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MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DE GOIÁS
INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA
PAULA ANDREIA SILVA
CARACTERIZAÇÃO MOLECULAR DE ASTROVÍRUS PROVENIENTES DE
AMOSTRAS FECAIS DE CRIANÇAS DA REGIÃO CENTRO-OESTE DO BRASIL
Orientadora:
Prof
a
. Dr
a
. Divina das Dôres de Paula Cardoso
Tese de Doutorado
GOIÂNIA-GO, 2005
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UNIVERSIDADE FEDERAL DE GOIÁS
INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PÚBLICA
PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA TROPICAL
PAULA ANDREIA SILVA
CARACTERIZAÇÃO MOLECULAR DE ASTROVÍRUS PROVENIENTES DE
AMOSTRAS FECAIS DE CRIANÇAS DA REGIÃO CENTRO-OESTE DO BRASIL
Orientadora:
Prof
a
. Dr
a
. Divina das Dôres de Paula Cardoso
Tese de Doutorado submetida ao
PPGMT/IPTDP/UFG como requisito parcial
para obtenção do Grau de Doutor em
Medicina Tropical, área de concentração -
Microbiologia.
Auxilio financeiro: CNPq nº 471968/03-0 e 520729/99-4
GOIÂNIA-GO, 2005
2
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(GPT/BC/UFG)
Silva, Paula Andreia.
S586c Caracterização molecular de astrovírus provenientes
de amostras fecais de crianças da região da região Centro-
Oeste do Brasil. - Goiânia, 2005.
58 f. : il.
Orientadora: Divina das Dôres de Paula Cardoso.
Tese (Doutorado) – Universidade Federal de Goiás,
Instituto de Patologia Tropical e Saúde Pública, 2005.
Bibliografia : f. 47.
1. Gastroenterite – Região Centro-Oeste 2. Vírus (As-
trovírus) - Gastroenterite I. Cardoso, Divina das Dores
de Paula II. Universidade Federal de Goiás. Instituto de
Patologia Tropical e Saúde Pública II. Título.
CDU: 616.34-002(817.3)
3
SUMÁRIO
Lista de Siglas e Abreviaturas..................................................................................................5
Lista de Figuras........................................................................................................................6
Resumo.....................................................................................................................................7
Abstract....................................................................................................................................9
1. Introdução..........................................................................................................................10
1.1. Gastroenterite..................................................................................................................10
1.2. Astrovírus........................................................................................................................11
1.2.1. Histórico.......................................................................................................................11
1.2.2. Classificação e Morfologia..........................................................................................12
1.2.3. Características Genômicas e Protéicas........................................................................13
1.2.4. Diagnóstico Laboratorial.............................................................................................17
1.2.5. Métodos de Genotipagem...........................................................................................19
1.2.6. Epidemiologia.............................................................................................................21
2. Justificativa.......................................................................................................................24
3. Objetivos...........................................................................................................................25
4 Artigo I...............................................................................................................................26
5. Artigo II.............................................................................................................................48
6. Conclusões Gerais.............................................................................................................62
7. Referências Bibliográficas................................................................................................63
4
LISTA DE SIGLAS E ABREVIATURAS
A
CaCo-2
DNA
EIE
GenBank
HastV-1 a -8
HEK
IME
Kb
kDa
LLC-MK2
ME
NASBA
nm
ORF
PLC/PRF/5
RNA
RT-PCR
ssRNA
%
Adenina
Linhagem celular Human Colon Adenocarcinoma (Adenocarcinoma de cólon humano)
Ácido desoxirribonucléico
Ensaio imunoenzimático
Banco de seqüências genéticas
Astrovírus humanos tipos 1 a 8
Linhagem celular Primary Human Embryonic Kidney (Primária de rim embrionário humano)
Imunomicroscopia eletrônica
Kilobases
Kilodaltons
Linhagem celular Rhesus Monkey Kidney Epithelial (Epitelial de rim de macaco rhesus)
Microscopia eletrônica
Nucleic Acid Sequence-Based Amplification (Amplificação baseada em seqüências de ácido
nucléico)
Nanômetro
Open Reading Frame (quadro aberto de leitura)
Linhagem celular Human Liver Hepatoma (hepatoma humano)
Ácido ribonucléico
Reverse Transcriptase-Polymerase Chain Reaction (Reação em cadeia pela polimerase pós-
transcrição reversa)
single strand RNA (RNA de fita simples)
Percentagem
LISTA DE FIGURAS
5
Figura 1 Microscopia eletrônica de astrovírus humano obtido de fezes de crianças
com gastroenterite.
4
Figura 2 Organização genômica de astrovírus. 6
Figura 3 Associação entre sorotipos e genótipos de astrovírus. Árvore filogenética
construída a partir de 348 nucleotídeos da região 5’ do ORF 2,
considerando amostras depositadas no GenBank.
7
RESUMO
6
Os astrovírus humanos (HAstV) são reconhecidos mundialmente como importante causa
de gastroenterite em crianças. O presente estudo apresenta a distribuição dos genótipos de
astrovirus em três cidades da região Centro-Oeste do Brasil (Brasília, Goiânia e Campo Grande),
além da análise filogenética de três regiões do genoma viral realizada em 20 amostras. Cinqüenta
e sete amostras de astrovírus provenientes de crianças menores de cinco anos de idade com
diarréia (n=56) e sem diarréia (n=1) foram identificados através da reação em cadeia pela
polimerase pós-transcrição reversa (RT-PCR). Todas as amostras foram submetidas à
genotipagem utilizando a nested RT-PCR e/ou seqüênciamento genômico da região 5’ da ORF 2,
tendo sido detectado a ocorrência de todos os genótipos de astrovírus, HAstV-1 (42.8 %), HAstV-
2 (23.2 %), HAstV-3 (3.6 %), HAstV-4 (14.3 %) e HAstV-5, -6, -7 e -8 (1.8 % cada), em
amostras diarréicas. A amostra positiva não diarréica era proveniente de Brasília e foi
caracterizada como HAstV-2. Em Goiânia e Campo Grande, o HAstV-1 foi o mais freqüente,
enquanto que em Brasília o HAstV-2 foi o genótipo predominante. Adicionalmente, os dados
mostraram ocorrência de mudança na predominância dos genótipos de astrovirus em relação aos
anos de estudo. Em Brasília, houve maior freqüência do HAstV-4 nos anos de 1994 e 1995, sendo
substituído pelo HAstV-2 em 1996, enquanto que em Campo Grande ocorreu predominância do
HAstV-2 em 2000 e 2002, sendo substituído pelo HAstV-1 em 2003. Os diferentes genótipos
foram detectados em crianças de todas as faixas etárias, não tendo sido observada correlação da
idade com qualquer genótipo. A análise filogenética de seqüências das ORFs 1b e 2 diferenciaram
os astrovírus em oito grupos filogenéticos, apesar da ORF 1b apresentar pequena distância
genética entre eles. Seqüências obtidas da ORF 1a classificaram todas as amostras analisadas em
genogrupo A. Foi realizado análise total do genoma de duas amostras pertencentes ao genótipo 4
e genótipo 5, as quais foram pela primeira vez depositadas no GenBank.
7
ABSTRACT
Human astroviruses (HAstV) are recognized as important agent of gastroenteritis in young
children worldwide. The present study describes the astroviruses genotypes distribution in three
8
locations of Central West region of Brazil (Brasília, Goiânia and Campo Grande) besides the
phylogenetic analysis of three different viral regions realized in 20 samples. Fifty seven
astrovirus samples from children up to five years old with (n=56) and without diarrhea (n=1)
were detected by RT-PCR. All samples were submitted to genotyping by nested RT-PCR and/or
genomic sequencing of 5’end region of ORF 2 and all HAstV genotypes, HAstV-1 (42.8 %),
HAstV-2 (23.2 %), HAstV-3 (3.6 %), HAstV-4 (14.3 %) and HAstV-5, -6, -7 and -8 (1.8 % each)
were observed, in diarrheic samples. The positive non-diarrheic sample was picked up in Brasília
and it was characterized as HAstV-2. In Goiânia and Campo Grande, HAstV-1 was the most
frequent genotype detected, while in Brasília HAstV-2 was predominant. Additionally, our data
showed the occurrence of a change in genotypes predominance in relation to the year. In Brasília
there was a high frequency of HastV-4 in the years of 1994 and 1995 with substitution of HastV-2
in 1996, while in Campo Grande HAstV-2 was predominat in 2000 and 2002 with substitution of
HastV-1 in 2003. The different astroviruses genotypes were detected in children of all age groups
and it was not observed correlation with any genotype. Phylogenetic analysis of ORF 1b and 2
sequences are used to differentiate astrovirus samples in eight groups, in spite of ORF 1b has
presented small genetic distance between them. Sequences from ORF 1a characterized all
analysed samples in genogroup A. Total genome analysis of two samples belonging to genotypes
4 and 5 were performed and, for the first time, their complete genome sequences were published
in GenBank.
1. INTRODUÇÃO
1.1 GASTROENTERITE
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A gastroenterite é um processo inflamatório da mucosa do trato gastrointestinal,
clinicamente, caracterizada por vômitos e/ou diarréia (Webb & Starr 2005). Os primeiros relatos
de doenças diarréicas em humanos remontam ao Egito antigo, através de hieróglifos datados de
3300 a.C., possuindo, até os dias atuais, grande importância para a saúde pública (Kapikian 1996,
Walter & Mitchell 2003).
Até o início da década de 70, a etiologia da grande maioria dos quadros de gastroenterites
permanecia desconhecida até que foi identificado o vírus Norwalk em amostras diarréicas,
utilizando-se a microscopia eletrônica (Kapikian et al. 1972). Posteriormente, outros vírus foram
identificados (Bishop et al. 1973) e, atualmente, considera-se que a maioria das gastroenterites
infantis seja causada por vírus pertencentes a quatro diferentes famílias: Reoviridae (rotavirus),
Caliciviridae (norovirus e sapovirus), Astroviridae (astrovirus) e Adenoviridae (adenovirus).
Outros vírus como coronavírus, torovírus, picobirnavírus e aichivírus também têm sido
relacionados com gastroenterite em humanos (Glass et al. 2001, Yamashita et al. 2001).
As doenças diarréicas são uma das mais importantes causas de mortalidade e morbidade
infantis, especialmente em países em desenvolvimento (Nikhil Thapar 2004). Em 1982, com base
em estudos realizados entre as décadas de 50 e 70, foi estimado que 4,6 milhões de crianças
morriam anualmente vítimas de diarréia (Snyder & Merson 1982). Após a introdução da terapia
de rehidratação oral e o aumento do conhecimento a respeito da patogênese da diarréia, uma
revisão de estudos conduzidos nos anos 80 sugeriu que mortes por diarréia teriam diminuído para
aproximadamente 3,3 milhões anuais (Bern et al. 1992). Estimativas mais recentes sugerem que
2,5 milhões de crianças ainda morrem todos os anos devido a gastroenterite (Kosek et al. 2003).
Considera-se, por outro lado, que o número de mortes infantis por gastroenterites é maior nos
países em desenvolvimento devido a alguns fatores característicos destas regiões, como a
precariedade de saneamento básico, a falta de água potável e higiene inadequada, bem como o
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atendimento médico inadequado (UNICEF 2002). Além disso, a desnutrição também foi
associada a 50% de todas as mortes dentre crianças destes países (Rice et al. 2000).
1.2 ASTROVÍRUS
1.2.1 HISTÓRICO
Em junho de 1975, Appleton e Higgins descreveram um pequeno vírus esférico que
possuía tamanho e morfologia diferentes dos previamente descritos para vírus Norwalk e
rotavírus. Tais partículas foram visualizadas, utilizando a microscopia eletrônica (ME), a partir de
amostras fecais provenientes de um surto diarréico (Appleton & Higgins 1975).
Em setembro do mesmo ano, Madeley e Cosgrove também descreveram partículas virais,
visualizadas por microscopia eletrônica, provenientes de amostras fecais de crianças com diarréia
e as nomearam de astrovírus. O termo astrovírus (do grego astron, que significa estrela) foi
utilizado para descrever um pequeno vírus esférico que apresentava cinco ou seis pontas
assemelhando-se a uma estrela (Madeley & Cosgrove 1975a). Posteriormente, esta pequena
partícula foi observada também em amostras fecais de crianças hospitalizadas com diarréia e em
surtos de gastroenterite em recém-nascidos (Madeley & Cosgrove 1975b).
Alguns anos depois, com o desenvolvimento de imunoreagentes específicos, foi possível
demonstrar que as partículas descritas por Appleton e Higgins eram as mesmas descritas por
Madeley e Cosgrove, ou seja, astrovírus (Caul & Appleton 1982).
Em 1981, foi observado que esses vírus multiplicavam-se em culturas celulares,
característica esta que também os distinguiram dos calicivírus (Lee & Kurtz 1981). Estes
experimentos proporcionaram, em 1984, o reconhecimento de cinco sorotipos de astrovírus
humanos (Kurtz & Lee 1984), bem como a produção, em 1988, de reagentes utilizados em reação
11
sorológica de ensaio imunoenzimático (EIA) (Herrmann et al. 1988) e ainda, em 1993, o
seqüênciamento total do genoma (Jiang et al. 1993). Em 1994, foi sugerida a existência de mais
dois sorotipos de astrovírus, sorotipos 6 e 7 (Lee & Kurtz 1994) e, em 1995, foi publicada a
seqüência de nucleotídeos da região do ORF 2 do sorotipo 8, no GenBank - banco de seqüências
genéticas, disponível em: http://www.ncbi.nlm.nih.gov sob o número de acesso Z66541l.
1.2.2 CLASSIFICAÇÃO E MORFOLOGIA
Os astrovírus pertencem à família Astroviridae, a qual é dividida em dois gêneros:
Mamastrovirus e Avastrovirus. O gênero Mamastrovirus inclui todos os astrovírus humanos
(HAstV 1 a 8) bem como astrovírus que infectam animais como bovinos (bovine astrovirus 1 e
2), felinos (feline astrovirus 1), suínos (porcine astrovirus 1), ovinos (ovine astrovirus 1) e martas
(mink astrovirus 1). O gênero Avastrovirus inclui astrovírus que infectam patos (duck astrovirus
1), perus (turkey astrovirus 1 e 2) e galinhas (avian nephritis virus 1 e 2) (Walter & Mitchell
2003).
Estes vírus apresentam morfologia esférica com diâmetro de 28 a 40 nm e não possuem
envoltório lipídico. São formados por um capsídeo icosaédrico com morfologia característica,
semelhante a uma estrela de 5 ou 6 pontas, observada em apenas 10% das partículas virais
(Matsui et al. 2001).
12
Figura 1: Microscopia eletrônica de astrovírus humano obtido de fezes de crianças com
gastroenterite. Disponível em: http://aapredbook.aappublications.org/week/010_01.jpg
1.2.3 CARACTERÍSTICAS GENÔMICAS E PROTÉICAS
O genoma dos astrovírus é constituído por uma molécula única de RNA, com polaridade
positiva, de aproximadamente 6,8 kilobases (kb) excluindo-se a cauda poli (A) na extremidade 3’.
Em ambas as extremidades do genoma existem regiões não codificantes que variam entre 80 e 85
nucleotídeos (nt), dependendo do sorotipo. A organização genômica dos astrovírus apresenta três
quadros abertos de leitura (ORF – open reading frame) denominados ORF 1a, 1b e 2 (Jiang et al.
1993). Os dois primeiros localizam-se na extremidade 5’ do genoma e codificam proteínas não
estruturais. O ORF 2 está localizado na extremidade 3’ e codifica proteínas do capsídeo viral
(Lewis et al. 1994) (Figura 2). O número de nucleotídeos de cada região, bem como o tamanho
das respectivas proteínas codificadas por elas, são variáveis dependendo de cada sorotipo de
astrovírus, definidos em reações sorológicas (Jiang et al. 1993, Willcocks et al. 1994, Mendez-
Toss et al. 2000, Oh & Schreier 2001).
13
O ORF 1a contém de 2763 a 2784 nt, sendo que 61 a 73 nt sobrepõem o ORF 1b. O ORF
1a codifica uma poliproteína de 920 a 935 aminoácidos, que contém motivos para serina protease,
além de quatro prováveis proteínas transmembranas, e uma outra que sinaliza a localização
nuclear (Jiang et al. 1993, Carter & Willcocks 1996).
A análise filogenética de uma região do ORF 1a contendo 289 nucleotídeos (posição 1182
a 1470, referente à seqüência depositada no GenBank sob o número de acesso L13745) definiu a
existência de dois genogrupos distintos, denominados A e B. Estes genogrupos foram
relacionados com os sorotipos, sendo que o genogrupo A inclui os sorotipos 1-5 e 8 e o
genogrupo B os sorotipos 6 e 7 (Belliot et al. 1997).
Figura 2: Organização genômica de astrovírus. A localização dos três ORFs e ainda, das
prováveis proteínas transmembranas (MB), serina protease (Pro), sinal de localização nuclear
(NLS), estrutura do frameshifting ribossomal (RFS) e a RNA dependente RNA polimerase (Pol)
estão indicados. Adaptado de Jiang et al. 1993.
14
O ORF 1b é constituído por 1548 a 1560 nt e também se sobrepõe ao ORF 2 em 8 nt. O
ORF 1b codifica uma poliproteína de 515 a 519 aminoácidos, a qual contém motivos para a RNA
polimerase RNA dependente. Esta poliproteína é expressa pelo mecanismo de frameshifting
ribossomal, que consiste em uma mudança de leitura dos códons no ribossomo, que ocorre no
final da tradução do ORF 1a, dando início à tradução do ORF 1b. Desta maneira, o produto da
tradução do ORF 1b é uma fusão de proteínas codificadas pelo ORF 1a e 1b (Jiang et al. 1993,
Lewis & Matsui 1996). Para que ocorra o frameshifting ribossomal é necessário que o genoma
apresente algumas características como uma determinada seqüência de sete nucleotídeos (A
6
C)
seguida de uma conformação estrutural do RNA em forma de grampo (Marczinke et al. 1994).
Tais características podem ser observadas na região de sobreposição entre o ORF 1a e ORF 1b do
astrovírus (Jiang et al. 1993).
Ademais, a análise filogenética de 316 nt da região 3’ do ORF 1b (posição 3724 a 4039,
referente à seqüência depositada no GenBank sob o número de acesso L13745) mostrou que,
embora a distância genética entre os diferentes tipos de astrovírus seja pequena, os astrovírus
podem ser agrupados em 8 tipos (Belliot et al. 1997).
O ORF 2 é constituído de 2316 a 2391 nt e pode ser encontrado sob a forma genômica ou
subgenômica (Sanchez-Fauquier et al. 1994). Esta região codifica uma poliproteína de 782 a 796
aminoácidos, a qual é processada dando origem a pelo menos três proteínas, de 34, 29 e 26
kilodaltons (kDa), que formam o capsídeo viral (Bass & Qiu 2000).
A análise filogenética de seqüências localizadas tanto na extremidade 5’ quanto 3’ do
ORF 2 (posição 4526 a 4974 e 6513 a 6781, respectivamente, referente à seqüência depositada no
GenBank sob o número de acesso L23513), tem mostrado que, em ambas extremidades, os
astrovírus apresentam diferenças genômicas que permitem sua caracterização em 8 genótipos
(Mitchell et al. 1995, Noel et al. 1995, Saito et al. 1995, Jakab et al. 2003, Nadan et al. 2003).
15
Além disso, tem sido observado que esses genótipos coincidem com os sorotipos definidos por
estudos sorológicos ( Nadan et al. 2003, Espul et al. 2004) (Figura 3).
Figura 3: Associação entre sorotipos e genótipos de astrovirus. Árvore filogenética construída a
partir de 348 nucleotídeos da região 5’ do ORF 2, considerando amostras depositadas no
GenBank (Banco de seqüências genéticas, disponível em: http://www.ncbi.nlm.nih.gov), cujos
números de acesso estão indicados na figura. O alinhamento das seqüências e análise filogenética
foi realizado utilizando os programas CLUSTAL W e Phylogeny Inference Package (PHYLIP),
versão 3.57c (Felsenstein 1985, Felsenstein 1993).
1.2.4 DIAGNÓSTICO LABORATORIAL
16
O diagnóstico da infecção por astrovírus tem sido realizado através de vários métodos
baseados tanto nas características morfológicas quanto antigênicas e genômicas do vírus. Dentre
esses, estão a microscopia eletrônica (ME) (Ong & Chandran 2005), a imunomicroscopia
eletrônica (IME) (Herrmann et al. 1990a), o ensaio imunoenzimático (EIE) (Herrmann et al.
1990b), a aglutinação em látex (Komoriya et al. 2003), o isolamento viral em culturas celulares
(Lee & Kurtz 1981) e a reação em cadeia pela polimerase pós-transcrição reversa (RT-PCR)
(Jonassen et al. 1995).
A técnica da ME foi a primeira a ser utilizada para detecção dos astrovírus e consiste na
observação direta da partícula viral. A característica morfológica semelhante a uma estrela,
devida a qual os astrovírus foram nomeados, é evidente, como referido, em apenas 10% das
partículas virais, o que torna a identificação viral difícil, além de diminuir a sensibilidade deste
método (Walter & Mitchell 2003). Pela IME, identifica-se o vírus por utilização de anticorpos
específicos, com a vantagem do aumento da sensibilidade do método (Berthiaume et al. 1981).
Recentemente, foi proposto um método de ME semi-automático que se baseia nas características
bidimensionais e no cálculo estimado da densidade da partícula viral para a sua identificação
(Ong & Chandran 2005).
O ensaio imunoenzimático é admitido ser 10 a 100 vezes mais sensível que a microscopia
eletrônica (Glass et al. 1996). Através dessa metodologia, é possível a detecção dos oito sorotipos
de astrovírus humano (Herrmann et al. 1988) e, atualmente, vários kits comerciais de diagnóstico
estão disponíveis.
Testes de aglutinação em látex têm sido desenvolvidos e são considerados procedimentos
simples, rápidos e menos dispendiosos, mas com menor sensibilidade que os ensaios
imunoenzimáticos (Kohno et al. 2000, Komoriya et al. 2003).
17
O isolamento viral em culturas celulares é uma metodologia que proporciona a
multiplicação do vírus, e deve ser seguida da identificação viral pela utilização de anticorpos
específicos bem como pela RT-PCR. Para o cultivo de astrovírus, algumas linhagens celulares
como as de células de hepatoma humano (PLC/PRF/5) (Taylor et al. 1997), células embrionárias
de rim humano (HEK) (Lee & Kurtz 1981), células embrionárias de rim de macaco Rhesus
(LLCMK2) (Herrmann et al. 1988) e células de carcinoma de cólon humano (CaCo 2) (Willcocks
et al. 1990) têm sido empregadas. Para o diagnóstico de rotina, esta técnica não é a de escolha,
pois é considerada trabalhosa e demorada, além da necessidade da presença de partículas virais
infecciosas nas amostras fecais, o que diminui sua sensibilidade.
Com o desenvolvimento de métodos moleculares como a RT-PCR, técnicas de
hibridização e NASBA (amplificação baseada em seqüências de ácido nucléico), que apresentam
alta especificidade e sensibilidade, houve um grande avanço na detecção e, principalmente, na
caracterização do genoma dos astrovírus (Willcocks et al. 1991, Gunson et al. 2003, Tai et al.
2003).
A técnica molecular da RT-PCR tem sido considerada, até o momento, a metodologia
mais sensível para a detecção dos astrovírus, por permitir a obtenção de resultados positivos a
partir de pequena concentração viral nas amostras fecais (Mitchell et al. 1995, Gunson et al.
2003). Esta técnica consiste na síntese de um DNA complementar a partir do RNA viral, seguido
da amplificação de uma região específica deste genoma. Várias regiões conservadas do ORF 1a
(Guix et al. 2002, Marie-Cardine et al. 2002), do ORF 1b (Lewis et al. 1994) ou da extremidade
3’ não codificante dos astrovírus (Monceyron et al. 1997) têm sido escolhidas como alvo para
amplificação pela técnica da RT-PCR. Outras regiões relativamente conservadas do ORF 2,
também têm sido utilizadas e, embora possam ser regiões com menor poder de sensibilidade para
18
a técnica, possibilitam a informação a respeito do genótipo viral (Palombo & Bishop 1996,
Mustafa et al. 2000, das Dores de Paula Cardoso et al. 2002).
As técnicas de detecção de astrovírus que utilizam a hibridização consistem basicamente
na ligação de sondas específicas ao RNA viral, seguido da revelação da reação que pode ser feita
utilizando enzimas ou radioisótopos (Willcocks et al. 1991). Estas reações de hibridização,
geralmente, apresentam a desvantagem de serem laboriosas e demandarem tempo para a sua
execução.
A técnica molecular NASBA consiste em também amplificar uma região do genoma viral,
mas com a peculiaridade de utilizar enzimas que permitem a amplificação de RNA em condições
isotermais. Esta técnica, quando comparada à RT-PCR, apresenta a vantagem de gerar o mesmo
número de cópias em menor espaço de tempo, além de não necessitar da etapa de “desnaturação”,
o que evita contaminações. Em contrapartida, a NASBA apresenta a desvantagem da
impossibilidade do controle do número de ciclos da reação e do aumento da temperatura,
utilizado para evitar reações inespecíficas (Tai et al. 2003).
1.2.5 MÉTODOS DE GENOTIPAGEM
Na década de 80, a caracterização antigênica dos astrovírus foi primeiramente realizada
através das técnicas de imunofluorescência, soroneutralização, bem como por imunomicroscopia
eletrônica (Lee & Kurtz 1982, Kurtz & Lee 1984, Hudson et al. 1989). A partir de então, o ensaio
imunoenzimático utilizando anticorpos específicos para cada sorotipo, a nested RT-PCR
utilizando iniciadores específicos e o seqüênciamento genômico também passaram a ser usados
como técnicas de soro/genotipagem de astrovírus (Noel et al. 1995, Mustafa et al. 1998).
Estudos comparativos, utilizando a técnica do ensaio imunoenzimático e do seqüênciamento
da região 3’ do ORF 2, mostraram que os grupos antigênicos dos astrovírus (sorotipos)
19
apresentavam correspondência com os grupos filogenéticos (genótipos) e, atualmente, baseado
nestes grupos, é considerada a existência dos oito sorotipos/genótipos de astrovírus (Noel et al.
1995, Mendez-Toss et al. 2000).
Por outro lado, cada região genômica de estudo gerou diferentes grupos filogenéticos, sendo
que análises destas diferentes regiões mostram que seqüências provenientes do ORF 1a
diferenciam os astrovírus em dois genogrupos e as provenientes do ORF 1b e 2 os diferenciam
em oito genótipos (Belliot et al. 1997). Em relação à distância genética entre seqüências do ORF
1b e 2, foi observado que o 1b apresenta maior homologia entre os genótipos e, portanto, a análise
do ORF 2 tem sido mais utilizada para a determinação dos genótipos de astrovírus devido a
maior variabilidade genética desta região (Belliot et al. 1997). Ainda nesse ORF, a análise de
seqüências das regiões 5’ e 3’ mostra que a região 3’ é a que apresenta maior distância genética
entre os genótipos (Matsui et al. 1998).
Atualmente, as técnicas mais utilizadas para a genotipagem de astrovírus são a nested RT-
PCR e o seqüênciamento genômico. Em 1998, a nested RT-PCR foi pela primeira vez utilizada
como metodologia para genotipagem de astrovírus pertencentes aos genótipos 1 ao 7 (Matsui et
al. 1998). Em 2000, o iniciador específico para o sorotipo 8 foi introduzido, permitindo então, a
caracterização de todos os genótipos de astrovírus (Sakamoto et al. 2000). Resumidamente, a
técnica consiste em, primeiramente, gerar fitas de DNA complementar a partir do RNA viral,
seguido pela amplificação da região transcrita utilizando um par de iniciador comum aos
diferentes genótipos. Na segunda amplificação, o iniciador antisense é comum aos genótipos,
enquanto o iniciador sense é específico. Assim sendo, a determinação dos genótipos é feita
através da observação do tamanho do produto de PCR esperado para cada genótipo (Sakamoto et
al. 2000).
20
O seqüênciamento genômico para a determinação dos genótipos de astrovírus tem sido
utilizado desde 1995 e a escolha da região a ser analisada é, como já referido, de fundamental
importância, pois cada região genômica apresenta diferentes grupos filogenéticos. O princípio da
técnica fundamenta-se basicamente na obtenção de vários fragmentos de DNA que, juntos,
representam todo o segmento a ser seqüenciado.
1.2.6 EPIDEMIOLOGIA
A importância médica dos astrovírus em humanos foi inicialmente comprovada por
Herrmann et al. (1991), que mostraram uma diferença estatisticamente significante em termos de
detecção viral em crianças com diarréia (8,6%) e sem diarréia (2%) (Herrmann et al. 1991).
Desde então, vários estudos foram realizados no intuito de detectar astrovírus como agentes
causadores de gastroenterites em crianças e dados de vários estudos têm demonstrado ser este
vírus o segundo agente mais comum dessa enfermidade (Giordano et al. 2001, Rodriguez-Baez et
al. 2002).
Em 1979, estudo realizado em voluntários comprovou a transmissão dos astrovírus pela
via fecal-oral (Kurtz et al. 1979). O contato pessoa a pessoa, associado ou não a fômites, bem
como a ingestão de água e alimentos contaminados, têm sido reportados como mecanismo de
transmissão desses vírus (Appleton 1987, Oishi et al. 1994, Abad et al. 2001, Oppermann et al.
2001, Pinto et al. 2001).
O aumento da ocorrência da infecção por astrovírus em determinada estação do ano pode ser
observado, mas há controvérsias entre os estudos quanto a uma possível sazonalidade, uma vez
que esta parece variar dependendo da região geográfica. De acordo com alguns estudos, a
infecção por astrovírus acontece com maior índice no inverno, similar à circulação de rotavírus
(Pang & Vesikari 1999, Qiao et al. 1999, Giordano et al. 2001). Em outros estudos, incluindo um
21
realizado em Goiânia-Goiás, os astrovírus apresentam um pico de detecção durante a primavera-
verão, diferente da circulação dos rotavírus (Guerrero et al. 1998, das Dores de Paula Cardoso et
al. 2002).
Estudos realizados em várias partes do mundo têm apresentado índices de detecção de
astrovírus que variam entre 2 a 26% dos casos analisados, dependendo da região e características
de cada estudo (Maldonado et al. 1998, Mustafa et al. 2000, das Dores de Paula Cardoso et al.
2002). Em países do continente asiático, como China, Japão e Paquistão, foi observado índice de
detecção viral de até 11,2%, embora um estudo realizado com crianças de uma creche no Japão
tenha mostrado percentual de 15,9% (Qiao et al. 1999, Liu et al. 2004, Phan et al. 2004, Akihara
et al. 2005). No continente europeu, investigações realizadas na Alemanha, Itália, Espanha,
França, bem como na Irlanda, Hungria e Finlândia relatam coeficientes de detecção de astrovírus
que variam entre 1,6 e 9% (Pang & Vesikari 1999, Foley et al. 2000, Dalton et al. 2002, Marie-
Cardine et al. 2002, Oh et al. 2003, Jakab et al. 2004, Galdiero et al. 2005). Nos Estados Unidos
da América, estudos têm associado os astrovírus com quadros diarréicos infantis em 3 a 10% dos
casos analisados, enquanto que no Canadá a estimativa é de menos que 1% (Shastri et al. 1998,
Waters et al. 2000, Denno et al. 2005).
Nos países africanos como África do Sul, Botsuana, Malaui e Nigéria, os índices de
positividade para os astrovírus têm sido estimados entre 1,9 e 6,7% (Steele et al. 1998, Cunliffe et
al 2002, Pennap et al. 2002, Basu et al. 2003). Dentre os países da América Latina, como
Argentina, Chile, Colômbia, Venezuela e México, os astrovírus têm sido detectados em 3,7 a
16,5% das amostras analisadas (Gaggero et al. 1998, Medina et al. 2000, Walter et al. 2001, Espul
et al. 2004). Por outro lado, em estudo realizado em crianças da área rural de Mayan, México,
reportou a associação dos astrovírus em 26% dos casos de gastroenterites analisados (Maldonado
et al. 1998)
22
No Brasil, a primeira publicação notificando infecções por astrovírus foi em 1991 e a
prevalência encontrada em crianças hospitalizadas provenientes da região de São Paulo foi de
4,8% (Stewien et al. 1991). Em 1993 e 2002, estudos semelhantes realizados também em São
Paulo e em Goiânia relataram prevalências de 3 e 2,8%, respectivamente (Stewien et al. 1993, das
Dores de Paula Cardoso et al. 2002,). Por outro lado, índice de 33% foi detectado durante um
surto em uma creche no Rio de Janeiro em 2001 (Silva et al. 2001).
Todos os oito sorotipos/genótipos de astrovírus têm sido detectados em diferentes países,
inclusive com circulação simultânea de vários deles na mesma região geográfica (Mustafa et al.
2000, Walter et al. 2001, Nadan et al. 2003). Na Holanda, um estudo de soroprevalência
utilizando anticorpos específicos para os sorotipos 1-7 mostrou que o sorotipo 1 foi o mais
prevalente, seguido pelos sorotipos 3 e 4, que apresentaram prevalência intermediária (50 a 70%),
os sorotipos 2 e 5 foram considerados como pouco prevalentes (30 a 40%), e os sorotipos 6 e 7
foram detectados com menor freqüência (10 a 20%) (Koopmans et al. 1998). Outros estudos
também relataram que o sorotipo 1 tem sido o mais comumente encontrado, seguido pelos
sorotipos 2, 3 e 4 que apresentaram baixas porcentagens de detecção, e pelos sorotipos 5, 6, 7 e 8,
que raramente foram identificados (Lee & Kurtz 1994, Noel & Cubitt 1994, Mustafa et al. 2000,
Nadan et al. 2003). Controversamente, em um estudo realizado na cidade do México foi
observado que o genótipo 2 foi o mais prevalente, seguido dos genótipos 4, 3, 1, 5 e 7 (Mendez-
Toss et al. 2004).
23
2. JUSTIFICATIVA
Como referido, os astrovírus são considerados importantes agentes causadores de diarréia
aguda em crianças menores de 5 anos de idade, o que indica a necessidade do desenvolvimento
de uma vacina eficaz contra este agente. Para tanto, é fundamental o conhecimento das
características moleculares das amostras circulantes em todo o mundo (Palombo & Bishop 1996).
No Brasil, existem poucos relatos a respeito da caracterização molecular dos astrovírus
humanos (Silva et al. 2001, das Dores de Paula Cardoso et al. 2002) e nenhum deles
proporcionaram informações que abrangessem a genotipagem a partir dos três ORFs virais. Por
outro lado, a determinação da seqüência total do genoma dos diferentes genótipos de astrovírus
tem sido publicada apenas para os genótipos 1, 2, 3 e 8 (Jiang et al. 1993, Willcocks et al. 1994,
Mendez-Toss et al. 2000, Oh & Schreier 2001).
Neste contexto, o presente estudo teve por finalidade a obtenção de informações referentes
à definição dos genótipos de astrovírus circulantes na Região Centro-Oeste do Brasil, além da
caracterização molecular realizada a partir do seqüênciamento de três diferentes regiões do
genoma dos astrovírus. Este trabalho teve ainda como proposta o seqüênciamento total de
amostras de astrovírus pertencentes a genótipos que ainda não foram depositados no GenBank.
Dessa forma, pretende-se que os dados obtidos no presente estudo tenham reflexo na
Saúde Pública e que, somados a outros, venham proporcionar informações que auxiliem nas
medidas de controle e prevenção da infecção por astrovírus na população.
24
3. OBJETIVOS
• Identificar os genótipos de amostras de astrovírus detectadas a partir de amostras
fecais provenientes de crianças da região Centro-Oeste do Brasil, através do
seqüênciamento genômico e/ou RT-PCR;
• Correlacionar os diferentes genótipos encontrados com a faixa etária da população
estudada;
• Analisar seqüências de nucleotídeos provenientes dos três ORFs do genoma do
astrovírus;
• Identificar os genogrupos das amostras virais analisadas;
• Obter a seqüência total do genoma de duas amostras de astrovírus pertencentes a
genótipos que ainda não foram publicados na literatura.
25
4. ARTIGO I – Artigo científico aceito para publicação na revista Archives of Virology.
Molecular characterization of human astroviruses isolated in Brazil, including
the complete sequences of astrovirus genotypes 4 and 5
P.A. Silva
1, 2*
, D.D.P. Cardoso
1
and E. Schreier
2
1
Laboratory of Virology, Institute of Pathology and Public Health, Federal University of Goiás,
Goiânia, Brazil
2
Robert Koch-Institute, Berlin, Germany
Running title: Characterization of human astroviruses isolated in Brazil
*Author’s address:
Paula Andreia Silva
Department of Molecular Epidemiology of Viral Pathogens
Robert Koch-Institute
Nordufer 20
13353 Berlin
GERMANY
e-mail: [email protected]; [email protected]
Phone: +49 1888 754 2379
Fax: +49 1888 754 2617
26
Summary
Human astroviruses (HAstV) are recognized as an important cause of gastroenteritis in
young children worldwide. This study describes the molecular characteristics of
astroviruses isolated in Brazil, using RT-PCR and molecular sequencing of segments of all
three viral ORFs. Genetic analysis of a 348-nucleotide segment from ORF 2 demonstrated
that the Brazilian isolates belong to HAstV genotypes 1 to 5 and 8. ORF 1b sequences
displayed a high degree of nucleotide identity even between different genotypes, which
disfavours HAstV genotyping in this region. ORF 1a sequence analysis classified all
Brazilian samples as genogroup A. The complete sequences of HAstV genotype 4 (putative
serotype 4) and genotype 5 (putative serotype 5) were determined for the first time.
Introduction
Gastrointestinal diseases belong to the most important causes of childhood morbidity and
mortality, especially in developing countries where they are the second most common cause of
death in children younger than 5 years [13, 25]. Enteric viruses, such as rota-, noro-, astro- and
adenoviruses have been recognized as important causes of acute gastroenteritis. Corona-,
picobirna-, and picornaviruses (enterovirus, Aichi virus) have also been shown to be an etiologic
agent of gastroenteritis [2, 9, 12, 38].
Human astroviruses (HAstV) were first described as a human pathogen in 1975 during an
outbreak of acute gastroenteritis among infants in the United Kingdom [18]. Since then, HAstV
infections have been shown to be a significant cause of gastrointestinal diseases [9, 34].
Epidemiological studies worldwide have reported astrovirus prevalence rates of 2 to 26 % among
children with acute gastroenteritis [5, 19, 23]
27
Astroviruses are spherical, non-enveloped 28 nm particles [18] belonging to the family
Astroviridae. Two genera have been described, Mamastrovirus and Avastrovirus [34]. The genus
Mamastrovirus includes all human astrovirus strains, as well as the feline, porcine, and ovine
astrovirus. The genus Avastrovirus includes turkey astrovirus and avian nephritis virus [34]. The
virus has a positive-sense, single-strand, polyadenylated RNA genome of approximately 6.8 kb
and consists of three sequential open reading frames (ORFs) designated ORF 1a, ORF 1b, and
ORF 2. ORF 1a and 1b, which are at the 5’ end of the genome, encode the non-structural viral
proteins, including a serine protease and a RNA-dependent RNA polymerase (RdRp),
respectively. ORF 1b is expressed as a fusion protein generated by a ribosomal frameshift [17,
20]. ORF 2 encodes a capsid precursor protein [16, 35]. The complete RNA sequences of the
serotypes/genotypes 1, 2, 3, and 8 have been characterized [14, 21, 27, 37].
Based on a 348-nucleotide (nt) region within ORF 2, HAstV has been divided into eight
genotypes (HAstV1 to HAstV8), correlating with the HAstV serotypes [22, 26, 33]. A similar
categorization has been observed for nucleotide sequences in the ORF 1b region, although the
distances between different genotypes are smaller. Phylogenetic analysis of a region in ORF 1a
demonstrates two distinct astrovirus genogroups, A and B. Genogroup A is composed of
serotypes 1 to 5 and 8, and genogroup B includes serotypes 6 and 7 [3].
In this work, we report the molecular characteristics of human astroviruses over segments
of all three ORFs, examining virus isolates from Brazil. In addition, the complete sequences of
HAstV genotype 4 and genotype 5 were determined for the first time.
28
Materials and Methods
Samples
Twenty fecal specimens, positive for astrovirus by nested-PCR, were included in this study. The
samples were obtained from children with acute gastroenteritis in three cities of the West Central
region of Brazil (Brasília-Distrito Federal, Goiânia-Goiás, and Campo Grande-Mato Grosso do
Sul) between December 1994 and July 2003. The dates of collection and geographic information
of the astrovirus-positive samples are given in Table 1.
Primers
Primers used to obtain partial sequences of the three astrovirus ORFs and the three overlapping
amplicons of the isolates, Goiania/GO/12/95/Brazil (BrG4) and Goiania/GO/12/94/Brazil (BrG5),
were either deduced from an alignment of published HAstV sequences or they were based on
oligonucleotides Mon 340, Mon 348 (ORF 1a), Mon 343, Mon 344 (ORF 1b), Mon 269, and
Mon 270 (ORF2) [3, 26]. All primers are listed in Table 2.
RNA Extraction and RT-PCR
Total viral RNA was extracted from 140 µl of a 20 % fecal suspension using the QIAamp viral
RNA Kit (Qiagen). The RT-PCR, first and second amplification conditions were the same as
described previously for astrovirus [28]. In order to avoid false-positive results, quality control
measures were taken as recommended by Kwok and Higuchu [15]. Negative sample controls
were included in each set of amplifications.
To determine the complete sequence of two isolates, BrG4 and BrG5, three fragments of
the full genome of each isolate were reverse transcribed using M-MLV, Thermo-X, or
PowerScript
TM
Reverse Transcriptases (Invitrogen, BD Biosciences) according to the
29
manufacturer’s recommendations. PCRs were carried out using either Platinum (94°C, 2.5 min;
35 cycles: 94°C, 30 sec; 55°C, 30 sec; 72°C, 1.5 min; final extension of 3 min at 72°C),
AccuPrime
TM
Pfx (95°C, 1 min 45 sec; 35 cycles: 95°C, 15 sec; 58°C, 30 sec; 68°C, 3 min; final
extension of 3’ at 68°C) or Phusion DNA polymerases (98°C, 30 sec; 35 cycles: 98°C, 10 sec;
60°C, 10 sec; 72°C, 1 min 40 sec; final extension of 3 min at 72°C). The enzymes were
manufactured by Invitrogen and Finnzymes.
Molecular Sequencing
PCR products and clones were sequenced using an ABI Prism 3100 Genetic Analyzer and Big
Dye Terminator Cycle Sequencing Mix (Applied Biosystems). All PCR products of three ORF
fragments (Fig. 1A) were sequenced directly in both directions, using the same primer pair as in
the nested PCR. The complete sequences of BrG4 and BrG5 were generated by amplifying 3
overlapping fragments of about 1.4, 3, and 2.4 kb (from 5’ to 3’ term) covering the complete viral
genome (Fig.1B and 1C). These fragments were cloned into pGEM®-T Vector (Promega)
according to manufacturer’s instructions, and sequenced using either primers of PCR or primers
derived from sequence data obtained during the characterization.
Phylogenetic Analysis
Phylogenetic analysis was performed over regions of 348 nt in ORF 2, 267 nt in ORF 1b,
and 198 nt in ORF 1a. For the complete sequences, additional analyses were performed over
ORF2 (2,353 nt), ORF 1b (600 nt), and over the full-length genome. Sequence alignments and
phylogenetic analysis were carried out using the CLUSTAL W program and the Phylogeny
Interference Package (PHYLIP), version 3.57c. Genetic distances were estimated using the
DNADIST program, and unrooted phylogenetic trees were constructed by the neighbour-joining
30
method. Bootstrap analysis of 100 replicate data sets was carried out using the SEQBOOT and
CONSENSE programs [7, 8].
Results
Phylogenetic Analysis
Nucleotide sequences of three genomic regions localized in ORF1a, ORF1b, and ORF2 were
obtained from 20 stool samples from Brazilian children with acute gastroenteritis. Phylogenetic
analysis was performed for all samples and eight reference strains published in GenBank (HAstV-
1 to HAstV-8; accession numbers are shown in Fig. 2), representing the eight genotypes.
Sequence analysis over an internal 348 nucleotides from the ORF 2 region showed that the
Brazilian isolates clustered into six different genotypes. Twelve isolates (60 %) clustered into
genotype 1, four (20 %) into genotype 2, and one (5 %) into each genotype 3, 4, 5, and 8 (Fig.
2A). Phylogenetic analysis exhibited intragenotypic identities of 89.4 – 97.4 % within genotype 1,
92 % within genotype 4, and > 96.6 % within the other genotypes. On the basis of nucleotide
sequence variation within the genotypes, we propose that the BrG1-1 to BrG1-5 (89.4 – 89.7 %
identity, 0.11 distance) and BrG4 isolates (92 % identity, 0.08 distance) represent subtypes of
HAstV genotype 1 and 4, respectively. An isolate is considered to be a subtype when it displays <
95 % identity and > 0.05 distance from the reference strain [11, 33].
Analysis of the reference sequences over the same region exhibited 74.7 – 84.5 % identity
among the genotypes, with the exception of genotypes 8 and 4, which presented 87.9 % identity
between them. The BrG4 isolate, which clustered with genotype 4, showed 92 and 89.7 %
identity compared to the reference sequences HAstV-4 and -8, respectively. The isolate that
clustered into genotype 8 (BrG8) showed an identity of 87.9 % with HAstV-4 and 98.6 % with
HAstV-8. These isolates had 89.9 % nucleotide identity among them and were identical on the
31
amino acid level (96.6 % identity with HAstV-4 and 100 % with HAstV-8). Based on the identity
to the reference sequence and the topology of the phylogenetic tree, we considered the BrG4
isolate to be genotype 4 and the BrG8 isolate to be genotype 8.
Predicted amino acid sequences (aa) of the ORF 2 fragment showed that there is no
divergence among the Brazilian genotype 1 isolates, with the exception of the BrG1-10 isolate,
for which a divergence of 0.9 % was observed. The four genotype 2 isolates and the genotype 3
isolate presented amino acid sequences identical to the HAstV-2 and HAstV-3 reference strains.
The genotype 5 isolate from Brazil showed 0.9 % amino acid divergence to HAstV-5.
Phylogenetic analysis of ORF 1b sequences showed that the identities among the
genotypes (80.6 – 96.3 %) were higher compared to ORF 2. Smaller divergences were observed
between genotypes 1, 2, 4, and 8 (3.8 – 9.7 %). 19 isolates clustered into the same genotypes as in
ORF 2 and one isolate (BrG4), grouped into genotype 4 in ORF 2, could not be clearly assigned
to any genotype in ORF 1b (Fig. 2B). This isolate showed a high nucleotide and amino acid
sequence identity with HAstV-1 (91.8 % nt, 96 % aa), HAstV-2 (90.3 % nt, 97 % aa), HAstV-4
(89.9 % nt, 94.9 % aa), and HAstV-8 (89.6 % nt, 94.9 % aa).
In order to eliminate the possibility of coinfection with other HAstV genotypes, a segment
spanning the ORF 1b / ORF 2 transition region (position 3742-4892 nt) of the BrG4 sample was
amplified and sequenced (data not shown). The nucleotide sequence of this segment was identical
with fragments obtained separately, and analysis of the 1150 nts showed that the BrG4 isolate
presented a higher identity (93 %) with HAstV-4 (AF292075) than with the other reference
sequences.
Analysis of the ORF 1a sequences demonstrated that all samples were classified as
genogroup A viruses (Fig. 2C). The nucleotide identity between viruses of the same genogroup
32
varied from 88.4 to 98.5 %, whereas the identity among isolates of different genogroups varied
from 78.4 to 82.9 %.
The accession numbers (acc. no.) of the Brazilian sequences deposited in GenBank are
shown in Table 3.
Molecular Characterization of the Complete Genome Sequences
Based on the phylogenetic analysis of 348 nt of the ORF 2 region, the BrG4 (acc. no.
DQ070852) and BrG5 (acc. no. DQ028633) isolates were classified into genotype 4 and genotype
5, which exhibited 92 % and 96.7 % identity with the respective reference strain. Pairwise
sequence comparison over two additional genome regions between the Brazilian isolates and
published sequences were performed.
1
Over the complete capsid protein precursor gene localized in ORF2 (approximately 2352
nt), pairwise sequence comparison demonstrated that the BrG4 and BrG5 isolates showed 91.7
and 95.8 % identity with HAstV4 and HAstV5, respectively. The maximum intergenotypic
identity (71 %) for BrG4 was observed with HAstV-8, and for BrG5 with HAstV-7. Considering
the predicted amino acid sequences, the isolate identities within the respective genotypes were
higher than 95 % but ranged between 65 and 77 % compared with different genotypes. The
nucleotide and amino acid data obtained from the region analysed suggested that the BrG4 and
BrG5 isolates belong to genotype/serotype 4 and 5, respectively.
Analysis of 600 nt from ORF 1b (position 3526-4126 of BrG4 and 3525-4125 of BrG5)
showed a high identity (91.2 – 94.2 %) among reference sequences of genotypes 1, 2, 4, and 8, as
already observed for the 267 nt ORF 1b segment . The BrG5 isolate clustered with the HAstV-5
reference sequence, presenting 95.2 % identity. The highest intergenotypic identity of this isolate
1
The GenBank accession numbers of the sequences compared to the Brazilian isolates are as follow: L23513,
L13745, AF141381, L23510, and L23512, AF260508 over ORF 2, and L23513, L13745, AF117209, Z33883,
U15136, Z46658, AF248738, Z66541 over the ORF 1b fragment.
33
was 85.3 % with HAstV-3. The BrG4 isolate did not cluster with any reference strain in the
phylogenetic tree, and presented 91.8, 90.7, 90.5, and 90 % nucleotide identities with HAstV-1,
HAstV-2, HAstV-4, and HAstV-8, respectively.
The complete sequences were obtained by molecular sequencing of three cloned
overlapping amplicons. The full-length genomic RNA of BrG4 and BrG5 isolates consisted of
6723 and 6762 nt, in each case, followed by a putative poly (A) tract. The isolates have a 5’ and
3’ non-translated region of 84 and 81 nt, and of 83 and 85 nt, respectively. The Brazilian isolates
displayed typical human astrovirus genomic features, comprising three sequential open reading
frames, which corresponded to ORFs 1a, 1b, and 2 [14]. ORF 1b overlaps ORF 1a by 64
nucleotides, containing a potential ribosomal frameshift signal.
Amino acid sequence analysis of the complete genome demonstrated that ORF 1a and 1b
encode the putative non-structural proteins, including a serine protease and a RdRp. ORF 2 of the
BrG4 isolate encodes a structural protein of 771 aa. The structural protein of the BrG5 isolate
consists of 783 amino acids. Comparison of the non-structural protein regions of BrG4 and BrG5
with reference sequences of genotypes 1, 2, 3, and 8 showed a high level of conservation (higher
than 93.5 % identity on the amino acid level). ORF 1a of both isolates contained a deletion of 21
and 45 nucleotides (position 2385 and 2456 of BrG4, and 2384 and 2457 of BrG5), compared
with HAstV-3 and HAstV-1, respectively.
The sequence identity between the full-length genome of the Brazilian isolates and
available complete sequences (HAstV-1 to -3 and -8) was less than 84 %, which implied the
involvement of different genotypes.
Discussion
34
Phylogenetic Analysis
Molecular characteristics of HAstV have been described for samples from different regions of the
world [1, 6, 11, 23, 24, 27, 33]. This study is the first report of the molecular characteristics of
astroviruses, encompassing three genome regions of Brazilian samples. Twenty astrovirus isolates
from stool samples of children with acute gastroenteritis were included in the study.
All astrovirus genotypes except HAstV-6 and HAstV-7 were detected in the central west
of Brazil, and HAstV-1 (60 %) was the most frequent genotype identified. These findings
reinforce data of other surveys carried out in several regions of the world [10, 11, 23, 26, 27, 30],
including Brazil that reported the prevalence of only HAstV-1 [5, 31]. The less common HAstV-2
to 5 and the rare HAstV-8 genotypes were also detected, demonstrating the simultaneous
circulation of different astrovirus genotypes in the same geographical area, which corroborates
the results of preceding studies [23, 24, 33].
Although strains and subtypes are not yet clearly defined, Walter et al. [33] considered an
isolate to be a subtype when it presents < 95 % sequence identity and > 0.05 distance to the
reference strain. Based on this definition, we have identified subtypes of genotypes 1 and 4
among the Brazilian isolates. Some authors have observed this variability within the genotypes
and related the occurrence of new HAstV-1 subtypes to time progression, suggesting a genetic
shift or introduction of a new strain [11, 29]. The majority of Brazilian HAstV-1 positive samples
were collected between 2000 and 2003, and we considered this period to be insufficient to
analyse the chronological order of the occurrence of different subtypes. We observed that among
5 viruses of the same subtype, 4 had been isolated in the same city (Campo Grande), suggesting a
regional subtype predominance.
Over the 348 nts of the ORF 2 fragment, the reference sequences HAstV-4 and HAstV-8
as well as the Brazilian isolates BrG4 and BrG8, classified as astrovirus genotype 4 and 8,
35
presented a high degree of nucleotide identity between them. Moreover, the predicted amino acid
sequences of both Brazilian isolates were identical, emphasizing a closer relationship between
these genotypes. This similarity between HAstV-4 and -8 has been reported by other authors [27,
32], including Espul et al. [6], who also described identical amino acid sequences in the same
genomic region. Analysis of the hypervariable 3’ end of the capsid region may be suited for
differentiating these genotypes [22, 32].
Brazilian and reference strains over 267 (for all isolates) and 600 (for BrG4 and BrG5
isolates) nucleotide segments of the ORF 1b region demonstrated a high degree of identity on the
nucleotide and amino acid levels. The similarity of the sequences in this region was high between
genotypes 1, 2, 4, and 8, which has also been reported in previous surveys [3, 16]. Although the
genotyping results over the ORF 1b and ORF 2 regions had coincided among them, with the
exception of one isolate (BrG4), we suggest that the ORF 2 region, which displays a higher
degree of variability, should be used for astrovirus genotyping.
Analysis of the BrG4 isolate in the ORF 2 region demonstrated its classification as
genotype 4, and sequence comparison over the ORF 1b region showed that it was highly similar
to reference sequences of other genotypes (1, 2, 4, and 8). This isolate, as already suggested by
the analysis of the 5’ end capsid region, appears to be a different HAstV-4 subtype, which would
explain the different characteristics over the ORF 1b region.
Of all of the sequences obtained in this study, 21 were submitted to GenBank. The
criterion adopted was a representative sequence from a group of sequences that were identical or
had higher than 99 % identity. This small variability means the exchange of less than 5
nucleotides, which may also be attributed to reverse transcriptase and polymerase error or to the
quasispecies population. In this context, we considered one sequence to represent a very similar
group and the publication of all sequences to be unnecessary.
36
Molecular Characterization of the Complete Genome Sequences
Complete genome sequences have so far been determined for 4 of the 8 human astrovirus
genotypes (1, 2, 3, and 8) [14, 21, 27, 37]. In this study, two more genotypes (4 and 5) were
characterized over the full genome, adding molecular information for each HAstV genotype.
The classification of the Brazilian isolates into genotypes was suggested by phylogenetic
analysis over a region at the 5’ end of the capsid gene, which has been confirmed to correlate with
the serotypes [22, 26, 33]. Considering this correlation, BrG4 and BrG5 isolates are the first
serotype 4 and 5 astroviruses to be molecularly characterized over the complete genome.
Since the 5’ end of the complete sequences was obtained using a primer based on the
reference sequence (L23513), the first 23 nucleotides resemble the reference sequence. The 3’
end was amplified using an oligo (dT) primer, which shows that HAstV contains a poly A tract,
as reported by other authors [4, 21].
Analysis of both complete genomes confirmed the organization of the astrovirus genomic
organization in three sequential ORFs (1a, 1b, and 2), with the two first ORFs being linked by a
conserved ribosomal frameshift signal [14, 16]. Predicted amino acid sequences of each ORF
suggested the expression of a serine protease, a RdRp, and a capsid protein, confirming the
observation of other groups [16, 37].
Sequences of ORF 1a demonstrated that BrG4 and BrG5 isolates contain deletions of 21
and 45 nucleotides, as compared to HAstV-3 and HAstV-1, respectively. The absence of 15
amino acids at position 790 has been associated with an astrovirus adaptation to HEK and
LLCMK2 cells [36, 37]. Controversially, some authors observed that a strain characterized
directly from fecal samples [27] or grown in CaCo-2 cells [21] also presented the deletion in the
same region. The Brazilian isolates, which were extracted from fecal samples, corroborate the
37
data observed for the German isolate [27], demonstrating that the occurrence of the deletion strain
is not limited to astrovirus adapted to cells and that the selection of the deletion strains can occur.
The deletion of 7 amino acids at position 768 could be observed only in comparison with HAstV-
3. To our knowledge, there is no report that addresses the cause or importance of this deletion,
and more detailed studies are needed to clarify these aspects.
The results of this study provide further molecular information on the astroviruses
genome. This information can contribute to the development of vaccines and other preventive
strategies for astrovirus infection.
Acknowledgements
P.A. Silva has been supported by a grant from DAAD (German Academic Exchange Service) and
CNPq (Brazilian National Council for Scientific and Technological Development).
We would like to thank our Brazilian collaborators, José M. S. Teixeira (Institute of
Health of Brasília-DF), Loreny G. Giugliano (Foundation of University of Brasília-DF) and
Márcia S. A. Andreasi (Federal University of Mato Grosso do Sul-MS), for providing the
samples. We also thank Djin-Ye Oh, Marina Höhne and Stefan Taube for the critical reading of
the manuscript and Guilherme Rangel for the support on graphics. We also thank colleagues from
FG15 in Robert Koch Institute-Berlin and from Laboratory of Virology in Goiânia-Brazil for the
constant aid and support.
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Table 1. Description of astrovirus isolates associated with gastroenteritis in Brazil
41
Isolate Collect location
(city, land
a
)
Date
(month/year)
HAstV
genotype
BrG1-1 Brasília, DF 01/1996 1
BrG1-2 Campo Grande, MS 08/2002 1
BrG1-3 Campo Grande, MS 05/2003 1
BrG1-4 Campo Grande, MS 07/2003 1
BrG1-5 Campo Grande, MS 07/2003 1
BrG1-6 Goiânia, GO 03/2001 1
BrG1-7 Goiânia, GO 09/2000 1
BrG1-8 Goiânia, GO 09/2000 1
BrG1-9 Campo Grande, MS 09/2003 1
BrG1-10 Goiânia, GO 01/2001 1
BrG1-11 Goiânia, GO 01/2001 1
BrG1-12 Brasília, DF 01/1996 1
BrG2-1 Brasília, DF 01/1996 2
BrG2-2 Campo Grande, MS 08/2002 2
BrG2-3 Brasília, DF 01/1996 2
BrG2-4 Brasília, DF 05/2001 2
BrG3 Brasília, DF 12/1994 3
BrG4 Brasília, DF 12/1995 4
BrG5 Brasília, DF 121994 5
BrG8 Goiânia, GO 08/1999 8
a
DF: distrito Federal; MS: Mato Grosso do Sul; GO: Goiás
Table 2. Astrovirus (RT-) PCR Primers
Primers
Sequence (5’ → 3’)
ORF or Fragment Location
Primers used in the amplification of three HAstV ORFs
AV1as
d
ACATgTgCTgCTgTTACTATITCg
AV2s
e
CgTCATTATTTgYTgTCATACT
AV3as* CCAGAAAAgAAACCTgT
AV4s* AgRTTYATACgIATggC
1a 1444-1467
a
1179-1200
1424-1440
1208-1224
AV5as ACWgTRAAgCCACAAAAIgATA
AV6s
f
gTAYAgACATgTRCATgA
AV7as
g
* CCRggYTTIACCCACAT
AV8s* CATgAITggTAYgTTgAYAAYCT
1b 4028-4049
3675-3692
3979-3995
3688-3710
AV9as
h
TCAgATgCATTRTCRTTIgT
AV10s
i
CAACTCAggAAACAgggTgT
AV11as* TTTgTgAgCCACCAgCCATC
AV12s* ggTgTCACAggACCAAAACC
2 4955-4974
4526-4545
4934-4953
4541-4560
Primers used in the amplification of three overlapping fragments (Fig. 1B and 1C) of the full
genome
AV13as ACATgTgCTgCTgTTACTATg
AV14s CCAAgAggggggTggTgATTggC
Fragment 1 1448-1468
b
1-23
AV15as CCTCTACTCCTggAATTgACTg
AV16s TgCATTATgTgTTATAgACAC
Fragment 2 (BrG5 only) 4438-4460
b
1388-1409
42
AV17as gATCgAggTCTggATTTgCTCC
AV18s gTTgTCATAAAACCAggTgCATTgTg
Fragment 2 (BrG4 only) 4385-4407
c
1372-1398
AV19as AgTCgACggATCCTTTTTTTTTTTTTTT
AV20s ggACCAAAgAAgTgTgATggCTAgC
(Fragment 3) > 6762
b
4310-4334
a
Genome location of AV1 to AV12 primers were based on HAstV-2 (GenBank acc.no. L13745).
b,c
Genome location
based on BrG5 (GenBank acc.no. DQ028633) and BrG4 (GenBank acc.no. DQ070852), respectively.
d-g
Derived from primers given by Belliot et al. [3],
h-i
derived/identical from primers given by Noel et al. [26].
*Primers used in the nested-PCR. s: Sense; as: Antisense
Table 3. Sequences of Brazilian HAstV ORFs published in GenBank
Isolate GenBank accession numbers
ORF 1a ORF 1b ORF 2
BrG1-5 DQ139817 DQ139819 DQ139825
BrG1-9 DQ139812 DQ139820 DQ139827
BrG1-10 DQ139815 -- DQ139828
BrG1-12 DQ139818 DQ139821 DQ139826
BrG2-1 -- -- DQ139830
BrG2-4 DQ139814 DQ139822 DQ139829
BrG3 DQ139816 DQ139823 DQ139831
BrG8 DQ139813 DQ139824 DQ139832
43
Legends
Figure 1
Schematic overview of the genomic astrovirus regions used for the phylogenetic analysis (A).
Overview of the three fragments used to determine the complete sequence of BrG4 (B) and BrG5
(C) isolates. The genome positions refer to HAstV-2 (acc. no. L13745), BrG4 (acc. no.
DQ070852) and BrG5 (acc. no. DQ028633), respectively.
Figure 2
Phylogenetic analysis of Brazilian isolates and selected reference strain (HAstV-1 to -8, shown in
bold letters) over 348 nt of ORF 2 (A), 267 nt of ORF 1b (B), and 198 nt of ORF 1a (C). Branch
lengths of the unrooted phylogenetic tree represent the genetic distance between sequences. The
scale indicates nucleotides substitutions per position. Bootstrap values >80 % are shown. The
corresponding genotypes are denoted to the right of each HAstV genotype (A and B). In Figure C
only the sequences deposited in the GenBank are shown. The corresponding GenBank accession
numbers for the reference sequences are as follows. L23513 (HAstV-1) and L13745 (HAstV-2)
were used for analysis of the three ORFs. AF292076 (HAstV-5), AF292977 (HAstV-6),
AF248738 (HAstV-7), and AF292073 (HAstV-8) were used for both ORF 2 and ORF 1b
analysis. L38505 (HAstV-3) and AF292075 (HAstV-4) were used for ORF 2 analysis, and
AF292074 (HAstV-3) and L23510 (HAstV-4) for ORF 1b. For analysis in ORF 1a, AF290504 to
AF290509 (HAstV-3 to HAstV-8) were used.
*Brazilian astroviruses sequences published in GenBank (acc. no. DQ139812 to DQ139832);
a
Complete genome sequences (acc. no. DQ070852 for BrG4 and DQ 028633 for BrG5).
44
Figure 1
45
Figura 2
46
5. ARTIGO II - Artigo científico submetido à publicação na revista Brazilian Journal of
Medical and Biological Research.
Circulation of human astroviruses genotypes in Central West of Brazil
P.A. Silva
1
, R.A.T. Santos
1
, P.S.S. Costa
1
, J.M.S. Teixeira
2
, L.G. Giugliano
3
, M.S.A. Andreasi
4
,
J.P.G. Leite
5
, E. Schreier
6
, D.D.P. Cardoso
1
1
Laboratório de Virologia, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal
de Goiás, Goiânia, Go, Brasil
2
Instituto de Saúde do Distrito Federal, Brasília, DF, Brasil
3
Laboratório de Microbiologia, Instituto de Ciências Biológicas, Universidade de Brasília,
Brasília, DF, Brasil
4
Centro de Ciências Biológicas e da Saúde, Universidade Federal do Mato Grosso do Sul, Campo
Grande, MS, Brasil
5
Laboratório de Virologia Comparada, Departamento de Virologia, Instituto Oswaldo Cruz-
FIOCRUZ, Rio de Janeiro, RJ, Brasil
6
Robert Koch-Institute, Berlin, Germany
Research supported by CNPq nº 471968/03-0 and 520729/99-4
Correspondence: D.D.P. Cardoso
IPTSP/UFG – Rua 235, esquina com 1ª Avenida, Setor Universitário
74605-050 Goiânia, Goiás, Brasil
Fax: +55-62-3521-1839
E-mail: [email protected]
Key words: astrovirus, genotyping, sequencing, Nested-PCR
47
Abstract
Human astroviruses (HAstV) are recognized as a common cause of diarrhea in
young children worldwide. Fifty seven astrovirus samples from children less than five years old
with (n=56) and without diarrhea (n=1) from three locations of the Central West region of Brazil
(Brasília, Goiânia and Campo Grande) were detected by RT-PCR. These samples were genotyped
by nested RT-PCR and/or genomic sequencing of the ORF 2 5’end region. It was detected
HAstV-1 (42.8%), HAstV-2 (23.2%), HAstV-3 (3.6%), HAstV-4 (14.3%) and HAstV-5, -6, -7
and -8 (1.8% each), in diarrheic samples. Five samples (8.9%) were untypeable. The positive non-
diarrheic sample was classified as HAstV-2. In Goiânia and Campo Grande, HAstV-1 was the
most frequent genotype detected, while in Brasília it was HAstV-2. Additionally, data from
Brasília and Campo Grande showed the occurrence of shift in astroviruses genotypes circulation
from HAstV-4 to HAstV-2 and from HAstV-2 to HAstV-1, respectively. The astroviruses
genotypes were detected in all age groups and there was no correlation between genotype and age
group. The results of this study reinforced data about the role of astroviruses in diarrhea and
provided information about viral genotypes circulation in Central West region of Brazil.
48
Introduction
Human astroviruses (HAstV) were first described in 1975 in fecal samples from children
with diarrhea, by electron microscopy [1]. Since then, the medical importance of these viruses has
been established and nowadays, in some studies, HAstV has been shown to be the second most
common cause of viral diarrhea in young children [2-5]. Epidemiological data have demonstrated
astrovirus prevalence rates between 2 and 26% in children with diarrhea [6, 7].
The astroviruses, family Astroviridae, genera Mamastrovirus, are non-enveloped spherical
particles with 29-30 nm of diameter [1]. Their genome is a single-stranded polyadenylated
positive-sense RNA of approximately 6.8 kb long [8, 9], which presents three open reading
frames (ORFs), 1a, 1b and 2 [10]. The ORF 1a and 1b encode non-structural proteins including a
serine protease and a RNA-dependent RNA polymerase, respectively, while the ORF 2 encodes
the viral capsid protein precursor [10, 11].
HAstV have been classified in eight serotypes/genotypes, according to their antigenic and
genomic characteristics [12, 13]. Previous studies have shown that HAstV-1 is predominant [12,
14, 15], however simultaneous circulation of different astrovirus types in the same geographical
area have been observed [15, 16].
In Brazil, there are few reports about human astroviruses genotypes circulation [17, 18].
This information is important concerning the future development of preventive therapies. In this
study, we describe the occurrence of genotypes of astroviruses isolated from children with and
without acute gastroenteritis in Central West region of Brazil, including previous data [17, 19].
49
Material and Methods
Fecal Samples
A total of 1,588 fecal samples (1,374 diarrheic and 214 non-diarrheic) were collected from
children under five years old from three location of Central West region of Brazil. This region
comprises by four states and in this study children from three of them were involved [Brasília-DF
(n=501), Goiânia-Go (n=743) and Campo Grande-MS (n=344)]. All samples were screened for
astroviruses by RT-PCR and 56 (4.1%) from diarrheic and 1 (0.4%) from non-diarrheic samples
were positive for this virus. Samples from Brasília were collected in two periods, 1994-1996 and
2000-2002. In Goiânia, samples were collected between 1998 and 2002, while in Campo Grande
it were between 2000 and 2003. The samples from Brasília, collected between 2000 and 2002,
were the majority (297/343) obtained by rectal swabs. Fecal suspensions (20%) were prepared in
PBS, pH 7.2 and stored at -20ºC until analysis.
The fecal samples were collected after written agreement of children’s parents or
guardians. This study was approved by the Ethics Committee on Human and Animal Research of
the Federal University of Goiás (004/2000).
Detection of astroviruses
Astroviruses were detected by RT-PCR assay using specific primers Mon269 (4526-4545
nt) and Mon270 (4955-4974 nt) directed towards the ORF 2 5’ end region, as described
previously [20]. The viral RNA was extracted from 200 µL of the 20 % fecal suspension by glass
powder method, using guanidinium thiocyanate as described by Boom et al [21]. The RT-PCR
and first amplification conditions were the same as described previously for astrovirus [17].
50
Genotyping of astroviruses
Nested RT-PCR - The astroviruses genotyping was carried out in 44 positive samples by
nested RT-PCR using primers that amplified the ORF 2 3’ end region, as described by Sakamoto
et al [12]. The RNA extraction, RT-PCR, first and second amplification were the same as
described previously [17].
Nucleotide sequencing and astroviruses genotyping - For determination of astroviruses
genotypes by genomic sequencing, phylogenetic analysis over the 348 bp of ORF 2 5’ end region
from 27 positive samples were performed. 14 samples were genotyped by both methodologies.
Total viral RNA was extracted from 140 µL of a 20% fecal suspension using the QIAamp viral
RNA Kit (Qiagen, Hilden, Germany). The products of nested RT-PCR, which were obtained
using the primer pair Mon269/Mon270 [20] in the first amplification and AV11as/AV12s
1
in the
second amplification [19], were sequenced directly in both directions using the same pair of
primer as in the nested-PCR, utilizing ABI Prism 3100 Genetic Analyzer and Big Dye Terminator
Cycle Sequencing Mix (Applied Biosystems). The RT-PCR and amplifications assays conditions
were the same as described previously for astrovirus [22]. Sequence alignments and phylogenetic
analysis were carried out using the CLUSTAL W program and the Phylogeny Inference Package
(PHYLIP), version 3.57c [23, 24].
Nucleotide sequences of astrovirus detected in this study have been submitted to GenBank
under accession number: DQ139825 to DQ139832, DQ070852 and DQ028633.
Results
1
AV11as (5’TTTgTgAgCCACCAgCCATC3’); AV12s (5’ggTgTCACAggACCAAAACC3’)
51
The genotyping of each astrovirus sample, carried out by nested RT-PCR and/or genomic
sequencing, showed that all known viral genotypes (HAstV-1 to HAstV-8) were detected in
Central West region of Brazil. Samples genotyped by both methodologies (n=14) presented
coincident results. Five samples (two from Brasília, two from Goiânia and one from Campo
Grande) submitted only to nested RT-PCR genotyping were untypeable.
HAstV-1, -2, -3 and -4 were detected in 42.8%, 23.2%, 3.6% and 14.3%, respectively,
while the HAstV-5, -6, -7 and -8 were detected each one in 1.8% of diarrheic samples.
Considering the origin location of the samples, it was observed that in Brasília HAstV-2 was the
most frequent genotype (37.5%), followed by HAstV-4 (25%), HAstV-1 (12.5%), HAstV-3
(8.3%), HAstV-5 and HAstV-6 (4.2% each). In Goiânia, HAstV-1 (66.6%) was the predominant
genotype, followed by HAstV-4 (9.5%), HAstV-2, -7 and -8 (4.8% each). In Campo Grande, only
two genotypes were detected, HAstV-1 (63.6%) and HAstV-2 (27.3%) (Table 1). The only one
non-diarrheic positive sample was collected in Brasília in 2001 and it was characterized as
HAstV-2.
Although it was not observed statistical significance between predominant genotype and
years of the collect, in Brasília, HAstV-4 was the most frequent in 1994 and 1995, while HAstV-
2 was detected in 71.4% of the samples in 1996. Between 2000 and 2002 astroviruses were not
detected in diarrheic samples from Brasília. In Goiânia, HAstV-1 was the predominant genotype
in all years of the study, but in 1999, only two positive samples were detected and one was
classified as HAstV-1 and the other HAstV-8. In Campo Grande, HAstV-2 predominated in 2000
and 2002, while HAstV-1 was the only one genotype detected in 2003 (Figure 1).
The different astroviruses genotypes were detected in children from all age groups up to
five years old and it was not observed correlation between genotype and age group (p> 0.1).
Discussion
52
Besides the knowledge about the genetic variability of the virus circulating in different
regions of the world, the large use of molecular techniques in epidemiological studies has
reinforced the importance of human astroviruses as cause of acute gastroenteritis [5, 9, 15]. The
present study reports the occurrence and distribution of astrovirus genotypes in Central West
region of Brazil.
Our data showed that HAstV-1 was the most frequent genotype in the studied region. This
finding corroborates data of other authors from several regions of the world, including Brazil [9,
17, 25, 26]. However, other genotypes considered less common (HAstV-2 to HAstV-5) or rare
(HAstV-6 to HAstV-8) were also detected. The circulation of different genotypes in the same
region is not uncommon and has also been reported in countries such as Germany, Spain,
Argentina, Mexico, and Egypt [9, 15, 16, 27, 28]. In Brasília and Goiânia the occurrence of at
least five astrovirus genotypes was observed, while in Campo Grande only two genotypes were
detected. This data could be explained by the fact that Brasília is a national and international
tourist destination beyond the high emigration rate that makes possible the occasional
introduction of different astroviruses genotypes. Similarly, Goiânia also received a significant
high number of tourists and emigrants that could introduce different viral genotypes in the native
population.
Considering the origin of the samples, in Goiânia and Campo Grande the most frequent
genotype was HAstV-1, while in Brasília it was HAstV-2. This variation in genotype
predominance from one location to another could suggest the occurrence of a regional
predominance of astroviruses genotypes. This fact was also observed in others studies [15, 29].
Analysis of data obtained from positive samples from Brasília and Campo Grande in
different years, showed a shift of astroviruses genotypes circulation from HAstV-4 to HAstV-2
and from HAstV-2 to HAstV-1, respectively. This event has been observed in some studies with
53
other viruses, including gastrointestinal viruses, and some authors suggested that this viral
genotypes shift could reflect a natural selection due to a lack of heterotypic immunity among the
different viral antigenic types [12, 27, 30-32].
From 44 positive samples that were submitted to nested RT-PCR genotyping, five were
untypeable. This fact has been reported in other study, which suggested that the failure of nested
RT-PCR typing is not related to sequence variation but could be due to mismatches, inhibitors, or
low RNA yield [33].
Enteric viruses usually have been detected directly from fecal samples in natura but other
collection methods, as rectal swabs also have been used [34]. In this study, we consider that the
absence of positive astrovirus samples from Brasília collected between 2000 and 2002 is due to
the method of fecal collection utilized because the majority of samples collected in this period
were obtained from rectal swabs that could impair the viral detection, fact also observed for
caliciviruses [Cardoso DDP et al., unpublished data].
In this study there was no correlation between age groups and astrovirus genotypes,
although previous data in Spain had indicated a predominance of HAstV-1 and -3 in children up
to 2 years old, and HAStV-4 and -8 in children older than 3 years [27].
In conclusion, the results of this study reinforce data about the role of astroviruses as an
important agent of diarrhea in children and provide the first data about viral genotypes circulation
in three location of Central West region of Brazil. This information is relevant for future
implementation of preventive strategies for the HAstV infection.
Acknowledgements
54
P.A. Silva was supported by a grant from DAAD (German Academic Exchange Service),
CNPq (Brazilian National Council for Scientific and Technological Development) and CAPES
(Coordination of Improvement of High Degree People).
We would like to thank Dr.Wilia Brito for the critical reading of the manuscript and
colleagues from Laboratory of Virology in Goiânia-Brazil and from FG15 in Robert Koch
Institute-Berlin for the constant aid and support.
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57
Table 1. Distribution of astroviruses genotypes detected in diarrheic samples of children from
three locations of Central West region of Brazil.
Genotypes Brasília
Nº (%)
Goiânia
Nº (%)
Campo Grande
Nº (%)
Total
Nº (%)
HAstV-1 3 (12.5) 14 (66.6) 7 (63.6) 24 (42.8)
HAstV-2 9 (37.5) 1 (4.8) 3 (27.3) 13 (23.2)
HAstV-3 2 (8.3) - - 2 (3.6)
HAstV-4 6 (25) 2 (9.5) - 8 (14.3)
HAstV-5 1 (4.2) - - 1 (1.8)
HAstV-6 1 (4.2) - - 1 (1.8)
HAstV-7 - 1 (4.8) - 1 (1.8)
HAstV-8 - 1 (4.8) - 1 (1.8)
Untypeable 2 (8.3) 2 (9.5) 1 (9.1) 5 (8.9)
Total 24 (100) 21 (100) 11 (100) 56 (100)
Figure 1. Distribution of HAstV genotypes detected in Central West region of Brazil, according
to the location and year of collection. (A) Brasília, (B) Goiânia and (C) Campo Grande.
58
(A) Brasília
HAstV-1
HAstV-1
HAstV-2
HAstV-2
HAstV-2
HAstV-4
HAstV-4
HAstV-5
HAstV-6
Untypeable
Untypeable
HAstV-3
0
20
40
60
80
100
1994 1995 1996
Years of collection
%
(B) Goiânia
HAstV-1
HAstV-1
HAstV-2
HAstV-4
HAstV-4
HAstV-8
Untypeable
HAstV-1
HAstV-1
HAstV-7
0
10
20
30
40
50
60
70
80
90
100
1998 1999 2000 2001
Years of collection
%
59
(C) Campo Grande
HAstV-1
HAstV-2
HAstV-2
HAstV-2
Untypaable
0
10
20
30
40
50
60
70
80
90
100
2000 2001 2002 2003
Years of collection
%
60
6. CONCLUSÕES GERAIS
• O resultado deste estudo mostra que os oito genótipos de astrovírus estão presentes na
região Centro-Oeste do Brasil, conhecimento importante para futuras implementações de
terapias preventivas contra o vírus. A distribuição desses genótipos foi diferente para cada
cidade estudada sugerindo predominância regional dos genótipos, embora tenham datas
diferentes de coleta de fezes. Nas cidades de Brasília e Campo Grande, os astrovírus
apresentaram mudança de genótipos predominantes em relação aos diferentes anos de
coleta;
• Não houve correlação entre qualquer genótipo viral e a faixa etária das crianças estudadas;
• Análise de seqüências da região do ORF 1b e 2 reforçam informações de que o ORF 2,
devido maior variabilidade genética, deve ser a região de escolha para a genotipagem dos
astrovírus;
• Todas as amostras analisadas foram classificadas como genogrupo A;
• Foi feita análise do genoma completo de duas amostras de astrovírus, genótipo 4 e 5, o
que aumenta o espectro de informações a respeito deste vírus, considerando ser estes
genótipos pela primeira vez, em termos mundiais, a serem totalmente analisados e
depositados no GenBank.
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