key: cord-0752793-gk4myx2o
authors: Lobiuc, A.; Dimian, M.; Sturdza, O.; Filip, R.; Covasa, M.
title: Emergence of first strains of Sars-CoV-2 lineage B.1.1.7 in Romania
date: 2021-02-01
journal: nan
DOI: 10.1101/2021.01.29.21250643
sha: 4e835ee8e932fc5c44de2909dd631338f09a7bb8
doc_id: 752793
cord_uid: gk4myx2o

United Kingdom reported the emergence of a new and highly transmissible SARS-CoV-2 variant B.1.1.7. that rapidly spread to other contries. The impact of this new mutation that occurs in the S protein, on infectivity, virulence and current vaccine effectiveness is still under evaluation. We have identified the first cases of the B.1.1.7 variant in samples collected from Romanian patients, of which one was traced to the UK region where the new variant was originally sequenced. Mutations in the Nsp3 protein, N844S and D455N and L15F in Orf3a were also detected, indicating common ancestry with UK strains as well as remote connections with strains from Nagasaki, Japan. These results indicate, for the first time, the presence and characteristics of the new variant B.1.1.7 in Romania and underscore the need for increased genomic sequencing in confirmed COVID-19 patients.

A new SARS-CoV-2 variant, with a N-Y substituion in the 501 position of the S (spike) protein, was detected in the UK in the fall of 2020. An initial version of the virus, termed 501 N, with fewer mutations, occurred in late September in Wales, followed by the current version (VUI-202012/01), giving rise to lineage B.1.1.7, which began to spread rapidly in the UK and then globally [1] . The great concern is the increased transmisibility and disease severity than older variants, raising questions on its potential avoidance of succesful nucleic acid amplification diagnostic or even vaccine effectiveness [2] . On January 8, 2021 Romania confirmed the first case of COVID 19 infection with the new strain, in a patient from Giurgiu, without a history of travel in the UK or contact with individuals from UK. On January 22, 2021, two additional individuals from Bucharest were identified with the new strain. They reported no travel history, good clinical condition, and were isolated at home under the supervision of family physician. A fourth case was reported in Suceava county, N-E of Romania, on January 25, 2021 in an indvidual who arrived from UK. A fifth reported case, at the time of writing this article, was confirmed on January 26, 2021 in a patient from Constanta, S-E of Romania, with no travel history or contact with individuals infected with the new strain. Considering the time of B.1.1.7 appearance in Europe, its fast spreading compared to earlier strains and the lack of genomic sequencing in Romania, there exists the possibility that the new variant is far more widespread in Romania. In this paper, we report the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 1, 2021. 

Twenty samples, collected from patients in the cities of Cluj, Craiova and Suceava counties from Romania were selected for analysis, including patients with possible contacts with UK infected individuals. Samples were quantified for viral titers and RNA amounts, through qPCR and Qubit methods, respectively. RNA extracts were reverse transcribed and libraries were prepared using Ampliseq SARS-CoV-2 primer panels and workflow. Automatic library templating was performed using Ion Chef equipment and sequencing was carried out on Ion GeneStudio S5 with Ion 540 chips. Sequencing reads and assemblies were checked for quality using Ion Torrent Suite software plugins. Aminoacid substitution analysis was performed using CoV-Glue platform . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250643 doi: medRxiv preprint Considering all Romanian GISAID accessions belonging to this clade, a synopsis of all mutations was constructed ( One such mutation is present only in the sample originating from Suceava (SV), affecting ORF8 protein, where a stop codon is gained, by changing C to T nucleotides in position 27945 in the genome. This mutation has been already encountered, according to GISAID, in over 500 samples from April to September 2020. Of these, 73% belong to UK collected specimens respectively, [5] , the hallmark N501Y mutation first appeared in Italy, in August 2020 [6] .

However, at this point, the Romanian strains bearing the particular ORF8 mutations described above clearly originated in the UK, which is also supported by the fact that the patient from Suceava county arrived in Romania from the UK.

Strains without a functional ORF8 protein are considered to present epitope loss, which may decrease the accuracy of serological testing, whereas ORF8 antibodies could offer information on both acute and convalescent antibody response. Furthermore, ORF8 truncated proteins decrease disease severity and asymptomatic or mildly diseased carriers might not be detected [7] . As such, the significance of ORF8 truncations in the context of B.1.1.7 strains should . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250643 doi: medRxiv preprint be promptly investigated, considering that mutations in S gene characteristic to this lineage, particularly the deletion at positions 69-70, may elude PCR diagnostic with certain kits, which have been used in the UK for a while [8] . This type of behaviour could be indirectly, but significantly, linked to increased transmissibility of the virus, as potentially infected population carrying this strain might have not been accurately identified as such.

Another noteworthy mutation is N844S within Nsp3 protein present in SV sample, which is recorded only in other 8 samples sequenced so far, most of them also from England Namely, the N50Y mutation of S gene significantly increases the force and number of interactions . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250643 doi: medRxiv preprint with the human receptor ACE2 [14, 15] . The two aminoacid deletion at positions 69-70 in the same S gene leads to systematically biased diagnostic tests and doubles the reproductive advantage and numbers of the virus [16] . Furthermore, the P681H mutation of S protein might influence the cleavage of S protein through effest on S1/S2 subunits furin cleavage site [17] . Identification of new mutations is crucial in designing correct diagnostic reagents [18] , slowing transmission and vaccine reconfiguration against new variants. Also, particular mutations, besides those specific to B.1.1.7, may, in the future, aid in tracing virus movements across Romania and worldwide. Many European countries, including Romania, lag in genomic sequencing and EU recommends increased focused sequencing based on epidemiological data, transmission rates, infectivity, treatment failure and S-gene "drop-out" in PCR testing. Therefore, thorough characterisation of strains circulating in Romania also contributes to developing useable diagnostic tests and vaccines, especially in the light of notable differences between strains belonging to the same clade and the evolutionary capacity of SARS-CoV-2.

This work was supported by a grant from Romanian Ministry of Education and Research, UEFISCDI, project number PN-III-P2-2.1-SOL-2020-0142.

AL, MD and MC contributed to the conception and design of the study. AL, OS, RF contributed to acquisition of data. AL and MC drafted the article. All authors revised the article for important intelectual content and aproved the final version of the manuscript.

The authors declare no competing interests . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250643 doi: medRxiv preprint

The study was aproved by the ethics committee of University Stefan cel Mare of Suceava, Romania (no. 11733/14.07.2020) and of Suceava County Emergency Hospital (no. 17669/13.07.2020). All participants signed individual informed consent.

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