Respiratory Viruses
SENTINELLE Newsletter
ReViLux Bulletin hebdomadaire
12 February 2021
Semaine: 5
Résultats cliniques de la sentinelle
La surveillance SENTINELLE des infections respiratoires aiguës est une collaboration entre le Laboratoire national de santé (LNS), la Direction de la Santé et un réseau de médecins-généralistes et de pédiatres répartis à travers tout le Luxembourg. Ce rapport hebdomadaire est publié par le départment de microbiologie du LNS dans le cadre de ses missions de surveillance, de vigilance et d’alerte dans les domaines de la santé publique, l'analyse des données rapportant l’activité clinique dans la Sentinelle et des données virologiques à l’appui.
Au cours de la semaine 5 (01/02/2021 - 07/02/2021), l'activité grippale est nulle.
Le taux de consultations avec ILI1 est de 0% (seuil épidémiologique à 3%).
Le taux de consultations avec ARI2 est de 20%.
- ILI: - Influenza-Like Illness: Signes respiratoires aigus <10 jours, Fièvre 38∘C, Signes systémiques (myalgie, malaise, maux de tête, …)
- ARI: - Infection Respiratoire Aiguë: Signes respiratoires aigus (bronchite, pharyngite, rhinite, pneumonie…) avec ou sans fièvre
| Number of Patients | Percentage | |
|---|---|---|
| ARI (acute respiratory infections) | 51 | 20% |
| ILI (influenza-like illness) | 0 | 0% |
| Consultations | 257 |
Respiratory viruses circulating in Luxembourg
| results | |
|---|---|
| Cumulative number of tests performed | 160 |
| Cumulative number of positive samples | 82 |
| Positivity rate | 51% |
Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2). We noticed a high positivity rate of HRV with 47 positive cases in 160 tests (29%). Moreover, there is a wave of Human Rhinovirus (HRV) circulating currently in Luxembourg.
SARS-CoV-2 Sequencing
Sequenced Samples
The national genomic surveillance program had maintained its capacity of 384 samples/week and reached since March a total of 4547 SARS-CoV-2 consensus sequences from clinical samples as of week 4/2021 (25/01/2021 - 31/01/2021). While the respiratory virus surveillance can address real-time data, the sequencing data have a delay of one week. This delay in reporting is represented by the technical process for sequencing samples upon reception to our premises which ranges 5-7 days. For data presentation purposes we use a window of 5 months so the plotted data are starting on 01/09/2020.
The frequency of variants reported in ReViLux is indicative of samples referred to LNS for sequencing with potential selection bias (e.g. requests by Inspection Sanitaire, positive samples form airport testing and cluster samples), with only low numbers of samples with full metadata. Therefore, the frequency of variants should not be extrapolated to prevalence of these variants in the community.
Referring to week 4/2021 the sequencing platform generated 159 genomes allowing lineage call, which represents 15.9% of new infections reported for this week. Among the 159 samples, 27% represent targeted sequencing on request by Inspection Sanitaire (e.g. contact tracing). These samples are therefore not representative for the general population.
In collaboration with Inspection Sanitaire, we are developing population surveillance to sequence 300 SARS-CoV-2 samples from different regions and age groups to offer a clear reflection of the circulation of different variants in Luxembourg on the basis of a data collection framework from partner laboratories. This will allow us to reach real-time epidemiology with accurate estimates of the frequency of circulating variants by end of February.
Circulating Lineages
Lineages have been assigned based on Rambaut et al by means of pangolin (v2.1.10, pangoLEARN version 2021-01-30), allowing for a lineage (=variant) assignment of viral genomes.
For the sampling time frame week 4/2021 18 circulating SARS-CoV-2 variants have been detected within our sequencing pool. The most prevalent lineage is B.1.1.7 (35.2%), followed by B.1.160 (18.9%) and B.1.177 (15.7%). The remaining variants, not considering variants of concern, mainly represent singular cases, besides B.1.221 (9.4%) and B.1.1.119 (3.8%, Northern Ireland lineage). B.1.160, B.1.177 and B.1.221 represent big and widely spread European Cluster.
Variants of Concern (B.1.1.7, B.1.351, P.1)
Referring to samples taken within week 4/2021, 56 cases of B.1.1.7 have been detected, representing 35.2% of strains in the sequencing pool (in comparison - week 3/2021 17.6%; including additional samples from retrospective catch-up sequencing). The total case count is set to 114 by week 4/2021 (additional cases have been identified retrospectively). The earliest sampling date remains 19/12/2020 and the latest is 31/01/2021.
For the sampling time frame of week 4/2021, 8 cases of the South African variant B.1.351 have been detected, representing 5% of strains in the sequencing pool (in comparison - week 3/2021 3.2%; including additional samples from retrospective catch-up sequencing). The total case count is set to 14 by week 4/2021. The earliest sampling date remains 11/01/2021 and the latest is 30/01/2021.
By retrospective sequencing one case of the Brazilian variant P.2 has been detected, with a sampling date of 08/01/2021. Lineage P.2 and P.1 have the same ancestor, namely B.1.1.28. It as well carries the E484k mutation, but lacking N501Y and K417N. By now P.2 is not defined officially as variant of concern, but being under close observation.
No cases have been detected for the Brazilian variant P.1 in Luxembourg by week 4/2021.
Lineage B.1.1.7 is characterized by several spike protein mutations including S:N501Y, deletion S:H69/V70 and S:P861H. The variant seems to have a considerable epidemiological impact, as it has a higher transmissibility (more efficient and rapid).
Lineage B.1.351 holds numerous spike protein mutations, wherein three are located in the receptor binding domain (K417N, E484K and N501Y), which is relevant for antibody binding. As for B.1.1.7, a higher transmissibility and viral load is in discussion. Due to K417N and E484K an impact on vaccination and possible reinfection is subject of scientific investigation.
Lineage P.1 (descendent of B.1.1.28) initially been found in the Amazon region has a similar mutation profile as the South African variant, including E484K and N501Y. Concerns are, as for the South African variant, higher transmissibility and a decreased protection by neutralizing antibodies.
It is to note, that additional samples are in preparation for sequencing, so that numbers might change with increasing representativeness of circulating strains and proportions.
Phylogenetic Analysis
Phylogenetic analysis has been done by means of nextstrain4 and is based on genetic distance including a timeline on the x-axis. The tree is rooted versus the first sequence obtained in Luxembourg (29/02/2020). Sequences obtained before 01/11/2020 are in grey and those obtained within the last 2 weeks from most recent sampling date referring to week 4/2021 (31st January) are represented in colour and by greater diameter. Due to the increasing number of sequences represented, we performed a random subsampling of 300 sequences over the complete sequencing time frame since 01/09/2020, adding on top the sequences representing B.1.1.7 and B.1.351 referring to week 4/2021.
The tree shows four visually well separated clusters. These clusters are represented by the lineages B.1.160, B.1.177, B.1.1.7 and B.1.221. Due to random selection of sequences and the explicit insertion of variants of concern, the size of clusters might be misleading and does not necessarily correlate with the prevalence of the respective variant.
Lineage B.1.1.7 (represented by light brownish dots) forms a very well distinguished cluster, due to its high amount of accumulated mutations. B.1.1.7 subtrees might direct to different subgroups, possibly related to different sources of infection.
The cases for B.1.351 (light orange dots, close to the bottom of the tree) are separating well from common circulating strains. Subtrees might direct to different subgroups, possibly related to different sources of infection.
- Nextstrain applied an additional filter on genome length > 27000, 353 sequences shown
Clinically relevant mutations
Currently genomic surveillance is - independently from lineage call - observing the occurrence of 13 different known SARS-Cov-2 mutations, being assumed to have a clinical or epidemiological relevance. The pool of observed mutations is updated continuously, depending on the development of SARS-CoV-2 variants and their prevalence.
Due to the increasing incidence of the variant B.1.1.7, the characterizing mutations N501Y, H69/V70del, Y144del and P681H are integrated in weekly reporting. Additionally, the mutation E484K is under observation, which is in combination with N501Y and K417N characteristic for the South African and Japanese/Brazilian variant.
The following table provides the overall frequency of these mutations in lineage assignable genomes between 01/09/2020 and –31/01/2021 January 2021 (N=3506), as well as a close-up of the last 14 days.
| Mutation | Gene | Genomic position in reference | Frequency overall | Frequency recent 14days | Characteristics | Reference |
|---|---|---|---|---|---|---|
| D614G | S gene | 23402 | 100.00% | 97.0% | Higher infectivity, higher case fatality rate, higher transmission; replaced original Wuhan strain, became globally dominant form of the virus | Eaaswarkhanth, al Madhoun, and Al-Mulla 2020, Becerra-Flores and Cardozo 2020, Hu et al. 2020, Plante et al. 2020 |
| E484K | S gene | 23012 | 1.00% | 6.0% | 501Y.V2 / possible impact on antibody neutralization activity (escape mutation), improved ACE2 binding affinity | Greaney et al, 2020 |
| G204R | N gene | 28883 | 11.00% | 39.0% | Fitness advantage for the virus | Leary et al. 2020 |
| H69/V70del | S gene | 21765-21770 | 5.00% | 29.0% | possible impact on antibody neutralization activity and reinfection; included in "mink" mutation | Kemp et al 2020, Kemp et al 2020 |
| K417N | S gene | 22813 | 0.00% | 4.0% | 501Y.V2 / possible impact on antibody binding affinity (escape mutation) | Kemp et al 2020 |
| L37F | Nsp6 | 11081 | 8.00% | 6.0% | Favored viral infection, higher severity | Aiewsakun et al. 2020 |
| N439K | S gene | 26143 | 3.00% | 1.0% | Improved ACE2 binding affinity | Zhou et al, 2020 |
| N501Y | S gene | 23063 | 4.00% | 31.0% | 501Y.V1/V2; Improved ACE2 binding affinity/higher transmissibility | Filip Fratev 2020 COVID-19 Genomics Consortium UK, 2020 |
| P323L | ORF1ab (RdRP gene, nsp12) | 14407 | 100.00% | 91.0% | Higher severity | Biswas and Mudi 2020 |
| P681H | S gene | 23604 | 7.00% | 31.0% | immediately adjacent to the furin cleavage site, a known location of biological significance | COVID-19 Genomics Consortium UK, 2020 |
| Q57H | ORF3a | 25561 | 46.00% | 38.0% | Higher severity | Biswas and Mudi 2020 |
| R203K | N gene | 28880 | 11.00% | 39.0% | Fitness advantage for the virus | Leary et al. 2020 |
| Y144del | S gene | 21991-21993 | 4.00% | 29.0% | possible impact on antibody binding affinity | Dawood et al 2020 |
Clinically relevant mutations frequencies over time
The D614G and P323L mutations have been and are continuously present in a vast majority of sequences. They mainly occur together.
In comparison to the previous week the frequency within the last 14 days increases for S:N501Y from 19% to 31%, for S:H69/V70 deletion from 16% to 29%, for S:P681H from 20% to 31%, for S:Y144del from 18% to 29%, for K417N from 3% to 4% and E484K remains on 6%.
Additional Information
For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html
References
Rambaut, A., Holmes, E.C., O’Toole, Á. et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol 5, 1403–1407 (2020). https://doi.org/10.1038/s41564-020-0770-5
Pangolin (pangolin assigns lineages to query sequences as described in Rambaut et al 2020); Áine O’Toole, JT McCrone, Emily Scher; github.com/cov-lineages/pangolin
Hadfield et al., Nextstrain: real-time tracking of pathogen evolution, Bioinformatics (2018)
Guangchuang Yu, David Smith, Huachen Zhu, Yi Guan, Tommy Tsan-Yuk Lam. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 8(1) 28-36 (2017). https://doi.org/10.1111/2041-210X.12628