 mice, however, complete viral clearance was not achieved in the brain and reproductive tract of the lethally challenged mice. We then tested a combination of two MV-based vaccines, the MV-E2 and a vaccine expressing NS1 (MV-NS1[2]), and we observed durable plasma cell responses, complete clearance of ZIKV from the female reproductive tract, and complete fetal protection in the lethal African challenge model. Our findings suggest that NS1 antibodies are required to enhance the protection achieved by ZIKV-E antibodies in the female reproductive tract.

Naturally acquired immunity among Kenyan adults suppresses the West African P. falciparum NF54 strain in controlled human malaria infection (CHMI)

We used controlled human malaria infection (CHMI) to study naturally acquired immunity of Kenyan adults. We administered 3.2x103 cryopreserved Plasmodium falciparum sporozoites (SPZ, NF54 West African strain) and undertook clinical monitoring and serial quantitative PCR (qPCR). Of the 142 volunteers who were eligible for analysis: 26 (18.3%) had febrile symptoms and were treated; 30 (21.1%) reached greater than or equal to 500 parasites/μl and were treated; 53 (37.3%) had parasitaemia without meeting thresholds for treatment and; 33 (23.2%) remained qPCR negative. We find that the immunity acquired by some Kenyan adults can completely suppress in vivo growth of a parasite strain originating from outside Kenya.

Uncovering the First Atypical DS-1-like G1P[8] Rotavirus Strains That Circulated during Pre-Rotavirus Vaccine Introduction Era in South Africa

Emergence of DS-1-like G1P[8] group A rotavirus (RVA) strains during post-rotavirus vaccination period has recently been reported in several countries. This study demonstrates, for the first time, rare atypical DS-1-like G1P[8] RVA strains that circulated in 2008 during pre-vaccine era in South Africa. Rotavirus positive samples were subjected to whole-genome sequencing. Two G1P[8] strains (RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1971/2008/G1P[8] and RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1973/2008/G1P[8]) possessed a DS-1-like genome constellation background (I2-R2-C2-M2-A2-N2-T2-E2-H2). The outer VP4 and VP7 capsid genes of the two South African G1P[8] strains had the highest nucleotide (amino acid) nt (aa) identities of 99.6–99.9% (99.1–100%) with the VP4 and the VP7 genes of a locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU1039/2008/G1P[8]. All the internal backbone genes (VP1–VP3, VP6, and NSP1-NSP5) had the highest nt (aa) identities with cognate internal genes of another locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU2344/2008/G2P[6]. The two study strains emerged through reassortment mechanism involving locally circulating South African strains, as they were distinctly unrelated to other reported atypical G1P[8] strains. The identification of these G1P[8] double-gene reassortants during the pre-vaccination period strongly supports natural RVA evolutionary mechanisms of the RVA genome. There is a need to maintain long-term whole-genome surveillance to monitor such atypical strains.

A Chimeric Zika Virus between Viral Strains MR766 and BeH819015 Highlights a Role for E-glycan Loop in Antibody-mediated Virus Neutralization

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which is of major public health concern. ZIKV infection is recognized as the cause of congenital Zika disease and other neurological defects, with no specific prophylactic or therapeutic treatments. As the humoral immune response is an essential component of protective immunity, there is an urgent need for effective vaccines that confer protection against ZIKV infection. In the present study, we evaluate the immunogenicity of chimeric viral clone ZIKBeHMR-2, in which the region encoding the structural proteins of the African strain MR766 backbone was replaced with its counterpart from the epidemic strain BeH819015. Three amino-acid substitutions I152T, T156I, and H158Y were introduced in the glycan loop of the E protein (E-GL) making ZIKBeHMR-2 a non-glycosylated virus. Adult BALB/c mice inoculated intraperitoneally with ZIKBeHMR-2 developed anti-ZIKV antibodies directed against viral proteins E and NS1 and a booster dose increased antibody titers. Immunization with ZIKBeHMR-2 resulted in a rapid production of neutralizing anti-ZIKV antibodies. Antibody-mediated ZIKV neutralization was effective against viral strain MR766, whereas epidemic ZIKV strains were poorly sensitive to neutralization by anti-ZIKBeHMR-2 immune sera. From our data, we propose that the three E-GL residues at positions E-152, E-156, and E-158 greatly influence the accessibility of neutralizing antibody epitopes on ZIKV.