key: cord-1036675-lr3qkxno
authors: Jelley, L.; Douglas, J.; Ren, X.; Winter, D.; McNeill, A.; Huang, S.; French, N.; Welch, D.; Hadfield, J.; de Ligt, J.; Geoghegan, J. L.
title: From elimination to suppression: genomic epidemiology of a large Delta SARS-CoV-2 outbreak in Aotearoa New Zealand
date: 2022-04-12
journal: nan
DOI: 10.1101/2022.04.04.22273376
sha: fce2dd762f4a46eca1260c76fae2b1c925bb2e9b
doc_id: 1036675
cord_uid: lr3qkxno

New Zealand's COVID-19 elimination strategy heavily relied on the use of genomics to inform contact tracing, linking cases to the border and to clusters during community outbreaks. In August 2021, New Zealand entered its second nationwide lockdown after the detection of a single community case with no immediately apparent epidemiological link to the border. This incursion resulted in the largest outbreak seen in New Zealand caused by the Delta Variant of Concern. Here we generated 3806 high quality SARS-CoV-2 genomes from cases reported in New Zealand between 17 August and 1 December 2021, representing 43% of reported cases. We detected wide geographical spread coupled with undetected community transmission, characterised by the apparent extinction and reappearance of genomically linked clusters. We also identified the emergence, and near replacement, of genomes possessing a 10-nucleotide frameshift deletion that caused the likely truncation of accessory protein ORF7a. By early October, New Zealand moved from elimination to suppression and the role of genomics changed markedly from being used to track and trace, towards population-level surveillance.

New Zealand's COVID-19 elimination strategy heavily relied on the use of genomics to inform contact tracing, linking cases to the border and to clusters during community outbreaks. In August 2021, New Zealand entered its second nationwide lockdown after the detection of a single community case with no immediately apparent epidemiological link to the border. This incursion resulted in the largest outbreak seen in New Zealand caused by the Delta Variant of Concern.

Here we generated 3806 high quality SARS-CoV-2 genomes from cases reported in New Zealand between 17 August and 1 December 2021, representing 43% of reported cases. We detected wide geographical spread coupled with undetected community transmission, characterised by the apparent extinction and reappearance of genomically linked clusters. We also identified the emergence, and near replacement, of genomes possessing a 10-nucleotide frameshift deletion that caused the likely truncation of accessory protein ORF7a. By early October, New Zealand moved from elimination to suppression and the role of genomics changed markedly from being used to track and trace, towards population-level surveillance.

Since early 2020, the World Health Organisation has monitored the global evolution of Severe acute respiratory coronavirus 2 (SARS-CoV-2). As new genomic variants emerged that posed an and other variants.

The Delta variant possesses at least 13 non-synonymous mutations compared to ancestral variants and its growth advantage can be explained primarily due to both immune evasion and a 40-60% increase in transmissibility compared to Alpha 3,4 . The astonishing speed at which Delta spread led to outbreaks in many countries that had previously achieved elimination. For example, Delta outbreaks in Australia, Fiji, Vietnam, Singapore and Taiwan demonstrated how previous public health measures used to control COVID-19 were less effective against this highly transmissible variant [5] [6] [7] [8] [9] .

New Zealand eliminated community transmission of SARS-CoV-2 by mid 2020 and continued to pursue a zero-COVID policy up until late in 2021 10 . Several small but quickly controlled SARS-CoV-2 outbreaks were detected in the community since the virus was first eliminated 11 . But for the most part, cases of COVID-19 were largely restricted to managed isolation and quarantine facilities at the border, where returning New Zealanders were required to undergo at least 14 days of quarantine. These border control measures averted numerous virus incursions and, as a result, New Zealand saw an increase in life expectancy over the first two years of the pandemic 12,13 .

In August 2021, following a brief period of reciprocal quarantine-free travel with Australia, New Zealand entered its second nationwide lockdown after the detection of a single COVID-19 community case in Auckland with no immediately apparent epidemiological link to the border.

Between 17 August and 1 December 2021 there were 8974 cases of COVID-19 reported in the community. In early October, due to the inability to eliminate Delta even under the most stringent lockdown measures, and with the aid of high vaccination rates, New Zealand moved from elimination to suppression, aimed at minimisation and protection 14, 15 . Stay-at-home orders persisted in Auckland and sporadically across various regions of the country until early December.

By the end of 2021 this Delta outbreak was the largest single-origin outbreak in New Zealand -the second largest occurred in August 2020 with fewer than 200 reported community cases 16 .

Here we describe a large-scale outbreak of SARS-CoV-2 in New Zealand and show how genomics was used in real-time to first help track and trace cases of COVID-19 in the community and, later, with the change to suppression, to monitor its spread and evolution. This genomic surveillance

identified numerous examples of cryptic virus transmission by the apparent extinction and reappearance of genomically linked clusters. Additionally, we identified the emergence and dominance of a lineage possessing a 10-nucleotide frameshift deletion likely rendering an accessory protein, encoded by the ORF7a gene, functionally impaired, within this outbreak.

Nasopharyngeal samples that had positive results for SARS-CoV-2 by real-time reverse transcription PCR were obtained from medical diagnostic laboratories located throughout New

Zealand. Under contract for the New Zealand Ministry of Health, the Institute of Environmental . CC-BY-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 April 12, 2022. ; https://doi.org/10.1101/2022.04.04.22273376 doi: medRxiv preprint Science and Research has approval to conduct genomic sequencing and phylogenetic analysis for surveillance of notifiable diseases.

For cases reported between 17 August and 1 December 2021 a random proportion of COVID-19 community cases were referred to the Institute of Environmental Science and Research, New Zealand for genome sequencing. In brief, viral extracts were prepared from respiratory tract samples in which SARS-CoV-2 was detected by rRT-PCR. Extracted RNA was subjected to whole-genome sequencing using the Oxford Nanopore Technologies R9.4 chemistry by following the Midnight protocol v6, designed by Freed et al. 17 , which contains a 1200-bp primer set tiling the SARS-CoV-2 genome. Consensus genomes were generated through a standardised pipeline Genomes were aligned using Nextalign 19 with Wuhan-Hu-1 (NC_045512.2) used as a reference. A maximum likelihood phylogenetic tree was estimated using IQ-TREE (v 1.6.8) 20 using the Hasegawa-Kishino-Yano (HKY+Γ) 21 nucleotide substitution model with a gamma distributed rate variation among sites (the best fit model was determined by ModelFinder 22 ), and branch support was assessed using the ultrafast bootstrap method 23 . The resulting phylogenetic tree was visualised and displayed using GrapeTree 24 and annotated by the location (i.e. District Health Board) from where genomes were sampled.

Genomes sampled between 17 August and 1 December 2021 were annotated based on whether they possessed a 10-nucleotide deletion within the Open Reading Frame (ORF) 7a between . CC-BY-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. 

We generated 3806 high quality genomes, designated as Delta VOC (B. New Zealand's 2021 Delta outbreak was genomically linked to a single introduction into the community (Figure 1 ). While no definitive epidemiological link to the border could be established, genome sequencing revealed four genomes sampled from a managed isolation and quarantine facility in Auckland that were genetically indistinguishable to the consensus genome of the first reported case in the community. These cases originated from two separate travel groups who had recently returned from New South Wales, Australia in the week prior to the community outbreak. In . CC-BY-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 April 12, 2022. ; https://doi.org/10.1101/2022.04.04.22273376 doi: medRxiv preprint addition, several genetically indistinguishable genomes were sampled from the New South Wales community during this time. Zealand. This was hampered by known genomic clades going unsampled before reappearing weeks later, suggesting substantial undetected community transmission. As case numbers rose and New Zealand moved to a suppression strategy the focus moved to monitoring active genomic . CC-BY-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 April 12, 2022. ; https://doi.org/10.1101/2022.04.04.22273376 doi: medRxiv preprint clades ( Figure 2 ). Cases reported from Auckland's metropolitan areas were evident in nearly all clades whereas cases reported from more regional localities showed much less genomic variation.

There was strong evidence of transmission 'seeding' from Auckland's metropolitan areas into other regions, resulting in the emergence of new, dominant clades that amplified, diverged and ultimately spread into further locations. An example is the Waikato region (purple in Figure 2 October genomes with this deletion accounted for over 90% of sequenced cases (Figure 3 ). There was little difference in the age distribution in cases with the deletion (mean age 31) and those without (mean age 27) suggesting viruses with the deletion was not more prevalent in any one particular age group (Figure 3) .

. CC-BY-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 April 12, 2022. ; We estimated the Reff of both lineages through time (Figure 4) . The wildtype ORF7a lineage peaked shortly after the first cases were discovered, with an Reff of 6.8 (and a 95% credible interval (CI) of 2.1-15). Following this, Reff declined rapidly to around 1 for the remainder of the outbreak, likely as a result of a stringent public health response and high vaccination rates (86.4% of the eligible (>12 years of age) population by 1 December). Similarly, the truncated ORF7a lineage peaked shortly after it was first detected, with a mean Reff of 2.1 (95% CI: 0.1-4.5). Although the truncated ORF7a lineage saw some initial success, its Reff gradually declined to around 1. There is no evidence that the wildtype nor truncated ORF7a lineages have any significant differences in transmissibility. vaccine. The nationwide change from elimination to suppression strategy is indicated, as are regional changes between mitigation settings in Auckland (the epicentre of the outbreak), with 'Alert level 4' being the most stringent 10 .

. CC-BY-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 April 12, 2022 

New Zealand's elimination strategy for controlling SARS-CoV-2 has been hailed a success, and the nation saw low levels of mortality during the first two years of the pandemic 12, 13 . Genomics Genomic surveillance showed that the New Zealand Delta outbreak was characterised by the emergence and subsequent increase in frequency of genomes containing a 10-nucleotide frameshift deletion in ORF7a. The polymorphic nature of this deletion provided greater resolution to the epidemiological picture, enabling cases to be linked to transmission chains and therefore aiding contact tracing efforts during the elimination period among otherwise relatively clonal genomes. The ORF7a gene itself encodes accessory protein 7a, which has strong sequence similarity with its ortholog within the SARS-CoV-1 genome 34 . Accessory protein 7a is a transmembrane protein with a seven-stranded β-sandwich fold 35 . Although considered nonessential, it is involved in a range of functions including antagonisation of the host interferon type 1 response 36 , binding to CD14+ monocytes in human peripheral blood 37 , involvement in protein trafficking 38 and regulation of the ORF7b peptide 39 , and is known to induce apoptosis when overexpressed 40, 41 .

Although highly variable in position and length, frameshift deletions that induce truncation of accessory protein 7a are not unique. There are numerous independent occurrences of such a . CC-BY-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 April 12, 2022. ; https://doi.org/10.1101/2022.04.04.22273376 doi: medRxiv preprint mutation across both A and B lineages of SARS-CoV-2, including among VOCs [42] [43] [44] [45] [46] [47] [48] [49] . Global genomic surveillance of SARS-CoV-2 has shown that mutations frequently occur at the downstream regions of ORF7a with such changes often leading to premature stop codons resulting in protein products with reduced functional activity. Such mutations in ORF7a appear to be associated with various viral mechanisms, including reduced viral suppression of the immune response 50 , an increase in viral progeny 46 , and a seemingly rapid increase in viral spread 44 .

Indeed, such "dispensable" accessory proteins are known to gain and lose functional activities quite rapidly in evolutionary time, across a wide range of viral taxonomies 51-53 -perhaps as a means to better explore their fitness landscapes and co-evolve with their host. This certainly appears to be the case for accessory protein 7a, which has repeatedly seen sweeping structural mutations across a range of geographical regions, while still maintaining a high level of fitness.

The truncated ORF7a lineage saw some initial success in New Zealand and quickly became the dominant variant in the community. Nevertheless, this may have simply been a founder effect that coincided with a relaxation of public health restrictions. Overall, there are too many confounding factors and an insignificant difference in reproduction number to determine whether the truncated variant had any enhanced transmissibility over the wildtype.

Overall, genomics has played a key role in New Zealand's science-led response to the SARS-CoV-2 pandemic, identifying links between cases in community outbreaks to help stamp out local transmission 54 . The Delta outbreak not only changed New Zealand's strategy for controlling SARS-CoV-2, but it also changed the role of genomics. Much like other parts of the world, genomic surveillance of SARS-CoV-2 should continue to inform public health responses in New Zealand, but at a different granularity.

. CC-BY-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 April 12, 2022. ; https://doi.org/10.1101/2022.04.04.22273376 doi: medRxiv preprint

A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology

Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi

Effectiveness of Covid-19 vaccines against the B.1.617.2 (Delta) variant

Transmission and containment of the SARS-CoV-2 Delta variant of concern in Guangzhou, China: A population-based study

Australia's struggle with the delta variant

COVID-19 in Fiji

First nosocomial cluster of COVID-19 due to the Delta variant in a major acute care hospital in Singapore: investigations and outbreak response

An observational study of breakthrough SARS-CoV-2 Delta variant infections among vaccinated healthcare workers in Vietnam

Rapid control of a SARS-CoV-2 B.1.617.2 (Delta) variant COVID-19 community outbreak: the successful experience in Pingtung County of Taiwan

COVID-19 in New Zealand and the impact of the national response: a descriptive epidemiological study

Real-time genomics for tracking Severe acute respiratory syndrome coronavirus 2 border Incursions after virus elimination

Reduced mortality in New Zealand during the COVID-19 pandemic

Mortality declines in Aotearoa NZ during the first two years of the Covid-19 pandemic

The next phase in Aotearoa New Zealand's COVID-19 response: a tight suppression strategy may be the best option

The end of the elimination strategy: decisive factors towards sustainable management of COVID-19 in New Zealand

Use of genomics to track coronavirus disease outbreaks

Rapid and inexpensive wholegenome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

Data, disease and diplomacy: GISAID's innovative contribution to global health

Nextclade: clade assignment, mutation calling and quality control for viral genomes

IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies

Dating of the human-ape splitting by a molecular clock of mitochondrial DNA

ModelFinder: fast model selection for accurate phylogenetic estimates

Improving the ultrafast bootstrap approximation

GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens

Protter: interactive protein feature visualization and integration with experimental proteomic data

Birth-death skyline plot reveals temporal changes of epidemic spread in HIV and hepatitis C virus (HCV)

An efficient coalescent epoch model for Bayesian phylogenetic inference

Adaptive dating and fast proposals: Revisiting the phylogenetic relaxed clock model

Omicron: a mysterious variant of concern

SARS-CoV-2 Accessory proteins in viral pathogenesis: knowns and unknowns

Transmission of Severe acute respiratory syndrome coronavirus 2 during border quarantine and air travel

Genomic epidemiology reveals transmission patterns and dynamics of SARS-CoV-2 in Aotearoa New Zealand

Phylodynamics reveals the role of human travel and contact tracing in controlling COVID-19 in four island nations

The proteins of Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the cause of COVID-19

Structural insights into SARS-CoV-2 proteins

Ubiquitination of SARS-CoV-2 ORF7a promotes antagonism of interferon response

Structural insight reveals SARS-CoV-2 ORF7a as an immunomodulating factor for human CD14+ monocytes

Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor

The coding capacity of SARS-CoV-2

Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway

Severe acute respiratory syndrome coronavirus gene 7 products contribute to virus-induced apoptosis

An 81-nucleotide deletion in SARS-CoV-2 ORF7a identified from sentinel surveillance in Arizona

Genome Sequences of Three SARS-CoV-2 ORF7a deletion variants obtained from patients in Hong Kong

Rapid spread of a SARS-CoV-2 Delta variant with a frameshift deletion in ORF7a. medRxiv, 2021

Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes

Replication kinetics of B.1.351 and B.1.1.7 SARS-CoV-2 variants of concern including assessment of a B.1.1.7 mutant carrying a defective ORF7a gene

Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8

Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a

Identification of eight SARS-CoV-2 ORF7a deletion variants in 2,726 clinical specimens. bioRxiv

SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression

Evolving the largest RNA virus genome

Studies of endogenous retroviruses reveal a continuing evolutionary saga

Evolutionary history of cotranscriptional editing in the paramyxoviral phosphoprotein gene

New Zealand's science-led response to the SARS-CoV-2 pandemic