Phylogenetic analysis
Phylogenetic analysis revealed that thirteen virus samples were situated within clade GH (GISAID classification), while two viruses were grouped with other viruses which belonged to clade GR, and one virus each that belonged to clade O and clade L (Figure 1). Three viruses from family cluster case-1 (YO-UGM-10001|EPI_ISL_576113, YO-UGM-10002|EPI_ISL_576114, and YO-UGM-10003|EPI_ISL_576115) formed a single group within clade GH, whereas viruses from family cluster-2 (YO-UGM-1004|EPI_ISL_576116, YO-UGM-1005|EPI_ISL_576128, and YO-UGM-1006|EPI_ISL_576130,) were separated into two different nodes within clade GH (Figure 1, top-right).
Molecular analysis
Ninety-four SNPs were detected throughout the ORP of the SARS-CoV-2 virus samples with 60% (54/94) of the nucleic acid changes resulting in amino acid substitutions (missense mutations) (Table 1, detailed in Table S.1). The types of nucleic acid base changes were more often detected as transitions (70%) compared to transversions (30%). Higher entropy values were observed more from nucleic acids that carried more frequent base changes; however, nucleic acid changes that caused missense mutation could have lower entropy values than those that resulted in synonymous mutation.
Table 1. Nucleic acid and amino acid mutations observed in seventeen SARS-CoV-2 virus genomes collected from Yogyakarta and Central Java provinces between June and September 2020.
No
|
NA
Position*
|
Gene/ Region
|
Number of SNP
|
NA change (SNP)
|
Type of base change
|
Entropy values
|
Frequency
|
Type of Mutation
|
AA changes (position)#
|
1
|
1154
|
NSP2
|
7
|
C -> T
|
Transition
|
0,21456
|
2
|
Missense
|
A205V
|
2
|
1280
|
C -> T
|
Transition
|
0,34883
|
2
|
Missense
|
V247A
|
3
|
1307
|
C -> T
|
Transition
|
0,45056
|
3
|
Missense
|
T256I
|
4
|
1501
|
C -> A
|
Transversion
|
0,34883
|
2
|
Missense
|
Q321K
|
5
|
1845
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
N435N
|
6
|
1998
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
T486T
|
7
|
2247
|
A -> G
|
Transition
|
0,21456
|
1
|
Synonymous
|
R569R
|
8
|
2772
|
NSP3
|
13
|
C -> T
|
Transition
|
0,45056
|
15
|
Synonymous
|
F106F
|
9
|
3264
|
T -> C
|
Transition
|
0,21456
|
1
|
Synonymous
|
D270D
|
10
|
3350
|
T -> C
|
Transition
|
0,21456
|
1
|
Synonymous
|
V299A
|
11
|
3819
|
C -> T
|
Transition
|
0,52971
|
4
|
Synonymous
|
G443G
|
12
|
4489
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P679S
|
13
|
4919
|
C -> T
|
Transition
|
0,66825
|
13
|
Missense
|
P822L
|
14
|
5519
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
T1022I
|
15
|
5541
|
C -> T
|
Transition
|
0,34883
|
2
|
Synonymous
|
C1029C
|
16
|
5990
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
A1179V
|
17
|
6047
|
C -> A
|
Transversion
|
0,21456
|
1
|
Missense
|
T1198K
|
18
|
6515
|
T -> G
|
Transversion
|
0,21456
|
1
|
Missense
|
F1354C
|
19
|
7374
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
F1640F
|
20
|
7448
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P1665L
|
21
|
8981
|
NSP4
|
1
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
A231V
|
22
|
9824
|
NSP5
|
4
|
A -> G
|
Transition
|
0,52971
|
4
|
Missense
|
K12R
|
23
|
9936
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
M49I
|
24
|
10242
|
C -> T
|
Transition
|
0,68696
|
8
|
Synonymous
|
N151N
|
25
|
10339
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P184S
|
26
|
10818
|
NSP6
|
1
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
L37F
|
27
|
11887
|
NSP8
|
2
|
G -> A
|
Transition
|
0,21456
|
1
|
Missense
|
A21T
|
28
|
12174
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
P116P
|
29
|
12544
|
NSP9
|
1
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
L42F
|
30
|
13465
|
NSP12 (RdRp)
|
12
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
A97V
|
31
|
13790
|
G -> T
|
Transversion
|
0,21456
|
1
|
Synonymous
|
L205L
|
32
|
13855
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P227L
|
33
|
13918
|
C -> T
|
Transition
|
0,52971
|
3
|
Missense
|
T248I
|
34
|
14027
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
D284D
|
35
|
14143
|
C -> T
|
Transition
|
0,45056
|
15
|
Missense
|
P323L
|
36
|
14429
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
D418D
|
37
|
15141
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
A656S
|
38
|
15278
|
G -> T
|
Transversion
|
0,21456
|
1
|
Synonymous
|
T701T
|
39
|
15500
|
A -> G
|
Transition
|
0,21456
|
1
|
Synonymous
|
L775L
|
40
|
15849
|
C -> T
|
Transition
|
0,34883
|
2
|
Missense
|
H892Y
|
41
|
15891
|
A -> G
|
Transition
|
0,21456
|
1
|
Missense
|
M906V
|
42
|
16130
|
NSP13
|
8
|
A -> T
|
Transversion
|
0,45056
|
3
|
Synonymous
|
P53P
|
43
|
16196
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
H75H
|
44
|
16351
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
T127I
|
45
|
16382
|
G -> T
|
Transversion
|
0,52971
|
4
|
Synonymous
|
T137T
|
46
|
16429
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
T153I
|
47
|
16476
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
V169F
|
48
|
16745
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
I258I
|
49
|
17699
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
M576I
|
50
|
18382
|
NSP14
|
4
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P203L
|
51
|
18479
|
C -> T
|
Transition
|
0,68696
|
8
|
Synonymous
|
Y235Y
|
52
|
18612
|
C -> T
|
Transition
|
0,59084
|
14
|
Synonymous
|
L280L
|
53
|
18737
|
A -> G
|
Transition
|
0,21456
|
1
|
Synonymous
|
L321L
|
54
|
19859
|
NSP15
|
2
|
T -> C
|
Transition
|
0,21456
|
1
|
Synonymous
|
I168I
|
55
|
20364
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
H337Y
|
56
|
20843
|
NSP16
|
2
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
F150F
|
57
|
21058
|
A -> G
|
Transition
|
0,21456
|
2
|
Missense
|
Y222C
|
58
|
21310
|
Spike (S)
|
12
|
C -> T
|
Transition
|
0,34883
|
2
|
Missense
|
L5F
|
59
|
21387
|
T -> C
|
Transition
|
0,21456
|
1
|
Synonymous
|
N30N
|
60
|
21477
|
C -> T
|
Transition
|
0,52971
|
4
|
Synonymous
|
S60S
|
61
|
21483
|
T -> C
|
Transition
|
0,21456
|
1
|
Synonymous
|
V62V
|
62
|
21544
|
G -> C
|
Transversion
|
0,21456
|
1
|
Missense
|
V83L
|
63
|
21935
|
T -> C
|
Transition
|
0,52971
|
4
|
Missense
|
V213A
|
64
|
22069
|
T -> C
|
Transition
|
0,21456
|
1
|
Missense
|
W258R
|
65
|
23138
|
A -> G
|
Transition
|
0,34883
|
16
|
Missense
|
D614G
|
66
|
23328
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
Q677H
|
67
|
23664
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
Y789Y
|
68
|
23729
|
A -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
N811I
|
69
|
23928
|
G -> T
|
Transversion
|
0,21456
|
1
|
Synonymous
|
L877L
|
70
|
25288
|
NS3
|
6
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
A54V
|
71
|
25298
|
G -> T
|
Transversion
|
0,59084
|
14
|
Missense
|
Q57H
|
72
|
25349
|
C -> T
|
Transition
|
0,34883
|
2
|
Synonymous
|
S74S
|
73
|
25422
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
A99S
|
74
|
25579
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
T151I
|
75
|
25791
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
D222Y
|
76
|
26470
|
M
|
3
|
C -> T
|
Transition
|
0,59084
|
14
|
Synonymous
|
Y71Y
|
77
|
26536
|
C -> A
|
Transversion
|
0,34883
|
2
|
Synonymous
|
L93L
|
78
|
26602
|
A -> G
|
Transition
|
0,66825
|
7
|
Synonymous
|
E115E
|
79
|
27345
|
NS7a
|
1
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
H73Y
|
80
|
27808
|
NS8
|
1
|
G -> A
|
Transition
|
0,21456
|
1
|
Synonymous
|
L60L
|
81
|
28046
|
N
|
14
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P13L
|
82
|
28363
|
G -> T
|
Transversion
|
0,52971
|
4
|
Missense
|
A119S
|
83
|
28470
|
T -> C
|
Transition
|
0,21456
|
1
|
Synonymous
|
N154N
|
84
|
28479
|
C -> T
|
Transition
|
0,21456
|
1
|
Synonymous
|
I157I
|
85
|
28487
|
A -> G
|
Transition
|
0,21456
|
1
|
Missense
|
Q160R
|
86
|
28586
|
G -> T
|
Transversion
|
0,52971
|
4
|
Missense
|
S193I
|
87
|
28593
|
A -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
R195S
|
88
|
28603
|
C -> T
|
Transition
|
0,21456
|
1
|
Missense
|
P199S
|
89
|
28616
|
G -> A
|
Transition
|
0,34883
|
2
|
Missense
|
R203K
|
90
|
28617
|
G -> A
|
Transition
|
0,34883
|
2
|
91
|
28618
|
G -> C
|
Transversion
|
0,34883
|
2
|
Missense
|
G204R
|
92
|
28710
|
G -> T
|
Transversion
|
0,21456
|
1
|
Missense
|
M234I
|
93
|
28944
|
A -> G
|
Transition
|
0,21456
|
1
|
Synonymous
|
S312S
|
94
|
29094
|
A -> T
|
Transversion
|
0,34883
|
2
|
Synonymous
|
T362T
|
* Nucleic acid numbering starting from ORF1ab start codon (ATG)
# Amino acid numbering starting from start codon of each gene
The majority of the virus samples (16/17) possessed D614G substitution on spike protein and 56% of these (9/16) showed other amino acid substitutions on this protein, including L5F, V83Ll V213A, W258R, Q677H, and N811I. Second amino acid mutations that were frequently detected were P232L substitution on NSP12 (RdRp) protein (15x), followed by Q57H substitution on NS3 (14x) and P822L substitution on NSP3 protein (13x). Furthermore, various amino acid mutations were also found in the other proteins of virus samples, including on NSP2 (A205V, V247A, T256I, Q321K), NSP3 (P679S, T1022I, A1179V, T1198K, F1354C, P1665L), NSP4 (A231V), NSP5 (K12R, M49I, P184S), NSP6 (L37F), NSP8 (A21T), NSP9 (L42F), NSP12/RdRp (A97V, P227L, T248I, A656S, H892Y, M906V), NSP13 (T127I, T153I, V169F, M576I, P203L), NSP15 (H337Y), NSP16 (Y222C), NS3 (A54V, A99S, T151I, D222Y), NS7a (H73Y), and N (P13L, A119S, Q160R, S193I, R195S, P199S, R203K, G204R, M234I).
COVID-19’s severity and spike protein mutations of COVID-19 samples
Based on the case definition of COVID-19 severity developed for this study, 3 of 17 virus samples (17.6%) were collected each from asymptomatic cases (people) and critical cases, 5 virus samples (29.4%) from mild cases, and 6 virus samples (35.3%) from moderate cases (Table 2). Two of the patients with critical stages eventually died. A range of Ct values was found amongst different stages of severity, nevertheless all the virus samples with D614G mutations, except one (YO-UGM-10004/2020|EPI_ISL_576116), showed lower Ct values (clade GH, GR, and O, Ct range 16.9 to 24.7) than those with no mutation in this position (clade L, Ct 27.9). Dual mutations of V213A and D614G on spike protein were detected in four patients, and two of these eventually died after a period of hospitalization.
Table 2. Severity and genetic data associated with SARS-CoV-2 viruses collected from seventeen COVID-19 patients in Yogyakarta and Central Java provinces, Indonesia from June-September 2020.
Patient No
|
Sex
|
Age (yo)
|
COVID-19 severity
|
CT value
|
Virus name
(GISAID Accession ID)
|
Average Coverage
|
Collection Date
|
Lineage (GISAID clade)
|
Spike mutations
|
1
|
Male
|
30
|
Mild
|
27.9
|
hCoV19/Indonesia/YO-UGM-200927/2020
(EPI_ISL_516806)
|
102x
|
16/05/2020
|
L
|
-
|
2
|
Female
|
49
|
Asymptomatic
|
22.31
|
hCoV-19/Indonesia/YO-UGM-00061/2020 (EPI_ISL_575331)
|
4655x
|
16/06/2020
|
GH
|
D614G
|
3
|
Male
|
77
|
Moderate
|
19.7
|
hCoV19/Indonesia/YO-UGM-202449/2020
(EPI_ISL_516800)
|
22088x
|
22/06/2020
|
GH
|
D614G
|
4
|
Female
|
55
|
Moderate
|
24.7
|
hCov19/Indonesia/JT-UGM-202538/2020
(EPI_ISL_525492)
|
347x
|
26/06/2020
|
GH
|
D614G
|
5
|
Male
|
28
|
Asymptomatic
|
21.05
|
hCoV-19/Indonesia/YO-UGM-107727/2020 (EPI_ISL_576145)
|
104x
|
30/07/2020
|
GR
|
D614G
|
6
|
Female
|
83
|
Moderate
|
16.9
|
hCoV19/Indonesia/YO-UGM-781481/2020
(EPI_ISL_516829)
|
3748x
|
10/08/2020
|
GH
|
D614G
|
7
|
Male
|
28
|
Critical
|
20
|
hCoV-19/Indonesia/YO-UGM-10002/2020 (EPI_ISL_576114)*
|
13x
|
13/08/2020
|
GH
|
V213A, D614G
|
8
|
Male
|
58
|
Critical (Died)
|
23
|
hCoV-19/Indonesia/YO-UGM-10003/2020 (EPI_ISL_576115)*
|
505x
|
16/08/2020
|
GH
|
V213A, D614G
|
9
|
Male
|
88
|
Critical (Died)
|
18.1
|
hCoV-19/Indonesia/YO-UGM-10001/2020 (EPI_ISL_576113)*
|
42888x
|
18/08/2020
|
GH
|
V213A, D614G
|
10
|
Male
|
8
|
Moderate
|
19.5
|
hCoV-19/Indonesia/YO-UGM-10006/2020 (EPI_ISL_576130)**
|
26x
|
18/08/2020
|
GH
|
L5F, D614G
|
11
|
Male
|
35
|
Mild
|
32
|
hCoV-19/Indonesia/YO-UGM-10004/2020 (EPI_ISL_576116)**
|
45x
|
20/08/2020
|
GH
|
D614G
|
12
|
Female
|
33
|
Mild
|
22
|
hCoV-19/Indonesia/YO-UGM-10005/2020 (EPI_ISL_576128)**
|
106x
|
21/08/2020
|
GH
|
L5F, D614G
|
13
|
Female
|
52
|
Asymptomatic
|
18
|
hCoV-19/Indonesia/YO-UGM-10007/2020 (EPI_ISL_632936)
|
2207x
|
22/08/2020
|
GH
|
D614G, N811I
|
14
|
Male
|
48
|
Moderate
|
19.64
|
hCoV-19/Indonesia/JT-UGM-47964/2020 (EPI_ISL_610162)
|
6291x
|
31/08/2020
|
O
|
W258R, D614G
|
15
|
Male
|
36
|
Mild
|
17.53
|
hCoV-19/Indonesia/JT-UGM-47906/2020 (EPI_ISL_576383)
|
653x
|
01/09/2020
|
GR
|
D614G
|
16
|
Female
|
64
|
Moderate
|
19.44
|
hCoV-19/Indonesia/JT-UGM-48660/2020 (EPI_ISL_610161)
|
22x
|
01/09/2020
|
GH
|
V213A, D614G
|
17
|
Male
|
41
|
Mild
|
21.24
|
hCoV-19/Indonesia/YO-UGM-48651/2020 (EPI_ISL_632937)
|
2867x
|
09/09/2020
|
GH
|
V83L, D614G, Q677H
|
Virus samples collected from family cluster-1 *) and from family cluster-2 **)
CT, cycle threshold; Ref. sequence: hCoV-19/Wuhan/Hu-1/2019 (NC_045512.2)
Disease outcomes of COVID-19’s family clusters
The epidemiological and clinical data of COVID-19’s family clusters, including clinical symptoms, date of first symptoms appeared, diagnostic results, pathological findings, comorbidity background are provided by timeline and tabulation in Figure 2 and Table 3, respectively.
Table 3. Characteristics of patients with COVID-19 from family cluster cases in Yogyakarta and Central Java
Patient No
|
Sex
|
Age (yo)
|
COVID-19 severity
|
Symptoms
|
Date of first symptoms appeared
|
Abnormal findings
|
Comorbidity
|
Virus name
(GISAID Accession ID)
|
1.1
|
Male
|
28
|
Critical
|
Fever, sore throat, nausea, cough, malaise
|
08/08/2020
|
CXR: typical COVID-19 bilateral pneumonia
NLR 12.2
ARDS PaO2/FIO2 260
|
Obesity
|
hCoV-19/Indonesia/YO-UGM-10002/2020 (EPI_ISL_576114)
|
1.2
|
Male
|
58
|
Critical (Died)
|
Fever, dyspnea, diarrhea
|
13/08/2020
|
Crackles in both lungs
NLR 8.2
5th days ARDS PaO2/FIO2 68 (intubated)
|
DM, Hypertension and obesity
|
hCoV-19/Indonesia/YO-UGM-10003/2020 (EPI_ISL_576115)
|
1.3
|
Male
|
88
|
Critical (Died)
|
Fever, dyspnea
|
19/08/2020
|
Crackles in both lungs
CXR: bilateral pneumonia and cardiomegaly
Electrolyte imbalance, PaO2/FIO2 123.3 (worsening ARDS)
Intubated on 7th day
|
Type 2 DM, Geriatric syndrome, History of infarction stroke
|
hCoV-19/Indonesia/YO-UGM-10001/2020 (EPI_ISL_576113)
|
2.1
|
Male
|
8
|
Moderate
|
Fever, cough, runny nose
|
16/08/2020
|
CXR: bilateral paracardial infiltrate
|
Autoimmune disorders (Henoch-Schönlein purpura)
|
hCoV-19/Indonesia/YO-UGM-10006/2020 (EPI_ISL_576130)
|
2.2
|
Male
|
35
|
Mild
|
Cold, headache, dry cough
|
20/08/2020
|
Eosinophilia (6.4%)
pH (7.45), PaO2 (83.2), PaCO2 (39.2), SaO2 (96.8), PaO2/FiO2 (416).
|
Hypertension
|
hCoV-19/Indonesia/YO-UGM-10004/2020 (EPI_ISL_576116)
|
2.3
|
Female
|
33
|
Mild
|
Cough, sore throat, runny nose
|
20/08/2020
|
No abnormality
|
None
|
hCoV-19/Indonesia/YO-UGM-10005/2020 (EPI_ISL_576128)
|
CXR, chest X-ray; ARDS, acute respiratory distress syndrome; NLR, neutrophil to lymphocyte ratio; DM, diabetes mellitus
In family cluster-1, all three patients showed critical COVID-19 and eventually, two died (YO-UGM-10001|EPI_ISL_576113 and YO-UGM-10003|EPI_ISL_576115) and one survived (YO-UGM-10002|EPI_ISL_576114). The disease began from patient-1.1, a 28-year-old male, who had a history of traveling from the local COVID-19 transmission area. He complained of fever, sore throat, cough and malaise on August 8th, 2020, and was tested for PCR three days afterward with the result of COVID-19 positive. His father (patient-1.2, 58-yo), who was living in the same house, showed fever, dyspnea and diarrhea on August 13th, then was followed by his grandfather (patient-1.3, 88-yo) who showed fever and dyspnea on August 18th. The PCR tests for both patients showed positive for COVID-19. All patients developed severe disease outcomes including bilateral pneumonia, cardiomegaly and ARDS. Several comorbidities were recorded from patient-1.1 (obesity), patient-1.2 (diabetes mellitus, and obesity), and patient-1.3 (type 2 diabetes mellitus, geriatric syndrome, history of infarction stroke). Patient-1.1 was uneventfully discharged from the hospital on day 29 of hospitalization, but sadly, patient-1.2 and patient-1.3 passed away in the hospital after 7 and 12 days of hospitalization, respectively.
Family cluster-2 involved three patients which were comprised of a son, 8-yo (patient-2.1), father, 35-yo (patient-2.2) and mother, 33-yo, with the following virus samples: YO-UGM-10006 (EPI_ISL_576130), YO-UGM-10004/2020 (EPI_ISL_576114), and YO-UGM-10005/2020 (EPI_ISL_576128), respectively. Prior to the index case, this family travelled from West Nusa Tenggara to Yogyakarta on August 2nd, 2020, in order to obtain medical treatment for patient-2.1 who had an autoimmune disorder in a hospital in Yogyakarta. Patient-2.1 had firstly exhibited symptoms of fever, cough, and runny nose on August 11th, 2020 and he was diagnosed COVID-19 positive on August 18th. His parents showed clinical signs of cold, headache and dry cough (patient-2.2) and cough, sore throat, and runny nose (patient-2.3) at the same day on August 20th and the PCR results of both patients were positive on August 21th. Patient-2.1 developed moderate severity with bilateral paracardial infiltrate, whereas patient-2.2 and patient-2.3 developed mild disease without any abnormalities in their chest X-rays and other laboratory findings, except eosinophilia (6.4%), increased levels in pH of the blood (7.45), PaO2 (83.2), PaCO2 (39.2) with SaO2 96.8% and PaO2/FiO2 value was 416 from the arterial blood glass analysis of patient-2.2. All three patients uneventfully recovered and were discharged from the hospital on September 2nd, 2020.
Molecular characterizations of virus samples collected from family clusters
Phylogenetic analysis revealed that the three viruses from family cluster-1 were grouped together from a single node. A matrix of nucleic acid difference showed that YO-UGM-10001|EPI_ISL_576113 and YO-UGM-10003|EPI_ISL_576115 were identical on their ORF (nucleic acid and protein levels) and both virus strains had differences of 2 nucleic acids and 1 amino acid in the NSP2 protein which correspond with V247A substitution in YO-UGM-10001|EPI_ISL_576113 and YO-UGM-10003|EPI_ISL_576115 and T256I substitution in YO-UGM-10002|EPI_ISL_576114, respectively (Table 4). Other unique mutations in the other viral proteins were detected in these three virus strains which were not shown in the other study viruses, including V213A (Spike), K12R (NSP5), T248I (NSP12/RdRp), A119S and S193I (N). The virus samples from family cluster-2 were separated in different nodes in the phylogenetic tree (Figure 3). The tree and the matrix sequence showed that YO-UGM-10005/2020|EPI_ISL_576128 and YO-UGM-10006|EPI_ISL_576130 were genetically identical. Both virus strains had 15 nucleic acid differences compared to YO-UGM-10004|EPI_ISL_576114 which resulted in amino acid variations detected in several viral proteins (Table 4).
Table 4. Amino acid mutations detected in SARS-CoV-2 viruses collected from two family cluster cases in Yogyakarta and Central Java provinces.
Virus (Accession No)
|
NSP2
|
NSP3
|
NSP4
|
NSP5
|
NSP12 (RdRp)
|
NSP13
|
NSP14
|
Spike
|
NS3
|
N
|
Amino acid position in each gene
|
247
|
256
|
321
|
299
|
822
|
231
|
12
|
248
|
323
|
892
|
|
|
5
|
213
|
614
|
57
|
119
|
193
|
Wuhan/19 (NC_045512.2)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
P
|
H
|
T
|
P
|
L
|
V
|
D
|
Q
|
A
|
S
|
YO-UGM-10001 (EPI_ISL_576113)*
|
A
|
T
|
Q
|
V
|
L
|
A
|
R
|
I
|
L
|
H
|
T
|
P
|
L
|
A
|
G
|
H
|
S
|
I
|
YO-UGM-10002 (EPI_ISL_576114)*
|
V
|
I
|
Q
|
V
|
L
|
A
|
R
|
I
|
L
|
H
|
T
|
P
|
L
|
A
|
G
|
H
|
S
|
I
|
YO-UGM-10003 (EPI_ISL_576115)*
|
A
|
T
|
Q
|
V
|
L
|
A
|
R
|
I
|
L
|
H
|
T
|
P
|
L
|
A
|
G
|
H
|
S
|
I
|
YO-UGM-10004 (EPI_ISL_576116)**
|
V
|
T
|
Q
|
A
|
L
|
V
|
K
|
T
|
L
|
H
|
I
|
L
|
L
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-10005 (EPI_ISL_576128)**
|
V
|
I
|
K
|
V
|
L
|
A
|
K
|
T
|
L
|
Y
|
T
|
P
|
F
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-10006 (EPI_ISL_576130)**
|
V
|
I
|
K
|
V
|
L
|
A
|
K
|
T
|
L
|
Y
|
T
|
P
|
F
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-10007 (EPI_ISL_632936)
|
V
|
T
|
Q
|
V
|
L
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-781481 (EPI_ISL_516829)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-202449 (EPI_ISL_516800)
|
V
|
T
|
Q
|
V
|
L
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
H
|
A
|
S
|
JT-UGM-202538 (EPI_ISL_525492)
|
V
|
T
|
Q
|
V
|
L
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-200927 (EPI_ISL_516806)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
P
|
H
|
T
|
P
|
L
|
V
|
D
|
Q
|
A
|
S
|
JT-UGM-47906 (EPI_ISL_576383)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
Q
|
A
|
S
|
JT-UGM-47964 (EPI_ISL_610162)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
P
|
H
|
T
|
P
|
L
|
V
|
G
|
Q
|
A
|
S
|
JT-UGM-48660 (EPI_ISL_610161)
|
V
|
T
|
Q
|
V
|
L
|
A
|
R
|
T
|
L
|
H
|
T
|
P
|
L
|
A
|
G
|
H
|
S
|
I
|
YO-UGM-00061 (EPI_ISL_575331)
|
V
|
T
|
Q
|
V
|
L
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-48651 (EPI_ISL_632937)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
H
|
A
|
S
|
YO-UGM-107727 (EPI_ISL_576145)
|
V
|
T
|
Q
|
V
|
P
|
A
|
K
|
T
|
L
|
H
|
T
|
P
|
L
|
V
|
G
|
Q
|
A
|
S
|
Family clusters are indicated in asterisks: Case-1 (*) and Case-2 (**)