CIRCULATING PROFILE OF ROTAVIRUS A, G AND P GENOTYPES BEFORE AND AFTER VACCINE INTRODUCTION IN THE BRAZILIAN MID-WEST 1986-2015

Abstract

Resumo

The purpose of this study was to perform a comparative analysis of Rotavirus A (RVA) G and P genotypes circulating in the Brazilian Mid-West in the period 1986-2015. Seven studies conducted from 1986 to 2009 were included, as well as fecal samples obtained in the period 2014-2015. RVA was screened by ELISA and/or PAGE; genotyping by conventional RT-PCR and/or genomic sequencing. A temporal variation in the predominance of G genotypes mainly G1 and G2 with G9 and G12 emergence was observed. Even with vaccination, RVA continues
to circulate in the population, requiring continuous virus monitoring.

Full text

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Rev Patol Trop Vol. 46 (1): 105-112. jan.-mar. 2017
doi: 10.5216/rpt.v46i1.46300
Departamento de Microbiologia, Imunologia, Parasitologia e Patologia Geral, Instituto de Patologia Tropical e Saúde Pública,
Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
Corresponding author: Dra. Divina das Dôres de Paula Cardoso. Rua 235 s/n, esquina com 1ª Avenida, Setor Universitário, CEP
74605-050 Goiânia, Goiás, Brazil. E-mail: dcardoso@ufg.br
Received for publication: 27/12/2016. Reviewed: 28/3/2017. Accepted: 29/3/2017.
short report
CIrCULAtING proFILe oF rotAVIrUs A,
G AND p GeNotYpes BeFore AND AFter
VACCINe INtroDUCtIoN IN the BrAZILIAN
MID-West 1986-2015
Tâmera Nunes Vieira Almeida, Teresinha Teixeira de Sousa, Menira Souza,
Fabíola Souza Fiaccadori, Kareem Rady Badr and Divina das Dôres de Paula
Cardoso
ABSTRACT
The purpose of this study was to perform a comparative analysis of Rotavirus A (RVA) G and
P genotypes circulating in the Brazilian Mid-West in the period 1986-2015. Seven studies
conducted from 1986 to 2009 were included, as well as fecal samples obtained in the period
2014-2015. RVA was screened by ELISA and/or PAGE; genotyping by conventional RT-PCR
and/or genomic sequencing. A temporal variation in the predominance of G genotypes mainly
G1 and G2 with G9 and G12 emergence was observed. Even with vaccination, RVA continues
to circulate in the population, requiring continuous virus monitoring.
KEY WORDS: Rotavirus infections; genotype; vaccination.
Rotavirus A (RVA) is an important causative agent of acute
gastroenteritis (AGE) mainly affecting infants. The viral particle is non-
enveloped and formed by three concentric protein layers that surround 11
segments of double-stranded RNA (dsRNA) (Estes & Greenberg, 2013).
Each RVA genomic segment has extensive variability, which may
reect in antigenic diversity of the respective proteins, mainly VP7 and VP4
proteins that comprise the outer layer. RVA has been classied in a binary
system represented by the combination of G genotypes (VP7) and P (VP4)
genotypes (Estes & Greenberg, 2013). There are, so far, 32G (G1-G32) and
47P (P[1]-P[47]) genotypes described (RCWG, 2016). In humans, the most
common combinations are G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and
G12P[8] (Santos & Hoshino, 2005, Dóró et al., 2014).

106 Rev Patol Trop Vol. 46 (1): 105-112. jan.-mar. 2017
Worldwide G1 and P[8] have been the predominant genotypes (Santos
& Hoshino, 2005), being replaced at regular intervals, especially by G2 and P[4]
(Dóró et al., 2014), as well as other emerging genotypes such as G5 and G9
(Santos & Hoshino, 2005). Many studies of the genotypic prole of RVA, in the
Brazilian Mid-West, have been conducted since 1986, detecting the occurrence
of distinct G and P genotypes with changes over time, notably G1P[8], G2P[4]
and G9P[8] (Cardoso et al. 2000, Souza et al. 2003, Costa et al. 2004, Andreasi
et al. 2007, Munford et al. 2009, Borges et al. 2011, Almeida et al. 2015).
Because of the severity of acute gastroenteritis caused by RVA,
especially in children under ve years of age, there have been two licensed
vaccines available for use in Brazil since 2006: RotarixTM (Glaxo Smith Kline)
and RotaTeqTM (Merck Sharp & Dohme). RotarixTM has also been included in
the Brazilian National Immunization Program (Dóró et al., 2014).
Studies conducted in the Brazilian Mid-West have shown a temporal
variation in the predominance of certain G and P genotypes, characterizing the
occurrence of an antigenic shift (Cardoso et al., 2000, Souza et al., 2003, Costa
et al., 2004, Andreasi et al., 2007, Munford et al., 2009, Borges et al., 2011,
Almeida et al., 2015).
Considering that the pediatric population in Brazil has access to both
RVA vaccines, this study presents the genotypic variability of RVA in the region,
in the pre- and post-vaccine periods.
In order to analyze the circulation of G and P genotypes in the Brazilian
Mid-West, data was compared from studies conducted in the Mid-West in the
pre-vaccine period (Cardoso et al., 2000, Souza et al., 2003, Costa et al., 2004,
Andreasi et al., 2007, Munford et al., 2009), and post-vaccine period (Munford
et al., 2009, Borges et al., 2011, Almeida et al., 2015), with data from samples
obtained in the period from 2014 to 2015.
In previous studies, fecal samples were collected from children with
AGE (Souza et al., 2003, Costa et al., 2004, Andreasi et al., 2007, Munford et
al., 2009, Almeida et al., 2015), without AGE (Borges et al., 2011), and from
children with or without AGE (Cardoso et al., 2000). These studies included
hospitalized children (Souza et al., 2003, Costa et al., 2004, Andreasi et al., 2007,
Munford et al., 2009, Almeida et al., 2015), hospitalized and seen in outpatient
clinics (Cardoso et al., 2000) and children that attended day-care centers (Borges
et al., 2011).
The study conducted during 2014-2015 included samples obtained from
children under ve years of age, with or without AGE symptoms hospitalized
in a child-care referral hospital in the state of Goiás (Hospital Materno Infantil
de Goiânia). One fecal sample was obtained from each child after the parent or
legal guardian signed the written consent form.
This study was approved by the Ethics Committee of Research in
the Hospital das Clínicas of the Universidade Federal de Goiás, (Protocol:
19948113.6.0000.5078).

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All the samples obtained in the period 1986-2009 were screened
by Enzyme-Linked Immunosorbent Assay (ELISA), according to the
manufacturer’s instructions and/or Polyacrylamide Gel Electrophoresis
(PAGE) using protocols previously described (Pereira et al., 1983).
RVA positive samples obtained in this period underwent G and P
genomic amplication by conventional RT-PCR. The dsRNA was extracted
using Trizol reagent (InvitrogenTM/Life Technologies, Foster City, USA),
following the manufacturer’s instructions. The reverse transcription of dsRNA
and the amplication reaction were performed using specic primer pairs for
the VP7 and VP4 encoding genes, previously described (Gouvea et al., 1990,
Gentsch et al., 1992). Negative (sterile water) and positive (RVA positive
samples) controls were also included in each run.
The amplication products were analyzed in 1.5% agarose gel,
0.5X TBE buffer (Tris-HCl-Borate-EDTA) with ethidium bromide staining,
compared to the 100 bp ladder (InvitrogenTM/Life Technologies, Foster City,
USA), and visualized under UV light (Vilbert Loumart). Fragments with an
expected size of 876 and 1.062 bp (VP4 and VP7, respectively) were considered
positive. All procedures were conducted in the proper environment in order to
avoid contamination.
For the samples obtained in 2014-2015, RVA screening was performed
using PAGE following a previously described protocol (Pereira et al., 1983).
RVA positive samples underwent genomic sequencing of genes encoding VP7
(G) and VP4 (P) proteins using the same primers and conditions of conventional
RT-PCR (Gouvea et al., 1990, Gentsch et al., 1992).
The genomic sequencing was performed using the BigDye Terminator
kit (Applied BiosystemsTM, USA) in automatic sequencer (ABIPrism 3130,
Applied BiosystemsTM, USA). The genotyping was performed by comparison
with sequences deposited in the National Center for Biotechnology Information
(NCBI) (https://www.ncbi.nlm.nih.gov).
RVA G and P genotypes were detected in previous studies conducted
in the Brazilian Mid-West, in 1986-2009, when G1 and P[8] genotypes proved
predominant among 474 RVA positive samples with positivity rate ranging
from 3.6 to 41.8% (Table 1).
Analyses regarding the collection period showed that in the pre-
vaccine period, G2 predominated in the years 1986-1995 and after that, G1
emerged. G5 was only detected in 1986-1995, and G9 emerged in 1998. Also
P[8] proved predominant in all studies and two P[9] samples were detected in
2005. In the post-vaccine period, G2 and P[4] were predominated until 2009.
In 146 samples with G and P combination, G1P[8] proved the most
frequent in the pre-vaccine period and after that G2P[4] samples emerged in
the post-vaccine period (Table 2).

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Table 1. Studies of Rotavirus A conducted in the Brazilian Mid-West, considering G and P genotypes from 1986 to 2009
Sampling
period
N%
Genotype G (VP7) Genotype P (VP4)
G1 G2 G3 G4 G5 G8 G9 MIX GNT P[4] P[6] P[8] P[9] MIX PNT Ref.
Pre-
vaccine
period
1986-1995 132 11.8 26 37 13 2 3 - - - 51 * * * * * * Cardoso et al. 2000
1998-2000 120 23.3 92 6 1 - - - 6 - 15 - 20 33 - - 67 Souza et al. 2003
2000-2002 77 37.2 38 - - 2 - - 21 - 16 - 3 23 - 16 35 Costa et al. 2004
2000-2004 85 23.2 49 - 1 5 - - 1 - 29 - - 37 - - 48 Andreasi et al. 2007
2005-2006a13 13.3 11 - - - - - 1 1 - - - 6 2 7 - Munford et al. 2009
Post-
vaccine
period
2005-2006b28 28.5 1 27 - - - - - - - 24 - 2 - - - Munford et al. 2009
2008-2008 08 3.6 - 7 - - - 1 - - - 6 - - - - 2 Borges et al. 2011
2008-2009 11 16.9 - 10 - - - - - - 1 4 - - - 1 6 Almeida et al. 2015
Total 474 217 87 15 9 3 1 29 1 112 34 23 101 2 24 158
a =data from 2005; b =data from 2006; N =total number of RVA positive samples; % =percentage of positive samples; MIX =co-detection of
genotypes; NT =non-typeable; * =not tested.

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Table 2. Combinations of G and P genotypes of Rotavirus A samples obtained from different studies conducted in the Brazilian
Mid-West from 1998 to 2009
Sampling
period
G (VP7) and P (VP4) combinations
N G1P[6] G1P[8] G2P[4] G2P[6] G2P[8] G3P[8] G4P[8] G8P[4] G9P[6] G9P[8] Ref.
Pre-
vaccine
period
1998-2000 46 13 25 - 1 2 - - - 5 - Souza et al. 2003
2000-2002 18 1 14 - - - - 1 - - 2 Costa et al. 2004
2000-2004 37 - 33 - - - 1 2 - - 1 Andreasi et al. 2007
2005-2006a 9 - 8 - - - - - - - 1 Munford et al. 2009
Post-
vaccine
period
2005-2006b26 - 1 24 - 1 - - - - - Munford et al. 2009
2008-2008 6 - - 5 - - - - 1 - - Borges et al. 2011
2008-2009 4 - - 4 - - - - - - - Almeida et al. 2015
Total 146 14 81 33 1 3 1 3 1 5 4
a = data from 2005; b = data from 2006; N = total number of RVA positive samples.

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In the study conducted in 2014-2015, in which 335 fecal samples were
analyzed, 134 were obtained from children with AGE and 201 from children
without AGE symptoms. Nine samples (2.6%) were RVA positive; eight were
from children with AGE and one sample from a child without AGE symptoms.
From the eight samples from children with AGE, four were G12P[8], one was
P[8], one G12 and the other two were non-typeable for G or P. The positive
sample from the child without AGE symptoms was P[8] and nontypeable G.
RVA vaccination has resulted in an important reduction in the number
of AGE cases and disease severity, and consequent hospitalization, around the
world (Kollaritsch et al., 2015). This situation has also been documented in
Brazil, including the Mid-West Region, where studies during the pre-vaccine
period showed detection rates of up to 37.2% (Cardoso et al., 2000, Souza et
al., 2003, Costa et al., 2004, Andreasi et al., 2007). In studies conducted after
the vaccine implementation, lower positive rates were noted (Borges et al.,
2011, Almeida et al., 2015).
It is noteworthy that lower positivity rates were observed in the study
performed in 1986-1995 (Cardoso et al., 2000) with children with and without
AGE and in the study performed in 2008 (Borges et al., 2011) which included
children without AGE, performed in the pre- and post-vaccine periods,
respectively.
In the 2009-2010 study (Munford et al., 2009) conducted with
hospitalized children with AGE, including samples from both periods, a higher
detection rate (41.8%) was observed. This data contrasts with the literature and
also the results from the study conducted in the period 2014-2015 when 2.6%
positivity for RVA was observed, even considering only the AGE population
(6.0%).
It was supposed that this study (2014-1015) would be limited due to the
use of a single technique (PAGE) to screen RVA. Although this methodology
presents good specicity, its sensitivity is lower than other techniques like
ELISA. Even so, the reduction of RVA detection may be related to vaccine
benets in the Mid-West Region.
Regarding G and P combinations, a predominance of G1P[8] samples
from the 1998-2004, pre-vaccine period was observed (Souza et al., 2003,
Costa et al., 2004, Andreasi et al., 2007), and the emergence of G2P[4] samples
between 2006 and 2009 in the post-vaccine period (Munford et al., 2009,
Borges et al., 2011, Almeida et al., 2015). Additionally, in spite of the time
lapse, the 2014-2015 sampling, reveals the G12P[8] combination. These data
corroborated previous studies in reinforcing the uctuation of RVA genotypes,
as well as the emergence of G12P[8] (Santos & Hoshino, 2005, Dóró et al.,
2014).
Additionally, considering samples collected between 2006 and 2009,
post-vaccine perid, it was observed the emergence of G2P[4] samples (Munford
et al., 2009, Borges et al., 2011, Almeida et al., 2015). In spite of the time lapse,

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the 2014-2015 sampling, reveals the G12P[8] combination, which corroborates
previous studies reinforcing the uctuation of RVA genotypes, as well as the
emergence of these genotypes (Dóró et al., 2014).
These data indicate a regular tendency to uctuation in RVA G and P
genotypes in the human population. This situation cannot be solely attributed
to vaccination since RVA may mutate over relatively short period of time
resulting in shifts in the circulation of predominant genotypes over time.
Therefore, despite the benets of vaccines, considering the reduction
of RVA positivity and hospitalization cases of AGE, the virus continues to
circulate, with the same vaccine genotypes or in a different one, such as
G12P[8]. Therefore, continuous monitoring of RVA samples circulating among
the human population remains important.
Finally, the continuity of studies that focus on RVA dynamics in the
human population may help to predict changes in the genotypic prole of
the circulating samples and in determining preventive measures such as the
development of vaccines from the predominant genotypes over time.
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