Near complete genome sequences from Southern Vietnam revealed local features of genetic diversity and intergenerational changes in SARS- CoV-2 variants in 2020–2021

Background Since its beginnings in 2019, the COVID-19 pandemic is still a problem of global medical concern. Southern Vietnam is one of the country's vast regions, including 20 provinces and the densely populated metropolis Ho Chi Minh City. A randomized retrospective study was performed to investigate the epidemiology and genetic diversity of COVID-19. Whole-genome sequencing of 126 SARS-CoV-2 samples collected from Southern Vietnam between January 2020 and December 2021 revealed the main circulating variants and their distribution. Methods Epidemiological data were obtained from the Department of Preventive Medicine of the Vietnamese Ministry of Health. To identify circulating variants, RNA, extracted from 126 nasopharyngeal swabs of patients with suspected COVID-19 were sequenced on Illunina MiSeq to obtain near complete genomes SARS-CoV-2. Results Due to the effectiveness of restrictive measures in Vietnam, it was possible to keep incidence at a low level. The partial relaxation of restrictive measures, and the spread of Delta lineages, contributed to the beginning of a logarithmic increase in incidence. Lineages 20A-H circulated in Southern Vietnam during 2020. Spread of the Delta lineage in Southern Vietnam began in March 2021, causing a logarithmic rise in the number of COVID-19 cases. Conclusions Pandemic dynamics in Southern Vietnam feature specific variations in incidence, and these reflect the success of the restrictive measures put in place during the early stages of the pandemic. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-023-08814-8.


Introduction
In late December 2019, a group of patients with pneumonia of unknown etiology was reported in Wuhan, Hubei province, China [1].From there, it spread massively to all 34 provinces of China.The number of confirmed cases increased rapidly, and thousands of new cases were identified daily.The highest average newly-confirmed cases per day reached 3,000 [2].Amidst this situation, the WHO declared the novel coronavirus outbreak a 'public health emergency of international concern' [3].On February 11, the International Committee on Taxonomy of Viruses designated the novel coronavirus 'SARS-CoV-2' , and associated illness was designated 'COVID-19' [4].In some cases, SARS-CoV-2 infection (COVID- 19) can lead to a severe chronic respiratory syndrome with pneumonia and/or a myriad of other complications.
After applying many COVID-19 control measures (shutting down Wuhan on the 23rd of January, blocking all travel to and from the city), the daily number of cases in China subsequently decreased.In contrast, large clusters were reported in an increasing number other countries.
In Vietnam, the first case was identified on January 23, 2020 [5].Since the beginning of 2020, Vietnam has experienced four waves of the COVID-19 epidemic: the first wave (January 23-July 24, 2020) with 415 cases, including 106 domestic cases and 309 imported cases; the second wave (July 25, 2020-January 27, 2021) with 1,136 cases, including 554 domestic and 582 imported; the third wave (January 28-April 26, 2021) with 1,301 cases, including 901 domestic and 391 imported; and a fourth wave (starting on April 27, 2021) which is still ongoing [6].By May 4, 2023, the national COVID-19 caseload had reached 10,433,530 with 43,057 fatalities [7].From March 2020 to December 2021, the strategy of COVID-19 control was tracing, quarantine, and PCR analysis of every single person who had been in contact with test-positive individuals.All people entering Vietnam were obligated to quarantine and underwent COVID-19 diagnostics (RT-PCR).
SARS-CoV-2 is an enveloped, positive-sense singlestranded RNA virus.Its 30 kilobase genome features a rapid mutation rate, and nucleotide changes accumulate over time.Identifying and following mutations helps to minimize adverse public health impacts.Southern Vietnam is one of three geographic regions which includes 20 provinces and a principal megalopolis, Ho Chi Minh, with a population of around 9 million people.Here, we studied the epidemiology of COVID-19, including wholegenome sequencing of SARS-CoV-2 samples from Southern Vietnam, during the period from January 2020 to December 2021.Identification of the main circulating variants during the pandemic in Vietnam and their succession is valuable information needed by scientists, policy makers, and health professionals.

Epidemiological data and study materials
All epidemiological data were obtained periodically from the Department of Preventive Medicine, Vietnamese Ministry of Health [7,8].During the period from January 2020 to December 2021, nasopharyngeal swabs of patients with suspected COVID-19 from twenty southern provinces of Vietnam (Bac lieu, An Giang, Bạc Liêu, Bac ninh, Binh Duong, Cà Mau, Da nang, Đồng Nai, Hau Giang, Ho Chi Minh City, Hung yen, Kiên Giang, Kom tum, Lam Dong, Long An, Quang Ninh, Tây Ninh, Tien giang, Tây Ninh, Vinh long), and nine imported cases, were collected and delivered to the Pasteur Institute in Ho Chi Minh City.Swabs were collected in 500 µL of viral transport medium, transported in tripled sealed containers, and stored under ultralow temperature conditions until further analysis.Viral RNA was extracted, and samples were tested for the presence of SARS-CoV-2 by realtime polymerase chain reaction (PCR) using a standard procedure at the Pasteur Institute in Ho Chi Minh City.

RNA purification, Real-time PCR
Total RNA from nasopharyngeal swabs samples were obtained by extraction and purification using the QIAamp Viral RNA Extraction Kit (QIAGEN, Germany) with the QIAcube Connect automatic station (QIAGEN, Germany) according to the manufacturers recommendations.Samples were eluted and stored at -70° C until further analysis.For SARS-CoV-2 detection and to assess viral load, swabs were thoroughly analyzed using Light-Mix ® Modular Wuhan CoV E-gene reagents (Roche SAP) according to manufacturer's recommendations.SARS-CoV-2-positive samples featuring Ct values of 25 or less were selected and studied further.

SARS-CoV-2 genome enrichment
To create NGS libraries, viral RNA was subjected to reverse transcription and subsequent PCR enrichment.Reverse transcription was performed using random hexamers with the Reverta L Kit (AmpliSens, Russia) following the manufacturer's protocol.Samples (cDNA) were stored at -20°C until amplification.To obtain a near complete genome sequences of SARS-CoV-2 (excluding the 5′ and 3′ ends), a total of 138 primer pairs covering the entire genome were applied according to a previously described multiplex PCR protocol [9].Reactions were performed in a total volume of 25 μl containing 2 μl cDNA, 0.1 μM of each primer, and 12.5 μl 2 × BioMaster HS-Taq PCR mix (Biolabmix, Novosibirsk, Russia).PCR were performed with following parameters: 95 °C for 3 min; 40 cycles (93 °C for 10 s, 57 °C for 30 s, 72 °C for 30 s); a final extension (72 °C for 5 min).Reactions were performed in a C1000 Touch thermal cycler (Bio-Rad, USA).The products were analyzed by electrophoresis on a 2.0% agarose gel in the presence of ethidium bromide.Reaction products were purified using the AMPure XP purification kit (Beckman Coulter, UK) in 1:1 ratio according to the manufacturer's instructions and equimolary pooled.The concentration of the PCR-fragment mixture was measured using the dsDNA HS Assay Kit (Invitrogen, USA) with a Qubit 4.0 Fluorimeter (Invitrogen, USA) and used for library preparation.

Library preparation and sequencing
Library preparation was performed using the TruSeq DNA CD Indexes Kit (Illumina Inc., USA) according to the Illumina TruSeq Nano DNA Kit protocol.Sequencing was performed on a MiSeq instrument using MiSeq V3 chemistry, generating 2 × 200 bp reads.

Variant annotation and phylogenetic tree reconstruction
Variant calling files (.vcf ) were processed for the effect of SNP variation with the SnpEff tool (http:// pcing ola.github.io/ SnpEff/).Unique S gene genotypes were extracted from the set of annotated.gvcffiles and used to make a variant map for each strain using the "Variant frequency plot" tool from Galaxy EU (www.usega laxy.eu).A global phylogenetic tree of SARS-CoV-2 variants was constructed using the tools implemented in Nextclade [15].

Epidemiology of SARS-CoV-2 in Southern Vietnam, 2020-2021
The first COVID-19 cases were confirmed in Southern Vietnam on January 23, 2020, when two Chinese individuals arrived in Ho Chi Minh City and tested positive for the virus [16].Viral dissemination in the community was prevented through effective public health strategies, which included mandatory quarantine for travelers from other countries, isolation of people with confirmed COVID-19, and isolation of contacts (those in contact with a confirmed COVID-19 case).The Vietnamese authorities took measures to close air borders and cancel all international flights in March 2020.Since then, only repatriates, foreign specialists, and highly skilled workers have been allowed entry into the country under the strictest quarantine conditions.In the context of the measures taken, incidence in Southern Vietnam in 2020 was sporadic.The incidence rate did not exceed 0.2 per 100,000 population (Fig. 1).Considering the situation in twenty provinces of Southern Vietnam, a total of 365 cases of COVID-19 were registered in 2020.Five provinces did not detect a single case: An Giang, Binh Phuoc, Hau Giang, Lam Dong, and Soc Trang.Almost half of all cases occurred in Ho Chi Minh City, Southern Vietnam's largest city (Table 1).
In January 2021, there was a relaxation of the ban on air travel, but only under certain conditions: if there was evidence of having completed a full course of COVID-19 vaccination (no later than 14 days before the intended visit to the country) as well as a negative test performed not more than 72 h earlier.During the first five months of 2021, the incidence rate remained at a consistently low level.In May 2021, the incidence rate was already 0.8 per 100,000 population.Despite previously successful public health measures, there has been a dramatic increase in cases since June 2021.The incidence rate rose from 16.8 in June to 851.9 by December 2021 (Fig. 2).The largest increase in COVID-19 cases was observed in Ho Chi Minh City; the total number of reported cases reached 504,558 in 2021 (Table 2).
The maximum increase in the number of COVID-19 cases in the second half of 2021 was observed in the three provinces closest to Ho Chi Minh City.Binh Duong province recorded 287,282 total cases in 2021.The southern districts of Binh Duong province are very urbanized and are within one of the districts of Ho Chi Minh City.The population is about 2.5 million people.Currently, Binh Duong is a zone of ecotourism, alongside a focus on historical and cultural relics.In the provinces of Dong Nai and Tay Ninh, 96,762 and 83,488 cases were detected, respectively.Due to their large populations, proximity to Ho Chi Minh City, and natural mobility of the population, these provinces experienced a high rate of increase in COVID-19 incidence (Table 2).

SARS-CoV-2 genetic diversity in Southern Vietnam, January 2020 to December 2021
Whole-genome sequencing was performed for SARS-CoV-2 isolated from 9 imported cases and 117 domestic cases from 14 provinces in Southern Vietnam from January 2020 to December 2021.Several sequences from Northern Vietnam were analyzed as well.Genomic analysis results based on Nextclade SARS-CoV-2 Clade Assigner showed 11 clades including the Alpha, Beta, and Delta VOCs (Fig. 3, Table 3).The maximum number of mutations among non-structural genes was noted in ORF1ab.Among structural genes, the spike protein gene was foremost (Table 4).One of the first imported cases of SARS-CoV-2 (from China to Ho Chi Minh City) belonged to clade 19A.

Mutations in the SARS-CoV-2 spike protein gene in samples from Vietnam
Of 126 genomes that underwent sequencing, 55 S gene sequence variants (SGSV) were identified based on SNP pattern.SGSV-1 was identical to the Wuhan-Hu-1/2019 (MN908947) reference strain without any SNPs or indels.The remaining 54 SGSVs had at least one SNP compared to the reference (Figs. 4 and 5).Within clade 19B, no mutations in the S gene were identified; all studied samples belonged to SGSV-1.In clade 19A, one strain had a non-synonymous SNP in the spike gene (S939F) and was attributed to SGSV-2.The other four strains of the clade had S gene sequences identical to that of the reference (Wuhan-Hu-1/2019).In clade 20A, apart from the defining D614G substitution, there was a unique nonsynonymous A1078S substitution.Based on S gene SNP patterns, strains of this clade belonged to four different SGSVs (Fig. 4).Clade 20B samples had only one defining Four different SGSVs were identified within the analyzed strains.Strains from clade 20C were identical by S gene sequence with only the single defining mutation (D614G), and they carried SGSV-9.Clade 20D samples had a single defining mutation (D614G) and a synonymous 23,731 C > T SNP.Clade 20E (EU1), apart from the defining A222V variant, had a synonymous 23,683 C > T (Fig. 4).Samples belonging to the Alpha VOC lineage (clade 20I), apart from defining mutations, gained a 24,109 C > T synonymous SNP.Beta VOC (20H) samples contained no defining D215G or E484K mutations, and no specific mutations.

Discussion
COVID-19 continues to be a pressing public health problem.Different countries have had varying levels of success in combating the COVID-19 pandemic, with some countries among the most successful in the world at containing the pandemic and others in serious jeopardy.In the East Asia and Pacific regions, the most successful are considered Singapore, New Zealand, South Korea, China, and Vietnam [17].Due to strict anti-COVID-19 measures and other actions of the authorities, the epidemic in Vietnam was somewhat under control, with a minimal number of registered cases spanning 1.5 years until July 2021.In early 2020, all passengers arriving on inbound flights to Vietnam underwent testing using real-time reverse transcription polymerase chain reaction (RT-PCR) (requirement expired 15 May 2022), alongside a mandatory 14-day quarantine in centralized facilities (requirement expired 1 January 2022) [18].Genomic surveillance for SARS-CoV-2 was announced by the WHO as a necessary measure for pandemic control.In June 2020, the WHO Virus Evolution Working Group was established with a specific focus on SARS-CoV-2 variants, their phenotype, and their impact on countermeasures [19].Genomic surveillance became the international standard for combating COVID-19 [20].The scale and strategy of SARS-CoV-2 sequencing in different countries were implemented differently, which led to uneven data coverage across regions in the GISAID database and GenBank.
The Pasteur Institute in Ho Chi Minh City has shown vigilance and readiness in terms of monitoring SARS-CoV-2 lineages using NGS methods.According to the data obtained, it can be said that the first imported cases were from China and belonged to 19A (lineage B).Variants of lineage 19B (lineage A) circulated in Southern Vietnam from February to March 2020.In China, lineages A and B circulated together for a short time and were quickly totally replaced by lineage B [21].Strains of lineage 19A (lineage B) were detected in Southern Vietnam until June 2020 and were further replaced by lineage 20 (A-E).Alpha and Beta (20I and 20H) variants from the VOCs list circulated in Southern Vietnam throughout 2020 without causing rises in the incidence or deterioration of the epidemiological situation.In neighboring ASEAN countries (Cambodia, Malaysia, the Philippines, Singapore, Indonesia, Thailand), lineage B variants widely replicated in 2020.
In addition to multiple introduced variants in 2020 [22], some countries have reported differences in the distribution and spread of local genetic variants.Indeed, local lineages have been reported in neighboring ASEAN countries [23][24][25][26].
The beginning of the pandemic was marked by two major lineages, denoted 'A' and 'B' , which are probably the result of two different spillover events.The first zoonotic transmission likely involved lineage B viruses and occurred from late-November to early-December 2019 (no earlier than early November 2019).Introduction of lineage A likely occurred within weeks of the first event [27].All published sequences from environmental samples taken at the Huanan Market also  fall in lineage B. The earliest lineage A genomes had no direct epidemiological connection with the Huanan market [28].
At the beginning of 2021, the Alpha and Beta variants (20I, 20H) continued circulation in Southern Vietnam.Like global COVID-19 pandemic trends, they were completely replaced by Delta lineages, followed by a sudden increase in incidence marking the beginning of the 4th pandemic wave in Vietnam.The Delta variant was represented by three phylogenetic lineages (21A, 21I, 21J), with the 21I lineage dominating.Intragenomic variability within the Delta lineage was observed as a set of synonymous and non-synonymous substitutions in addition to the defining ones.
The first Delta variant sequences available in GISAID for ASEAN countries came from Malaysia [24] and Singapore in February 2021 [22].From early 2021, Delta became the predominant variant in the Philippines, as well as in Singapore, Indonesia, Malaysia [25] and Thailand, causing a new wave of morbidity [29].During 2021, the Delta variant, which included the original B.1.617.2Pango lineage and descendants of the AY lineage, became widespread in Malaysia.Lineage B.1.617.2 was identified as an early dominant strain circulating throughout the country, but was later superseded by lines AY.59 and AY.79 [30].In Southern Vietnam according to Pango classification, lineage AY.57 was dominating.The limitation of the study is that we observed and described only part of the genetic variation among SARS-CoV-2 lineages in Southern Vietnam in 2020-2021.The primary diversity, associated with multiple independent imports in 2020, was superseded by the Delta variant by mid-2021.Delta then became the main circulating variant in Vietnam, causing a new wave of incidence.Similar dynamics were observed in neighboring Asian countries.Watching subsequent waves of growth in case numbers associated with the easing of anti-COVID restrictions demonstrates the effectiveness and necessity of containment measures.Monitoring of circulating and emerging SARS-CoV-2 strains is essential to understanding the mechanisms of the pandemic and developing effective responses.

Conclusion
Pandemic dynamics in Southern Vietnam feature specific variations in incidence, and these largely reflect the success of the substantial, ongoing restrictive measures put in place during the early stages of the pandemic.Tracking of circulating lineages revealed major variants from the list of variants-of-concern, including intragenomic variability within circulating lineages.Further evaluation of epidemiological features and the circulation of SARS-CoV-2 variants is an essential part of COVID-19 surveillance in Vietnam.

Fig. 1
Fig.1COVID-19 incidence rate in Southern Vietnamese regions in 2020.Due to restrictions on movement and border crossing under strict quarantine, the incidence in 2020 in Southern Vietnam was sporadic.The incidence did not exceed 0.2 per 100,000 population

Fig. 2
Fig.2COVID-19 incidence rate in Southern Vietnamese regions in 2021.During the first five months of 2021, the incidence rate remained at a consistently low level.In May 2021, the incidence rate increased to 0.8 per 100,000 population.Since June 2021, there has been an increase in the number of cases from 16.8 in June to 851.9 by December 2021

Fig. 3
Fig. 3 SARS-CoV-2 phylogenetic tree reconstruction based on Nextclade tools.Branches are labeled by lineage according to Nextstrain nomenclature (legend top left).Sequences obtained in this study are colored red.The scale bar at the bottom indicates the number of nucleotide differences between each sample and the Wuhan-Hu-1/2019 reference sequence (GenBank: MN908947)

Fig. 4
Fig. 4 Phylogenetic comparison and heatmap analysis of variations observed in clades 19A-B and 20A-H.The figure shows the phylogenetic diversity and analysis of variations observed in lineages 19AB and 20A-H based on S gene sequence.Mutations were found relative to the Wuhan-Hu-1/2019 (GenBank: MN908947) reference strain.SGSV -S gene sequence variants.Clades in which this pattern occurs are indicated in parentheses.The number of strains of the specified variant in the studied group is indicated by a colon

Table 1
COVID-19 case numbers by province in 2020

Table 3
Geographic distribution of successfully sequenced samples

Table 4
Number of SNPs in each ORF in each lineage