Background
Methods
Mosquito collection
Cell culture and virus isolation
Indirect immunofluorescence assay
RT-PCR and nucleotide sequencing
Primer | Positiona | Sequence, 5'-3' | Polarity |
---|---|---|---|
CxFV-SD-1-F | 21-39 | TGGTTACACCGCAGATTG | Sense |
CxFV-SD-1-R | 1198-1217 | GATTGTAGGGCTGGGTTGAG | Reverse |
CxFV-SD-2-F | 1034-1049 | AACGGACTTCTTGAGTTTCGC | Sense |
CxFV-SD-2-R | 2197-2217 | GCCTTGGTGTAGACAAAGTATC | Reverse |
CxFV-SD-3-F | 2002-2020 | GCAAGGTTGGAGAATGGC | Sense |
CxFV-SD-3-R | 3238-3256 | GACCTTGAAGTGAAATACCC | Reverse |
CxFV-SD-4-F | 3034-3055 | GTCTAAAGCAAACCACATTCC | Sense |
CxFV-SD-4-R | 4233-4255 | CGGTCCGTAAGTTCCTTCTAAT | Reverse |
CxFV-SD-5-F | 3880-3989 | CTATGCTGTGACGCATCCTG | Sense |
CxFV-SD-5-R | 6232-6254 | AAGCCACAAATAGTCCATACAG | Reverse |
CxFV-SD-6-F | 6048-6069 | ACTCAGTGCTGTTTCAAGG | Sense |
CxFV-SD-6-R | 7204-7226 | CTCGTCCTGTTATCTCATCGTC | Reverse |
CxFV-SD-7-F | 7021-7039 | GATTTCCTGGGAGACGAT | Sense |
CxFV-SD-7-R | 8319-8337 | TCTCGCAACCTCTTCACG | Reverse |
CxFV-SD-8-F | 8012-8033 | TTCTGTTGTAAGGTGCTGTCT | Sense |
CxFV-SD-8-R | 9326-9344 | GGTTCTTCCCATTCGTCA | Reverse |
CxFV-SD-9-F | 9140-9159 | CTGGACCAAGTGACTGACC | Sense |
CxFV-SD-9-R | 10332-10350 | TGGCTGCGAGGGTGACTT | Reverse |
CxFV-SD-10-F | 10016-10038 | AGTTTGATTGGTGAGCGGGACA | Sense |
CxFV-SD-10-R | 10794-10815 | CCCGCAACAAGTCTCCTAACG | Reverse |
Sequence analysis and phylogenetic comparisons
No. | Strain | Year | Geographic Location | GenBank accession no. | ||
---|---|---|---|---|---|---|
Country | Location | Source/Host | ||||
1 | SDDM06-11* | 2006 | China | Dongming |
Culexpipiens
| JF938690 |
2 | Tokyo | 2003 | Japan | Tokyo-Shinjuku |
Culexpipiens
| NC_008604/AB262759 |
3 | NIID-21-2 | 2004 | Japan | Tokyo-Shinjuku |
Culexpipiens
| AB377213 |
4 | Narita | 2003 | Japan | Chiba-Narita |
Culexpipiens
| AB262760 |
5 | Toyama | 2003 | Japan | Toyama |
Culexpipiens
| AB262761 |
6 | Hokkaido | 2003 | Japan | Hokkaido-Abashiri |
Culexpipiens
| AB262762 |
7 | Osaka | 2003 | Japan | Osaka |
Culexpipiens
| AB262763 |
8 | Mie-Cp | 2004 | Japan | Mie-Tsu |
Culexpipiens
| AB262764 |
9 | Morioka | 2003 | Japan | Iwate-Morioka |
Culexpipiens
| AB262765 |
10 | Mie-Ct | 2004 | Japan | Mie-Tsu |
Culextritaeniorhynchus
| AB262767 |
11 | Surabaya | 2004 | Indonesia | Surabaya |
Culexquinquefasciatus
| AB262766 |
12 | Izabal | 2006 | Guatemala | Puerto Rico Izabal |
Culexquinquefasciatus
| EU805805 |
13 | HOU24518 | 2008 | USA | Texas-Harris county |
Culexquinquefasciatus
| FJ502995 |
14 | HOU24519 | 2008 | USA | Texas-Harris county |
Culexrestuans
| FJ502996 |
15 | HOU24284 | 2008 | USA | Texas-Harris county |
Culexrestuans
| FJ502997 |
16 | HOU24471 | 2008 | USA | Texas-Harris county |
Culexrestuans
| FJ502998 |
17 | HOU24516 | 2008 | USA | Texas-Harris county |
Culexquinquefasciatus
| FJ502999 |
18 | HOU24559 | 2008 | USA | Texas-Harris county |
Culexquinquefasciatus
| FJ503001 |
19 | TR3115 | 2008 | West Indies, Trinidad | Champs Fleur |
Culexquinquefasciatus
| FJ503002 |
20 | HOU24522 | 2008 | USA | Texas-Harris county |
Culexquinquefasciatus
| FJ503000 |
21 | TR3116 | 2008 | West Indies, Trinidad | Champs Fleur |
Culexquinquefasciatus
| FJ503003 |
22 | CxFV-Mex07 | 2007 | Mexico | Yucatan State |
Culexquinquefasciatus
| EU879060 |
23 | 1064 | 2007 | USA | Iowa |
Culexpipiens
| FJ663026 |
24 | 380 | 2007 | USA | Iowa |
Culexpipiens
| FJ663027 |
25 | 383 | 2007 | USA | Iowa |
Culexpipiens
| FJ663028 |
26 | 635 | 2007 | USA | Iowa |
Culexpipiens
| FJ663029 |
27 | 318 | 2007 | USA | Iowa |
Culexpipiens
| FJ663030 |
28 | 377 | 2007 | USA | Iowa |
Culexpipiens
| FJ663031 |
29 | 599 | 2007 | USA | Iowa |
Culexpipiens
| FJ663032 |
30 | 657 | 2007 | USA | Iowa |
Culexpipiens
| FJ663033 |
31 | Iowa07 | 2007 | USA | Iowa |
Culexpipiens
| FJ663034 |
32 | Uganda08 | 2008 | Uganda | Entebbe |
Culexquinquefasciatus
| GQ165808 |
33 | T955 | 2008 | Mexico | Yucatan Peninsula |
Culexinterrogator
| GU289683 |
34 | M2168 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289684 |
35 | M2313 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289685 |
36 | M2605 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289686 |
37 | M2614 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289687 |
38 | M2617 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289688 |
39 | M2618 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289689 |
40 | M2630 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289690 |
41 | M2635 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289691 |
42 | M2636 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289692 |
43 | M2637 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289693 |
44 | M2644 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289694 |
45 | M2645 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289695 |
46 | M2648 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289696 |
47 | M2650 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289697 |
48 | M2656 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289698 |
49 | M2663 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289699 |
50 | M2665 | 2008 | Mexico | Yucatan Peninsula |
Culexquinquefasciatus
| GU289700 |
Results
Virus isolation and identification
Analysis of the E gene sequence
GENOTYPE 1 | GENOTYPE 2 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
China | Japan | Indonesia | Texas | Iowa | Mexico | Guatemala | Trinidad | Uganda | ||
China
| -- |
94.6
|
94.8
|
94.5
|
94.4
|
89.4
|
89.7
|
89.6
|
90.2
| |
Japan
| 98.6 | -- |
97.5
|
97.4
|
97.4
|
89.1
|
89.3
|
89.3
|
90.2
| |
GEN 1
|
Indonesia
| 98.4 | 99.3 | -- |
97.4
|
97.3
|
89.9
|
90.1
|
90.1
|
90.9
|
Texas
| 98.6 | 99.5 | 99.3 | -- |
99.6
|
89.8
|
89.9
|
89.5
|
90.6
| |
Iowa
| 98.6 | 99.5 | 99.3 | 100 | -- |
90
|
90.2
|
89.8
|
90.8
| |
Mexico
| 97.9 | 97.4 | 97.2 | 97.4 | 97.4 | -- |
99.2
|
98.4
|
97.9
| |
Guatemala
| 97.9 | 97.4 | 97.2 | 97.4 | 97.4 | 100 | -- |
98.4
|
97.9
| |
GEN 2
|
Trinidad
| 97.9 | 97.4 | 97.2 | 97.4 | 97.4 | 100 | 100 | -- |
98
|
Uganda
| 97.9 | 97.4 | 97.2 | 97.4 | 97.4 | 99.5 | 99.5 | 99.5 | -- |
Genotype1 | Residue2 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
E-60 | E-64 | E-117 | E-125 | E-185 | E-236 | E-326 | E-340 | E-381 | E-407 | E-414 | E-423 | |
G1 | V (Val) | F (Phe) | G (Gly) | I (Ile) | H (His) | V (Val) | R (Arg) |
V (Val)
| H (His) |
F (Phe)
| F (Phe) |
V (Val)
|
G2 |
A
3
(Ala)
|
Y (Tyr)
|
E (Glu)
| I (Ile) |
Y (Tyr)
|
I (Ile)
|
K (Lys)
| I (Ile) |
Y (Tyr)
| L (Leu) | F (Phe) | I (Ile) |
SDDM06-11 | V (Val) | F (Phe) | G (Gly) |
V (Val)
| H (His) | V (Val) | R (Arg) | I (Ile) | H (His) | L (Leu) |
V (Val)
| I (Ile) |
Analysis of the ORF gene sequence
SDDM06-11 | C | PrM | E | NS1 | NS2a | NS2b | NS3 | NS4a | NS4b | NS5 | Total |
---|---|---|---|---|---|---|---|---|---|---|---|
417(139)a | 429 (143) | 1281 (427) | 1107 (369) | 690 (230) | 381 (127) | 1779 (593) | 567 (189) | 771 (257) | 2667 (889) | 10089 (3363) | |
Tokyo
| 94.5 (95.0)b | 95.3 (98.6) | 94.6 (98.6) | 94.7 (99.2) | 98.3 (97.8) | 96.9 (94.5) | 94.3 (97.5) | 94.0 (98.4) | 94.0 (97.3) | 94.7 (98.7) | 94.9 (98.0) |
NIID-21-2
| 94.7 (95.0) | 95.6 (99.3) | 94.6 (98.6) | 94.8 (99.2) | 98.3 (97.8) | 96.9 (94.5) | 94.5 (97.6) | 94.0 (98.4) | 94.0 (97.3) | 95.0 (99.1) | 95.0 (98.2) |
Iowa07
| 95.0 (94.2) | 95.6 (99.3) | 95.0 (98.8) | 94.8 (99.2) | 98.6 (98.3) | 96.6 (94.5) | 94.5 (97.5) | 94.0 (98.4) | 94.2 (97.3) | 95.1 (99.3) | 95.1 (98.2) |
HOU24518
| 95.0 (95.0) | 94.6 (98.6) | 94.5 (98.6) | 94.4 (99.2) | 98.3 (98.3) | 96.1 (95.3) | 94.2 (97.5) | 94.4 (99.5) | 94.4 (96.9) | 94.6 (98.7) | 94.8 (98.1) |
CxFV-Mex07
| 91.8 (89.9) | 92.8 (98.6) | 89.4 (97.9) | 91.3 (98.1) | 94.8 (93.9) | 92.4 (93.7) | 88.8 (96.5) | 87.5 (96.3) | 88.8 (95.7) | 89.8 (97.5) | 90.2 (96.6) |
UGANDA08
| 92.3 (91.4) | 93.0 (97.9) | 90.2 (97.9) | 92.0 (98.1) | 94.8 (94.3) | 92.9 (94.5) | 89.3 (96.5) | 88.4 (96.3) | 87.7 (94.2) | 89.8 (97.2) | 90.5 (96.5) |