key: cord-0961506-24a66ndd
authors: Dao, Manh H.; Phan, Lan T.; Cao, Thang M.; Luong, Quang C.; Pham, Hang T. T.; Vu, Nhung H. P.; Khuu, Nghia V.; Nguyen, Thinh V.; Nguyen, Long T.; Nguyen, Hieu T.; Nguyen, Anh H.; Huynh, Loan Kim Thi; Huynh, Thao P.; Nguyen, Quan H.; Truong, Hieu C.; Nguyen, Hang Minh; Trinh, Tung Xuan; Nguyen, Dung T.; Nguyen, Trieu B.; Do, Hung T.; Pham, Quang D.; Nguyen, Thuong V.
title: Genome‐wide analysis of SARS‐CoV‐2 strains circulating in Vietnam: Understanding the nature of the epidemic and role of the D614G mutation
date: 2021-06-06
journal: J Med Virol
DOI: 10.1002/jmv.27103
sha: 493d3c8e00eb491cd7dcc88e370f813594815e34
doc_id: 961506
cord_uid: 24a66ndd

Genome‐wide analysis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) strains is essential to better understand infectivity and virulence and to track coronavirus disease 2019 (COVID‐19) cases and outbreaks. We performed whole‐genome sequencing of 27 SARS‐CoV‐2 strains isolated between January 2020 and April 2020. A total of 54 mutations in different genomic regions was found. The D614G mutation, first detected in March 2020, was identified in 18 strains and was more likely associated with a lower cycle threshold (<25) in real‐time reverse‐transcription polymerase chain reaction diagnostic tests than the original D614 (prevalence ratio = 2.75; 95% confidence interval, 1.19–6.38). The integration of sequencing and epidemiological data suggests that SARS‐CoV‐2 transmission in both quarantine areas and in the community in Vietnam occur at the beginning of the epidemic although the country implemented strict quarantine quite early, with strict contact tracing, and testing. These findings provide insights into the nature of the epidemic, as well as shape strategies for COVID‐19 prevention and control in Vietnam.

which is presumed to have originated in horseshoe bats. 3 Efficient and widespread human-tohuman transmission has subsequently triggered a global coronavirus pandemic, resulting in more than 100 million cases of COVID-19 with nearly 2.5 million deaths worldwide during the past year. 4 Early in the pandemic, Vietnam closed its borders with China to prevent importation of COVID-19 cases after the first COVID-19 case was detected in Ho Chi Minh City on January 23, 2020. 1 Quarantine measures were enforced to travelers from China beginning in February 2020, and for travelers from South Korea, Japan, Europe, the United States, South East Asia, and all countries since March 21, 2020 . Electronic health declaration forms were made mandatory for all passengers entering Vietnam to assist in detecting suspected cases for testing and quarantine.

As of February 24, 2021, there were 2412 confirmed cases, and more than half of these cases were identified among travelers entering Vietnam. 5 Several COVID-19 outbreaks which have occurred as a result of local person-to-person transmission of SARS-CoV-2 may give the viruses chances to develop mutations for a further efficient adaption and evolution and a wider circulation in humans. 6 Many mutations have been found amongst SARS-CoV-2 strains circulating around the world, particularly the D614G which has been shown to increase infectivity. 7, 8 The relevance of the mutations to virulence or virus replication is still unknown. Therefore, it is necessary to elucidate the nature of the COVID-19 epidemic and to explore the characteristics of circulating SARS-CoV-2 strains. In this study, we isolated and generated whole virus genome sequences from 27 SARS-CoV-2-infected patients in southern Vietnam. Findings from epidemiological and sequencing data of SARS-CoV-2 provide information for building better strategies of COVID-19 prevention, control, and research in Vietnam. 

Between January and April 2020, swab specimens (both NP and OP specimens were collected and combined in a single virus transport medium tube to maximize test sensitivity) were taken from more than 24,000 persons who traveled to Vietnam and were close contacts with confirmed cases. SARS-CoV-2 was detected in 71 samples by real-time reverse-transcription polymerase chain reaction (RT-PCR). 9 Viral isolation was successful in 33 SARS-CoV-2-positive samples. Whole-genome sequencing was performed for 27 SARS-CoV-2 strains from imported cases and three clusters in southern and central Vietnam.

Vero E6 cells (ATCC) were used for virus isolation, as previously 

The raw sequencing data for each sample were trimmed for quality and assembled by mapping to the reference genome from Wuhan (MT019529) using the CLC Genomics Workbench v10.1.1. Viral sequences were deposited in NCBI. We downloaded 233 full genome sequences of SARS-CoV-2 from the GISAID database based on two steps: (1) location and time that patients came from or went through;

(2) randomly selecting 10 full genome sequences with high coverage from each location mentioned in step 1 at 1 month before patient's onset day. Then, we aligned the obtained sequences with 206 reference sequences by the Mafft software (v7.452). Phylogenetic trees were constructed by the maximum-likelihood method with 1000 bootstrap replicates in MEGA (v10.0.5). Variant analysis was done using CLC (v10.1.1) and Nextclade (https://clades. nextstrain.org).

To deal with a small sample size, we applied Poisson regression analysis, using a prevalence ratio (PR) as a conservative, consistent, and interpretable measure, to look for the predictors, including two cases of a family cluster originating in a man from Wuhan, China, 11 two cases of a cluster sharing a room in a quarantine area (travelling back from United States and Australia, respectively) and 17 imported cases from European countries, the United States, and Australia ( Figure S1 ). Of the 27 individuals from whom SARS-CoV-2 was isolated and sequenced, the median age was 28 years (range:

16-73 years), and 51.9% were males. Symptoms included fever (44.4%), cough (40.7%), sore throat (18.5%), and shortness of breath (11.1%) ( Table 1 ). 

Complete genome sequences of 27 SARS-CoV-2 isolates were aligned with 206 SARS-CoV-2 sequences obtained from GenBank or GISAID. The directionality of alterations was inferred based on the SARS-CoV-2 strain from Wuhan (accession number: MT019529).

The 54 different variants included 30 missense mutations, 19 synonymous mutations, and 5 mutations in the noncoding region (Tables S1, S2, and S3).

A total of 29 variants were found in ORF1ab, which roughly occupied two-thirds of the whole genome, including 19 missense and 12 synonymous mutations (Table S2 ). The largest numbers of missense alterations were recognized in the NSP3, NSP4, and NSP13

regions. Particularly, G8388A in NSP3 and A8987T in NSP4 which lead to amino acid change including S2708D and I2907F were the most common (27/27 samples). We also found an alteration in the nonstructural protein RNA-dependent RNA polymerase (RdRp) that resulted in amino acid changes, including P314L (18/27 samples) (Table S3) (Table S3 ). We also found that the appearance of the ORF1ab-14408 mutation was inversely correlated with D614G (Table S4) .

Finally, there were 13 transversions and 40 transitions ( Figure S2 ). As expected, transition mutations were generated at a higher frequency than transversions. The most popular base change transition was C→T (n = 28). There were 19/40 transitions and 11

transversions that led to amino acid alterations. It is assumed that nonsynonymous transversions hardly conserve biochemical characteristics of the initial amino acid. 13 

A multivariate generalized Poisson regression model with the E gene (C t < 25) as the dependent variable showed that the presence of the D614G mutation in the SARS-CoV-2 spike protein was more likely to have a lower C t (C t < 25) in real-time RT-PCR than the original (D614) (prevalence ratio = 2.75; 95% confidence interval, 1.19-6.38) (Table S5 ).

Our study showed that the integration of sequencing and epidemiological data could elucidate the nature of the COVID-19 epidemic in Vietnam. Travelers from outside countries are quarantined for 14 days and must have a negative RT-PCR test before entering the community. This is useful to prevent imported cases, particularly in the early phase of the epidemic. 14 D614G mutation has detected in many areas of the world. 7 This mutation located in the S1-S2 junction region near the furin recognition site (R667) for the cleavage of the S protein when the virions enter the host cells. 20 Moreover, D614G mutation was found in the six SARS-CoV-2 strains isolated from the outbreak occurring in a bar. In our study, those with D614G mutation in the SARS-CoV-2 spike protein were more likely to have a lower C t (<25) in real-time RT-PCR tests than the original (D614). This might indirectly reflect a higher viral load in nasal and pharyngeal membranes in people infected with G614 than D614. The finding that the G614-mutation strain is more contagious than D614 has been shown in a number of studies. 7, 8, 21 Therefore, it is possible that D614G facilitates the transmission of SARS-CoV-2 and may cause larger outbreaks.

Despite the limited number of samples sequenced, our findings are essential to inform on the circulation of variant strains of SARS-CoV-2 during the beginning of the COVID-19 epidemic in Vietnam.

Evidence of SARS-CoV-2 transmission in the quarantine setting suggests the need for implementing extremely strict preventive measures. SARS-CoV-2 was also found to be silently transmitted in the community as early as March 2020, when the D614G strain was first detected and became dominant. Several mutations found in the current study need further exploration to understand their roles in viral infectivity, virulence, and even vaccine development. Until now, Vietnam has experienced and controlled a few waves of epidemics, the "silent" cases may appear through illegal border crossing.

Therefore, the current measures of quarantine, case isolation, rigorous contact tracing, testing, and wearing face masks, which have been well applied in the country, need to be maintained and strengthened until an effective COVID-19 vaccine programme can be implemented.

Importation and human-tohuman transmission of a novel coronavirus in Vietnam

Pattern of early human-to-human transmission of Wuhan

The origin of SARS-CoV-2

World Health Organization. COVID-19 Weekly Epidemiological Update 22. World Health Organization

Ministry of Health-Newsletter about acute respiratory infections COVID-19

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2

Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus

Spike mutation D614G alters SARS-CoV-2 fitness

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Clinical features, isolation, and complete genome sequence of severe acute respiratory syndrome coronavirus 2 from the first two patients in Vietnam

In the interest of public safety: rapid response to the COVID-19 epidemic in Vietnam

Evidence for the selective basis of transitionto-transversion substitution bias in Two RNA viruses

The role of asymptomatic class, quarantine and isolation in the transmission of COVID-19

Superspreading event of SARS-CoV-2 infection at a bar

Tracking the COVID-19 pandemic in Australia using genomics

A SARS-CoV-2 vaccine candidate would likely match all currently circulating variants

Experimental and in silico evidence suggests vaccines are unlikely to be affected by D614G mutation in SARS-CoV-2 spike protein

D614G spike mutation increases SARS CoV-2 susceptibility to neutralization

Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

Global spread of SARS-CoV-2 subtype with spike protein mutation D614G is shaped by human genomic variations that regulate expression of TMPRSS2 and MX1 genes

How to cite this article

Genome-wide analysis of SARS-CoV-2 strains circulating in Vietnam: Understanding the nature of the epidemic and role of the D614G mutation

The authors would like to thank Dr Richard Yanagihara with the 

The authors declare that they have no conflict of interest.