Full-length genome characterization and phylogenetic analysis of SARS-CoV-2 virus strains from Yogyakarta and Central Java, Indonesia

Introduction

In December 2019, an outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus Disease 2019 (COVID-19) was detected in Wuhan, China and has become a global pandemic, including Indonesia (World Health Organization, 2020a; Phelan, Katz & Gostin, 2020).

In Indonesia, the first two COVID-19 cases were reported on 2 March 2020. Since then, the confirmed cases have been continuously increasing although several public health measures, involving isolation of confirmed patients and community wide containment in addition to strictly enforced personal health protocols, were conducted to halt transmission events (World Health Organization, 2020b). Tragically, on 19 November 2020, Indonesia recorded 478,720 COVID-19 infections and 15,503 deaths (World Health Organization, 2020b). This situation means that Indonesia has reported the second most confirmed COVID-19 cases in the South East Asia countries after the Philippines, yet has the highest number of deaths caused by COVID-19 among other South East Asia countries (World Health Organization, 2020b).

Just as many other countries, the detection of SARS-CoV-2 in suspected people is mainly based on reverse transcriptase real-time polymerase chain reaction (RT-PCR). The supply of PCR reagents, trained lab personnel and the availability of laboratories with sufficient biocontainment levels are major challenges of SARS-CoV-2 detection in developing countries, such as Indonesia (Younes et al., 2020). Therefore, it is not surprising that the tested people per week is still lower than the World Health Organization (WHO) standard (World Health Organization, 2020b).

Recently, SARS-CoV-2 with the D614G mutation became the most frequently detected globally, including South East Asia region (Korber et al., 2020; Nguyen et al., 2020). Interestingly, SARS-CoV-2 with the G614 variant had significantly higher infectious titers than the original D614 virus, and COVID-19 patients with the G614 variant had a higher viral load than patients without the mutation (Korber et al., 2020). A recent study showed that the SARS-CoV-2 with the G614 variant revealed increased infectivity, competitive fitness, and transmission than the wild-type D614 virus in human airway epithelial cells and hamster (Hou et al., 2020). However, this mutation was not associated with the severity of COVID-19 (Korber et al., 2020; Nguyen et al., 2020). Here, we aimed: (1) to report full-length genome sequences of SARS-CoV-2 collected from four COVID-19 patients in the Special Region of Yogyakarta and Central Java provinces, Indonesia; (2) to compare the clade distribution of full-length genome sequences from Indonesia (n = 60) from March to September 2020; and (3) to perform phylogenetic analysis of SARS-CoV-2 complete genomes from different countries, including Indonesia.

Materials and Methods

Results

Clade distribution of full-length genome sequences from Indonesia

Whole genome sequencing revealed that one virus (hCoV19/Indonesia/YO-202449/2020, EPI_ISL_516800) had a complete SARS-CoV-2 genome (29.903 nt). Although the other three virus samples were shorter due to incomplete UTRs at either the 5′ or 3′, they possessed full-length and complete open reading frames (ORFs) with a size of 29.409 nt consisting of 11 genes (ORF1ab, S, ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10).

Next, we compared the clade distribution of full-length genome sequences from Indonesia (n = 60) from March to September 2020. Based on the collection data, most (39/60, 65%) virus genomes contained the D614G mutation representing clade G (2), GR (7) and GH (30) (Fig. 1). From March to April 2020, clade L was dominant. On the other hand, there has been an increase in the detection of clade GH since April 2020 until now.

Phylogenetic analysis

Phylogenetic analysis of whole genome sequencing showed that three virus samples (EPI_ISL_525492, EPI_ISL_516800 and EPI_ISL_516829) clustered amongst viruses from the clade GH from multiple countries across Asia, Middle East and Europe (Fig. 2). In particular to EPI_ISL_516829, this virus showing an extended branch length to the ancestral human SARS-CoV-2 virus, hCoV-19/Wuhan/Hu-1/2019. On the other hand, one virus sample (EPI_ISL_516806) was situated between clade L viruses mainly from Asia (China, Malaysia, Indonesia, India, United Arab Emirates and Japan) (Fig. 2)

Discussion

The present study reports four full genomes of SARS-CoV-2 from patients with COVID-19 in Yogyakarta and Central Java Provinces, Indonesia. Phylogenetic analysis showed that three of four samples collected in June and August 2020 were clustered within SARS-CoV-2 viruses belonging to GH clade. One of these (EPI_ISL_516829) displayed a longer branch length compared to the other viruses indicating a greater evolutionary distance to the ancestral virus, hCoV-19/Wuhan/Hu-1/2019. This was confirmed by the molecular characterization showing that more SNPs and amino acid mutations were found within this virus genome. The phylogenetic analysis also revealed that one virus sample collected in May 2020 (EPI_ISL_516806) was in evolution closely related to the other SARS-CoV-2 viruses belonged to the L clade, including those from Indonesia which were collected in the early months of disease transmission during the pandemic between March and April 2020.

Our finding corresponds with the situation in Indonesia, showing that during the early pandemic only two clades, O and L, were detected, and the latter clade was more dominantly found from COVID-19 cases. However, since the first detection of clade GH in April 2020, this virus was more frequently detected than the previous circulating clades. Based on the data available in GISAID, 60 virus samples representing five clades have been detected from COVID-19 cases in Indonesia up to September 2020 (based on full-length genome and submission date): L (20), O (1), G (2), GR (7) and GH (30) (GISAID, 2020). Whether this pattern correlates with the increase in the number of COVID-19 cases recently in Indonesia has to be investigated further. Interestingly, a similar situation was found in some countries in North America (Mercatelli & Giorgi, 2020) and Africa (Wruck & Adjaye, 2020), which also detected more SARS-CoV-2 virus strains belonging to clade GH than to the other clades. An increase in SARS-CoV-2 detection conveys the D614G mutation concurrent with the recent global situation of COVID-19 (GISAID, 2020).

The D614G mutation dominates globally approximately 77,818/96,215 (~81%) full genomes submitted at GISAID until 18 September 2020 (GISAID, 2020). Three of four (75%) SARS-CoV-2 in our case series also consisted of D614G. According to phylogenetic tree and sequence distribution analysis, it has been suggested that the dominating D614G globally is caused by a positive selection (Korber et al., 2020), while the dominating D614G in Europe is due to a founder effect (Dearlove et al., 2020). Whether which mechanism occurs in Indonesia is difficult to conclude since only limited full genomes were submitted to GISAID until the submission date of the end of September 2020 (n = 60) (GISAID, 2020). The virus with the D614G mutation in Indonesia was first detected in April 2020 in Surabaya, East Java (GISAID, 2020), followed by other provinces, including Yogyakarta, Central Java, West Java and Banten. Clade L was mostly detected in Jakarta (7/20) and Surabaya, East Java (7/20), followed by Papua (3/20) (Fig. 1) (GISAID, 2020).

It has been reported that COVID-19 patients with the D614G mutation have a higher viral load than patients infected by SARS-CoV-2 without mutations (Korber et al., 2020). The patients with D614G had a C_T value lower than one patient without the mutation (Table 1).

Interestingly, patients infected with SARS-CoV-2 bearing D614G mutations showed moderate COVID-19, while the patient without mutations suffered from mild symptoms. These differences might be associated with the small sample size of our study (n = 4) compared with previous studies (n = 999 (Korber et al., 2020), 175 (Wagner et al., 2020) and 88 (Lorenzo-Redondo et al., 2020)). Moreover, the severity of COVID-19 is affected by many factors, including age, sex, presence of comorbidities, and patients’ immune responses (Beeching, Fletcher & Fowler, 2019; Zou et al., 2020). Further study with a larger sample size and involving risk factors for COVID-19 severity is mandatory to determine the association between the D614G mutation and the severity of COVID-19, particularly in Indonesia.

Among GH clades, they also consisted of different mutations in addition to the variants that determine the clade name (Table 1). It has already been reported that the D614G variant is almost always accompanied by three other variants: a C–T change in the 5’UTR, a silent c.3307C > T variant, and P323L (Younes et al., 2020). All GH clade samples in the present study also contained P323L (Table 1).

Notably, whole genome sequencing is of practical importance to determine virus variants and clades and is associated with particular geographic disseminations to decide clinical and political approaches at the regional and local levels (Mercatelli & Giorgi, 2020). Moreover, whether the differences in the case fatality rate and viral spread or transmission among different countries/regions are affected by differences in the virus clade (Brufsky, 2020) needs to be further studied.

Our study has some limitations, including that the samples sequenced are only 0.02% (60/290,000) of all confirmed cases in Indonesia. These facts should be considered in the interpretations of our findings, especially about epidemiological patterns (e.g., increase vs. decrease of frequency) of a particular clade in Indonesia. Another limitation of our study is we do not have any data from epidemiological tracing, nor data concerning detection (how far, how fast) to determine what is the size of the cluster where each patient belongs, or what is the transmission pattern of the cluster.

Conclusions

We report the full-genome sequence characterization and phylogenetic analysis of SARS-CoV-2 from Indonesia. SARS-CoV-2 with the D614G mutation appears to become the major circulating virus in Indonesia, which is concurrent with the COVID-19 situation worldwide. Further study with a larger sample size is necessary to investigate whether the dominating SARS-CoV-2 bearing the D614G mutation is due to a positive selection or a founder effect or some other mechanism and to explore the role of the D614G mutation in the pathogenesis and virulence of SARS-CoV-2.

Supplemental Information