Arch Virol (2010) 155:525–533 DOI 10.1007/s00705-010-0617-0
ORIGINAL ARTICLE
Predominance of rotavirus G2P[4] and emergence of G12P[9] strains in Asuncio´n, Paraguay, 2006–2007 Magaly Martı´nez • Alberto A. Amarilla • Maria E. Galeano Victor H. Aquino • Norma Farin˜a • Graciela Russomando • Gabriel I. Parra
•
Received: 3 November 2009 / Accepted: 4 February 2010 / Published online: 7 March 2010 Ó Springer-Verlag 2010
Abstract Rotavirus is the most common cause of severe diarrhea in children worldwide. Monitoring the diversity of rotavirus strains is of great importance for current and future vaccination programs. To determine the diversity of rotavirus circulating in Asuncion, Paraguay, between 2006 and 2007, we carried out a molecular characterization of rotaviruses detected in children \5 years old and adults ([18 years old). We found that the most common M. Martı´nez and G. I. Parra contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-010-0617-0) contains supplementary material, which is available to authorized users. M. Martı´nez A. A. Amarilla M. E. Galeano N. Farin˜a G. Russomando G. I. Parra Molecular Biology Department, IICS, National University of Asuncion, Rı´o de la Plata y Lagerenza, 2511 Asuncio´n, Paraguay A. A. Amarilla V. H. Aquino Virology Research Center, Ribeira˜o Preto School of Medicine, USP, Av. Bandeirantes, Ribeira˜o Preto, SP 3900, Brazil N. Farin˜a Clinical Laboratory of San Roque Private Hospital, Asuncio´n, Paraguay Present Address: G. I. Parra (&) Laboratory of Infectious Diseases, NIAID, National Institute of Health, 9000 Rockville Pike, Bldg 50, Room 6316, Bethesda, MD 20892, USA e-mail:
[email protected];
[email protected] Present Address: V. H. Aquino Clinical, Toxicological and Bromatological Department, Faculty of Pharmaceutical Sciences of Ribeira˜o Preto, USP, Ribeira˜o Preto, Brazil
circulating strain was G2P[4] (69/143), followed by G9P[8] (37/143). The temporal distribution of strains showed that, in children, G2P[4] was predominant in 2006, and that G2P[4] and G9P[8] were co-predominant in 2007, whereas in adults, G2P[4] was predominant in both years. Additionally, one G9P[6] and three G12P[9] strains were found in adult samples, making this the first report of these strains circulating in Paraguay. Sequence analysis of the G12P[9] strains suggests across-border migration of this strain within the southern cone of America.
Introduction Group A rotaviruses are the leading cause of severe gastroenteritis in infants and young children worldwide, resulting in more than half a million deaths per year. The rotavirus incidence has increased in the last decade when compared with the previous two decades. This phenomenon has been attributed to improved hygiene and sanitation, which has had a greater impact in reducing diarrheas associated with bacteria and parasites, but not those associated with rotavirus. Therefore, mass vaccination is considered the best strategy to decrease the severity of this infection [19, 20]. The rotavirus genome contains 11 segments of doublestranded RNA (dsRNA), which are packaged within a triple-layer capsid, and codes for six structural proteins and six non-structural proteins. These segments can be separated by polyacrylamide gel electrophoresis (PAGE), showing a characteristic migration pattern termed ‘‘electropherotype’’. In humans, two main electropherotypes (long and short) can be detected, based on the mobility of the different sizes of the NSP5 gene. The two outermost capsid proteins elicit neutralizing antibody responses and specify the G (VP7) and P (VP4)
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types. At least 23 G- and 31 P-types have been detected, with G1, G2, G3, G4, G9, P[4], P[6] and P[8] being the most prevalent types found in humans [30, 31, 37]. Due to the segmented nature of their genomes, rotaviruses can reassort their genes, leading to different combinations; however, some biases toward specific clustering of genes (also termed genogroups) have been shown [14]. Three genogroups, represented by the strains Wa, DS-1, and AU-1, have been found in nature. The strains belonging to the genogroup ‘‘Wa’’ are characterized by the association of types G1, G3 and G4 with P[8], a VP6 gene of subgroup II, an NSP4 gene of genotype B and a long electropherotype, while the strains ‘‘DS-1’’ are characterized by the association of G2 with P[4], VP6 subgroup I, NSP4 genotype A and a short electropherotype. The last genogroup is represented by the strain AU-1, which has a G3 associated with P[9], subgroup II, NSP4 genotype C and a long electropherotype and is believed to have a close evolutionary relationship with feline rotaviruses [16]. After the first licensed rotavirus vaccine (RotaShieldÒ) was withdrawn from the market, new promising products were developed to overcome the high burden that rotavirus represents to worldwide health. Two of them are already licensed for use in vaccination programs. These are RotarixÒ, a monovalent, human, live attenuated vaccine, which has G1P[8] specificity and RotateqÒ, a multivalent, human–animal reassortant, live vaccine, which has G1, G2, G3, G4 and P[8] specificity [17]. Both vaccines are highly efficient against any severe rotavirus episodes; however, their protection against G2P[4] strains is not as good as against strains from the ‘‘Wa’’ genogroup [17]. In addition, their protection against uncommon or animal strains (e.g. G5, G8, G12) has not yet been tested on a large scale. Thus, a concern among scientists and policy makers is whether or not massive worldwide vaccination is going to change the epidemiological patterns of the sero/genotype distribution and ultimately result in the failure of the available vaccines [12]. We have previously described rotavirus epidemiology and diversity in Paraguay from 1998 to 2005 [1, 22, 23]; thus, the objective of this work was to continue the monitoring of rotavirus strain diversity in this country during 2006 and 2007.
rotavirus by using an immunochromatographic (IC) test (SD Bioline One Step Rotavirus Antigen Test). Of these, 501 samples were collected for further molecular analysis: 186 from children (up to 5 years of age) and 315 from adults (18 years old and up). The samples collected represent *90% (292/322) of the total positive samples and *16% (209/1307) of the negative samples initially screened by IC. Rotavirus characterization Rotavirus dsRNA was extracted by TRIZOLÒ (Invitrogen, Inc., Carlsbad, CA, USA) from 10% fecal suspensions according to the manufacturer’s instructions, or the method of Boom et al. [4] when inhibitors impaired the ability to detect the genotype. Rotavirus dsRNA electropherotypes were determined by PAGE using a 4.5% stacking gel and a 7.5% running gel followed by silver staining [5]. Reverse transcription (RT)-PCR of the VP4 and VP7 genes was carried out using consensus primers Con2–Con3 and Beg–End, respectively [10, 11], and the G- and P-types were determined using a heminestedmultiplex PCR, as described previously [7, 10]. The amplicons of the NSP4 and NSP5 genes were obtained by using consensus primers of the 50 and 30 ends of the gene [33, 39]. Reaction conditions are available from the authors upon request. The PCR products were resolved by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized under long-wave UV light. The NSP4 and NSP5 genes and first-round amplicons from the VP7 and VP4 genes were purified from agarose gels using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA), and sequencing was performed using an automatic sequencer, ABI Prism 3100 (Applied Biosystems, Foster City, CA, USA). Sequence data were submitted to the GenBank database under the accessions numbers FJ941092–FJ941115. The phylogenetic relationships between strains were reconstructed by the neighbor-joining and maximum-parsimony methods using PAUP 4.0b10 [35] and MEGA v4.0 [36].
Results Electropherotyping and genotyping
Materials and methods Sample collection A total of 1,629 samples from out- and in-patients with acute diarrhea who were admitted to two main private hospitals in Asuncion, Paraguay, San Roque Hospital and La Costa Children Hospital, were initially screened for
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The rotavirus electropherotype was defined in 257 out of the 261 samples positive by molecular methods, 179 of which (88 from children and 91 from adults) presented a short electropherotype and 78 of which (41 from children and 37 from adults) presented a long electropherotype. In the four remaining samples, the amount of sample was not enough to obtain a full characterization of the
Rotavirus strains in Paraguay
527
Table 1 Age group distribution of rotavirus strains circulating in Paraguayan children and adults with acute gastroenteritis during 2006–2007 (a) Children Age group (months)
Electropherotype ratiob
Electropherotype Short
Long
Total rotavirus-positives
NDa
0–5
6
4
1.5
10
6–11
10
7
1.4
17
12–23
31
11
24–35
18
11
36–47
13
48–60
10
Total
88
41
2.8
42
1.6
31
6
2.2
19
2
5.0
12
2.1
131
Electropherotype ratio
Total rotavirus-positives
2
2
(b) Adults Age group (years)
Electropherotype Short
Long
18–30
26
14
ND
1
1.9
40
31–40
25
7
3.6
33
41–50
7
7
1.0
14
51–60
14
7
2.0
21
[60
19
2
1
9.5
22
Total
91
37
2
2.5
130
a
Rotavirus-positive samples with no determined electropherotype
b
Short/long electropherotype ratios from a specific age group that are above the average are shown in bold
electropherotype. In 179 samples, only one short electropherotype pattern was detected; however, two different long electropherotype patterns, one found in 75 samples and the other in three adult samples, were detected (Supplementary Fig. 1). Interestingly, the age-group distribution of the electropherotypes shows that children older than 2 years old and adults older that 60 years are more likely to be infected by strains presenting short electropherotype (Table 1). A total of 84 samples from children (47 from 2006 and 37 from 2007) and 56 from adults (29 from 2006 and 27 from 2007) were randomly chosen for genotyping. In addition, the three samples from adults that presented a different long electropherotype were also chosen for genotyping. We found that, overall, the most common circulating strain was G2P[4] (69/143), followed by G9P[8] (37/143). Mixed infections and uncommon G and P combinations were detected in 13 samples; additionally, one strain showed the combination G9P[6] (Table 2). The yearly distribution showed that, in adults, the G2P[4] strains predominated in both years, whereas in children, the G2P[4] strains predominated during 2006 and then the G2P[4] and G9P[8] strains co-predominated during 2007 (Fig. 1).
Table 2 G- and P-type combinations of rotavirus strains circulating in Paraguayan children and adults with acute gastroenteritis during 2006–2007 (a) Children G-types
P-types P[4] P[6] P[8] P[9] P[4] ? [8] P untypeable Total
G2 G9 G2 ? G9 G untypeable Total
44
2
1 5
22
1
3 53
0
24
0
1
1
47
2
26 5
3
6
6
84
(b) Adults G-types
P-types P[4] P[6] P[8] P[9] P[4] ? [9] P untypeable Total
G2
25
G9 G12
1
1
1
G2 ? G9 G untypeable Total
15 1
1 2 28
1
16
1
1
3
28
1 1
18 3
1
2
6
8
12
59
G12 and P[9] types, as well as one G2, two G9, two P[4] and one P[8], were detected by nucleotide sequencing
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528
number of samples
35 30 25 20
Children
15 10 5
number of samples
0 20 15 10
Adults
5
from different lineages were compared. It is worth mentioning that differences in the amino acid sequence within the VP8* fragment of the VP4 protein were also found when strains from different lineages are compared (Supplementary Fig. 2). It has been shown that the T152 strain, a G12P[9] strain detected more than 10 years ago in Thailand, presents a AU-1 gene constellation [16, 27]; thus, to gain insight into the genome characteristics of the G12P[9] strains detected in Paraguay, the partial nucleotide sequences of the NSP4 (nt 42–569) and NSP5 (nt 80–623) genes were obtained. Phylogenetic analysis of these genes grouped the Paraguayan strains with the clusters C (E3) and H6, respectively, in close association with the T152 strain (Fig. 4c, d) [16].
0 2006
G2
G9
2007
G12
G2+G9
G untypeable
Fig. 1 Yearly distribution of G types from rotavirus isolated in Paraguayan children and adults
Sequence analysis Since the G2P[4] strains have been circulating at a high level in Paraguay and the rest of Latin America over the last 4 years [1, 15, 34], the partial sequences of the VP4 (nt 43–846) and VP7 (nt 88–996) genes were analyzed to increase our knowledge about their diversity and potential causes of their high incidence over the last years. Phylogenetic analysis showed that the Paraguayan G2P[4] strains clustered together with the most commonly detected G2P[4] strains, i.e. lineage II from the VP7 gene [18] and lineage V from the VP4 gene (Fig. 2) [9]. Since only one rotavirus G2P[4] (Py99406) has been reported to be circulating in Paraguayan children during 1998–2004 [21], its VP7 gene was sequenced and included in the phylogenetic analysis for comparison with the ones circulating after 2004. The rotavirus Py99406, which was detected in 1999, also clustered with lineage II (Fig. 2a). The amino acid identity of this strain with the ones isolated during 2006– 2007 ranged from 97.9 to 98.9%, and none of the amino acid substitutions were found within the antigenic regions of the Paraguayan strains over time (Fig. 3). The partial sequences of the VP7 (nt 49–977) and VP4 (nt 33–750) genes of the G12P[9] strains showed 99% and [98% identity, respectively, with the G12P[9] strains isolated in Argentina and Brazil. Phylogenetic analysis showed that the G12P[9] Paraguayan strains clustered into lineage II of the VP7 gene [28], while analysis of the VP4 gene showed that they clustered with a new lineage of the P[9] genotype (Fig. 4a, b). This new proposed lineage for the P[9] genotype, also observed recently by Castello et al. [6], is supported by 10.4% nucleotide distance when strains
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Discussion In Paraguay, acute diarrheal disease is the third leading cause of mortality in children between 1 and 4 years of age. Epidemiological studies carried out in children with acute diarrhea in Asuncio´n, Paraguay, have shown the presence of rotavirus in 20–30% of these cases [1, 5, 22]. Since the two currently used rotavirus vaccines show a low degree of protection against the G2P[4] strains, most recent studies have focused in the presence of these strains in the human population [12, 13, 15, 24]. Whereas no G2P[4] strains were reported to be circulating in Paraguayan children from 2000 to 2004, *39% of the strains characterized in 2005 were shown to have a short electropherotype and genotype G2, making this strain the second most prevalent one circulating that year. This coincided with the predominance of these strains in the adult population [1]. During the first year of the present study, the G2P[4] strains were predominant in children, with a frequency of 63.8%, which decreased to 46%—a value similar to that of the G9 strains—in 2007. In adults, the G2P[4] strains were predominant during both years. Since the population under study did not receive the rotavirus vaccines, and high incidences of these strains have been reported recently in countries neighboring Paraguay [15, 34], the yearly shift of the predominant strains seems to reflect a natural pattern of rotavirus epidemiology. Interestingly, in this study, the presence of G2P[4] strains was associated with older children and adults, who would have been previously infected with the predominant Wa-like strains [22]. In addition, no major differences were found between the VP4 and VP7 genes of the Paraguayan G2P[4] strains, even when strains circulating 7 years apart were analyzed. This suggests that the same strain has cycles of increased circulation in the human population, which might constitute a mechanism by which these viruses evade the immune
Rotavirus strains in Paraguay
529
(a)
(b) FJ492767-Human-PSAL977-Brazil-2006 FJ492765-Human-PSAL818-Brazil-2006 FJ492766-Human-PSAL906-Brazil-2006 FJ492770-Human-PSAL1655-Brazil-2006 Py1138ASR07-P4 Py1105ASR07-P4
87
Py1494SR07-P4 FJ492769-Human-PSAL1581-Brazil-2006 FJ492771-Human-PSAL1904-Brazil-2006
EU045251-Human-Py05SR1134-2005-G2
FJ492764-Human-PSAL704-Brazil-2006
EU045252-Human-Py05SR1297-2005-G2
Py1452SR06-P4
EU045250-Human-Py05SR1124-2005-G2
82
Py1073ASR07-P4
FJ492780-Human-PSAL1967-Brazil-2006-G2
Py1033ASR06-P4
FJ492776-Human-PSAL977-Brazil-2006-G2
Py1399SR06-P8
FJ492778-Human-PSAL1581-Brazil-2006-G2
DQ904511-Human-J-4787-Japan-2006 AF401754-Human-KO-2-Japan-2001
FJ492779-Human-PSAL1904-Brazil-2006-G2 83
DQ904513-Human-VN-19-VietNam-2006
FJ492777-Human-PSAL1581-Brazil-2006-G2
99 DQ904512-Human-VN-11-VietNam-2006
Py1528SR07
DQ904519-Human-CH-188-China-2006 Py40699-P4
Py1138ASR07
Lineage I I
Py1093ASR07
AJ293722-Human-Sc27-India
77
EF199724-Human-CMH017/03-Thailand-2003
Py1105ASR07
AY707784-Human-CMH277-Thailand-2004
Py1157ASR07
AY261349-Human-KY3103/99-SouthAfrica-1999
AF401755-Human-KO-2-Japan-2000-G2
AF480268-Human-Mvd9707-Uruguay-1997
71
DQ109975-Human-98TW762-Taiwan-1998-G2
72
AF480269-Human-Mvd9704-Uruguay-1997
98
DQ109982-Human-00TW1959-Taiwan-2000-G2
AF480267-Human-Mvd9701-Uruguay-1997
X95371-Human-312-India
DQ109978-Human-97TW967-Taiwan-1997-G2
X95370-Human-253-India 81
DQ097014-Human-02TW569-Taiwan-2002-G9
96
U36241-Human-E210-Australia
DQ172842-Human-Ita3-Italy-2004-G2
L11605-Human-M48-India
94 DQ172841-Human-Ita1-Italy-2003-G2
X95273-Human-IS2-India
92
AY261348-Human-BS3585/99-SouthAfrica-1999 86
AB081593-Human-116E3D-India-2002
FJ492773-Human-PSAL1967-Brazil-2006 99
70
AY261338-Human-514GR/87-SouthAfrica-1987
81 99 99
AJ540227-Human-DS-1-USA-1976-G2 91
Lineage I
AY261337-Human-405GR/87-SouthAfrica-1987
75
DQ857954-Human-rj5619/02-Brazil-2002
99
99
Lineage II I
Lineage I
Lineage III
DQ857927-Human-RJ5619/02-Brazil-2002-G2 DQ857926-Human-RJ5323/02-Brazil-2002-G2
AY261341-Human-64SB/96-SouthAfrica-1996 AY261340-Human-1831GR/93-SouthAfrica-1993
M32559-Human-RV-5-Australia-G2
M58292-Human-L26-Philippines-1987/88-G12
DQ857953-Human-rj5323/02-Brazil-2002 97
EU045253-Human-Py05ASR60-2005-G2
DQ172838-Human-H41-Italy-1993-G2
EF672581-Human-DS-1-USA-2008 AF106280-Human-TA3-Taiwan-1981
EU045249-Human-Py04ASR42-2004-G2 100
FJ492772-Human-PSAL1920-Brazil-2006
AY787646-Human-TB-Chen-China-2008
99
EU045254-Human-Py05AP98-2005-G2 EU045248-Human-Py99406-1999-G2
98
AF260956-Human-97S43-China-2000 89
Lineage V
Py1096ASR07
AF254140-Human-CIT-220RV-Ireland-2000 73
DQ172840-Human-I200-Italy-1997-G2
Lineage IV
86 DQ172839-Human-H93-Italy-1996-G2
AF450292-Human-T79-China-2001
AY855067-Human-R291-Brazil-G8
U73955-Human-95A-Australia-1996
96
AJ278256-Human-MW333-Malawi-1997-G8
Lineage II
U73947-Human-92A-Australia-1996
M33516-Porcine-Gottfried-G4P6
AY766085-Porcine-34461-4-Spain-2004
0.02
Fig. 2 Phylogenetic analysis of the VP7 (a) and VP4 (b) genes from G2P[4] strains detected both in Paraguay and other countries worldwide. The trees were constructed using the Kimura 2-parameter model as a nucleotide substitution model and neighbor joining for tree reconstruction. Bootstrap values are shown at the branch nodes (values \70% not shown). The Paraguayan strains isolated in 1999
0.05
are marked with a filled square, and the ones isolated from 2004 to 2007 with a filled circle. Strains isolated in neighboring countries of Paraguay are marked with a filled triangle. For each strain (where available), the GenBank accession numbers, host species, country of origin, and year of isolation are shown
Fig. 3 Amino acid substitutions in the variable regions 3, 4, 5 (antigenic region A), 7 (antigenic region B) and 8 (antigenic region C) of the VP7 genes of Paraguayan G2P[4] strains
response to previous infections [23]. It is worth mentioning that Brazil and other Latin American countries have already introduced the G1P[8] vaccine in their vaccination programs [8]; therefore, further studies will be needed to clarify this phenomenon. During the late 1990s, the presence of G9P[6] strains in South America was reported at high frequencies in Brazil
and Argentina, but during the 2000s, G9P[8] strains have been shown to have displaced the former ones. Interestingly, both strains have been shown to group within the phylogenetic cluster of G9 strains that emerged worldwide during mid 1990s [2, 3, 29, 33]. This work constitutes the first report of rotavirus G9P[6] in Paraguay, and even though several attempts were made to obtain the VP4 and/or
123
M. Martı´nez et al.
530
(a)
(b) 87 EF059916-Human-CAU-195-G12 100
EF059917-Human-CAU-214-G12
AB008669-Human-512B
DQ995173-Human-SI-264-G12P8-2006
100 AB008668-Human-512A
AM397926-Human-BP875-G12P8-2005 82
D14615-Human-AU228-G3
AB269689-Human-MD844-G12P8-2004
94
D14613-Human-AU1115-G3 AB008289-Human-02-92-G3
AM397928-Human-BP1503-G12P8-2005
84 D14614-Human-AU125-G3
DQ146643-Human-B4633-G12P8-2003
D10970-Human-AU-1-G3
90 DQ490556-Human-RV176-G12P6-2000
D14620-Human-AU938-G3
DQ490550-Human-RV161-G12P6-2000
78
D10971-Feline-FRV-1-G3
DQ146687-Human-N26-G12P6-2002
AB008666-Human-O265
AJ311741-Human-Se585-G12P6-1998 72
D14619-Human-AU785-G3
Lineage III
AB264004-Human-04S010-G12-2004
83
D14618-Human-AU720-G3
DQ099754-Human-ISO26-G12
91
81
91
AB008667-Human-M318 D14616-Human-AU379-G3
DQ146654-Human-Dhaka25-G12P8-2002
74
D14617-Human-AU387-G3
DQ062125-Human-ISO21-G12
AJ311736-Human-Se584-G6
DQ062124-Human-ISO19-G12
71
EF559260-Human-RV024-G12-2006
86
DQ099751-Human-ISO16-G12
AJ311734-Human-CC425-G3 AJ488137-Human-Hun2-G3
99 99
DQ062129-Human-ISO29-G12
99
AJ311735-Human-KC814-G3
99
90 78
AJ488136-Human-Hun1-G3 AJ488138-Human-Hun3-G6
DQ017650-Human-ISO14-G12
AY855066-Human-BrzR239-G10
EU284730-Human-SA3370JHB-G12P6-2004 73
100
D90260-Human-K8-G1
DQ146665-Human-Dhaka12-G12P6-2003 98 91 93
D14624-Human-PCP5-G3
76
DQ146676-Human-Matlab13-G12P6-2003
D13403-Feline-Cat2-G3
95
Py1037ASR07-G12
D14623-Human-PA151-G6
Py1135ASR07-G12P9
93
AY855065-Human-BrzHC91-G12P9
100
93
EU496259-Human-Kor588-G12-2002
AJ488142-Human-Hun8-G6 AJ488141-Human-Hun7-G6
DQ111868-Human-Arg720M-G12P9-1999 EU259895-Human-GJ0612314-G12
D14622-Human-MZ58-G3
AJ488140-Human-Hun6-G6
Py942ASR06-G12P9 97
EU513175-Human-Arg721-G12
Lineage I I
99 100
Py1135ASR07-G12P9 Py942ASR06-G12P9
AB125853-Human-CP1030-G12P9-2001
AB125855-Human-CP1030-G12
EU496256-Human-Arg721M-G12P9-1999
EU513177-Human-Kor588-G12 EU259893-Human-GJ0612314
AB071404-Human-T152-G12P9-1998
AB077766-Human-T152-G12
AB186120-Human-K12-G12-1999
AB125854-Human-CP727-G12
DQ204743-Porcine-RU172-G12
EU513174-Human-Arg720-G12
Lineage I
0.002
Lineage II
EU513176-Human-K12-G12
AB125852-Human-CP727-G12P9-2001
EF672595-Human-L26-1987
Lineage I
AB008665-Human-O264
DQ062128-Human-ISO28-G12
90
DQ923802-Human-CMH134/04-G3 DQ923798-Human-CMH120/04-G3
GU250828-Human-RV98670-Brz-G12P6-2008
U32167-Human-429-S4
0.05
Fig. 4 Phylogenetic analysis of the VP7 (a), VP4 (b), NSP4 (c), and NSP5 (d) genes from Paraguay, G12P[9] strains and rotavirus strains detected worldwide. The trees were constructed using the Kimura 2parameter model as a nucleotide substitution model and neighbor joining for tree reconstruction. Bootstrap values are shown at the
branch nodes (values \70% not shown). The Paraguayan G12P[9] strains are marked with a filled circle. Strains isolated in countries neighboring Paraguay are marked with a filled triangle. For each strain (where available), the GenBank accession numbers, host species and associated G- and P-types are shown
VP7 sequences from this sample, all of them were unsuccessful, and we thus have no data that will help us to identify the potential origin of this strain. Initially, by using RT-PCR, we were unable to type 22.3% (32/143) of the rotavirus-positive samples; however, after using the RNA extraction method of Boom et al. [4], nine rotavirus-positive samples were positive for the firstround amplicon of the VP4 and/or VP7 genes and were thus then subjected to nucleotide sequencing. One sample was shown to bear genotype G2; two, genotype G9; two, genotype P[4]; and one, genotype P[8]. It is worth mentioning that no nucleotide differences were found at the primer-binding site between the strains correctly genotyped and those untypeable by RT-PCR (data not shown). Therefore, the presence of PCR inhibitors could explain the failure to obtain the genotype from these samples. Surprisingly, three rotavirus-positive samples—all of which
were from adults (23, 49 and 60 years old)—showed the genotype G12 and/or P[9]. It is of note that these three samples showed a long electropherotype that differed from the ones of the G9P[8] strains. The presence of G12 was first reported in South America in 1999 in Buenos Aires, Argentina, and in 2003 in Parana, Brazil, both of which are neighboring countries of Paraguay [6, 25]. It is worth mentioning that Parana is a Brazilian state located in the south of the country that borders both Paraguay and Argentina, a fact that could indicate the spread of this strain within the southern cone of America by across-border migration. The phylogenetic clustering of the VP4 and VP7 genes from the G12 strains detected in these three countries supports this hypothesis. Interestingly, the VP7 gene from two G12P[6] strains detected in 2008 in the north of Brazil has been reported recently in the public databases, and phylogenetic analysis has grouped them
123
Rotavirus strains in Paraguay
531
(c)
(d) 99 AB091355-Human-H085/92-G2
96 EF059918-Human-CAU195-G12P6
AB091359-Human-H102/92-G2
EF059919-Human-CAU214-G12P6
AY803730-Human-RMC437
80 FJ152125-Human-US6597-G12P6
AY841126-Human-RMC61
DQ995177-Human-SI264/06-G12P8 89
95 DQ995178-Human-SI403/06-G12P8 100
98
AB091354-Human-H185/88-G1
FJ152114-Human-US6588-G12P8
99
AB091353-Human-H134/87-G1
Lineage B (E1) Wa like
EF672617-Human-ST3/75-G4P6
DQ189253-Porcine-221-20/04-G4P6
AF093199-Human-Wa-G1P8-virulent 95
DQ189249-Porcine-ES51/04-G4P6
AF093200-Human-Wa-G1P8-attenuated
99 DQ189245-Porcine-134-10/04-G3P6
U42628-Human-RV3
DQ189246-Porcine-134-11/04-G3P6
HRU59109-Human-M37/82-G1P6
DQ189248-Porcine-ES51/03-G4P6
AB008252-Human-Odelia/84-G4P8
DQ204739-Porcine-RU172-G12
97 AB008251-Human-Hochi/80-G4P8
DQ189243-Porcine-134-7/04-G4P6
DQ629927-Porcine-P21 5-G1P27
DQ189252-Porcine-221-19/04-G5P6
DQ534017-Porcine-CMP034-G2P27
100
Lineage E9
DQ189250-Porcine-221-7/04-GNP6
AY353725-Human-120-G4
DQ189251-Porcine-221-13/04-G3P6
DQ146691-Human-N26/02-G12P6
Lineage E6
100
DQ189247-Porcine-ES51/02-G5P6
D88833-Feline-FRV64-G3P3
U54772-Human-Mc323/89-G9P19 DQ189254-Porcine-221-21/04-G9P6
AB048197-Feline-FRV72-G3P3
U92797-Porcine-CN86
AB048196-Feline-FRV70-G3P3
81
AB048199-Feline-FRV303-G3P3
98
100
U92798-Porcine-CC86 AY770974-Human-RMCG60
AF144806-Canine-CU1-G3P3
AY769695-Human-RMCG7
AB048200-Feline-FRV317-G3P9
U54773-Human-Mc345/89-G9P19
100 AB048202-Feline-FRV381-G3P9 91
AB048203-Feline-FRV384-G3P9
98
84
98
92
AB091356-Human-H116/88-G4
100
AF306494-Human-Wa-G1P8
D88832-Canine-RS15
80
AF338244-Human-M0-G1
Py1135ASR07-G12P9
99 89
96 AF338245-Human-M2-G1
Py1037ASR07-G12
AF306493-Simian-SA11
Py942ASR06-G12P9
AB232700-Human-India-R152
DQ146706-T152-G12P9
100
Py1135ASR07-G12P9
100
AJ311732-Human-L26-G12P4
Py1037ASR07-G12
EF672582-Human-DS1-G2P4 100
HRU59103-Human-RV5/77-G2P4
AB091358-Human-H242/88-G3 AB008656-AU-1-G3P9
Lineage A(E2) DS-1 like 93
Lineage H3 AU-1 like
AY787650-Human-TBChen/96-G2P4
98
HRU59104-Human-S2/82-G2P4
85 100
DQ205231-Lapine-30/96-G3P14 99
AY740731-Human-B4106/00-G3P14
AJ311731-Human-Se585-G12P6
AY787651-Human-TBChen/96-G2P4
AJ400638-Human-US1206-G9P6
AF508732-Human-NR1
AF079358-Human-US1205-G9P6
98
AJ400644-Human-US430/96-G9P6
U96336-Murine-EHP 98
M28378-Human-B37-G8
100
Lineages D(E7)
X76782-Human-v51
U96335-Murine-EW
AB065287-Avian-Ch1
AY769694-Human-RMCG66 AB091727-Human-KUN-G2
U96337-Murine-EC 100
90
Lineage F(E10)
98
AB065286-Avian-Ty3
Lineage H2 DS-1 like
X76780-Human-v252 X76777-Human-v115
77
AB065285-Avian-Ty1
100
Lineage H6
Py942ASR06-G12P9
100
AY527226-Bovine-Hg18-G15P21
94
Lineage H5
AF306492-Rhesus-RRV-G3P3
AF087678-Simian-SA11-G3P1
99
X76779-Human-v183
DQ146705-Human-T152-G12P9
99
99
AB022773-Human-KU-G1P8 U96698-Human-Z10262/95-G1
D89873-Human-AU1-G3P9 AB048201-Feline-FRV348-G3P3
99
AB091357-Human-H069/92-G1
70
Lineage C(E3) AU-1 like
D89874-Feline-FRV1-G3P9 100
Lineage H1 Wa like
DQ189244-Porcine-134-8/04-GNP6
AB048198-Feline-FRV73-G3P3 100
AY601548-Human-RMC83 AF373605-Human-RMC100
FJ152136-Human-US6668-G1P8
X76778-Human-v158 Lineages E(E4/ 11)
X76783-Human-v61
AB009627-Avian-PO13
0.1
75
X76781-Human-v47
0.02
Fig. 4 continued
within lineage III (Fig. 4a), suggesting the recent introduction of new G12 strains into South America. To date, most of the G12 strains circulating worldwide belong to the VP7 lineage III and are associated with the P[6] and/or P[8] types [27]; however, the G12 strains that are circulating in Korea, Japan, Argentina, Brazil and Paraguay are associated with P[9] and cluster into lineage II of the VP7 gene [6, 25, 26, 32]. Based on information from epidemiological studies, i.e. the fact that the P[9] type is highly prevalent in the feline population [30], results obtained by hybridization analysis, sequence analysis of the VP4, VP7, NSP1, NSP4 and NSP5 genes [6, 25, 26, 38], and analysis of the complete genome of the T152 strain (a G12P[9] strain isolated from humans but with
feline characteristics [27]), it appears that the emergence of these G12P[9] strains can be explained by interspecies transmission from the feline to the human population. However, since no strains bearing the G12P[9] combination have been found in the feline population so far, the origin of the G12 strains circulating in the human population worldwide still remains obscure. Thus, improvements in the surveillance of animal rotavirus strains may help to elucidate this. Acknowledgments We thank Maria Victoria Gonzalez Eusevi for her critical reading and editing of the manuscript. Conflict of interest statement interest to declare.
The authors have no conflicts of
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532
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