Rotavirus G2P[4] and G2P[4]+[6] infections during norovirus gastroenteritis outbreak: summer season 2010, Brazil

Abstract

Introduction

This study aimed to monitor the seasonality of rotavirus infection, and gain insight into the variability of Brazilian strains.

Methods

A total of 28 stool samples were analyzed from 698 revised cases of gastroenteritis during a norovirus outbreak in the summer of 2010 in Guarujá, Brazil. Diagnosis was performed using enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and sequencing.

Results

Rotavirus infection was detected in 17.9% (5/28) of samples; 4 samples were G2P[4] genotype, and one G2P[4]+P[6] genotype. G2 and P[4] sequences showed a genetic relationship to strains from India and Russia, respectively.

Conclusions

The seasonal pattern of rotavirus may be a consequence of human activity apart from climate factors.

Rotavirus; Summer; Seasonality

A key characteristic on rotavirus (RV) epidemiology is its distinct seasonal pattern¹; however, the mechanism responsible for this seasonality is not clear². In temperate climates, RV disease occurs in the cooler, dryer months of the years, and far less often in tropical areas². In Brazil, the seasonality is distinct, with positive RV specimens appearing to peak during the winter or dry season in central and southern states that exhibit a temperate-like climate, but not in north and northern areas, where RV infections occur equally over the year³.

During summer 2010, the State of São Paulo (SP), located in southern area of the country, experienced a large gastroenteritis outbreak due to norovirus (NoV)⁴, a common viral pathogen associated with diarrhea, which has a marked seasonality during summer. The coastline area of SP was most greatly affected⁴, likely due to the increase of population density during the summer vacations and flooding in conjunction with the waterborne and seafood components of NoV transmission⁵.

In the popular seashore City of Guarujá, atypical RV infections were observed at the time of a large NoV outbreak. The aims of this study were to contribute to the knowledge of seasonality of RV infection in tropical countries, and to perform sequence analyses to gain insight into the variability of Brazilian strains.

The increase in acute gastroenteritis cases from Guarujá was detected by the Epidemiological Surveillance Center of São Paulo (CVE)⁵, which receives weekly reports from sentinel sites of the Acute Diarrhea Disease Monitoring Program. An epidemiological retrospective investigation was conducted by CVE, and a total of 698 cases were analyzed⁵. The Enteric Diseases Laboratory tested 28 stool specimens from the 698 Guarujá patients.

Rotavirus was detected using a commercial immunoenzymatic assay (RIDASCREEN® Rotavirus, R-BiopharmAG, Darmstadt, Germany), performed according to the manufacturer's instructions. RV-positive stool samples were typed after reverse transcription (RT) followed by semi-nested polymerase chain reaction (PCR)⁶ and sequencing on an ABI 3100 thermocycler (Applied Biosystems, Foster City, CA, USA). Sequence data was aligned and edited with the BioEdit Sequence Alignment Editor (version 7.0.5.2) software (Ibis Therapeutics, Carlsbad, CA). A genetic tree was constructed using molecular evolutionary genetics analysis (MEGA) software version 4.0 by the neighbor joining (NJ) method⁷.

Rotavirus infection was detected in 17.9% (5/28) of samples. The median age of the patients was 14.9 years; twenty percent of patients were female and 80% were male. According to the Enteric Diseases Laboratory records, NoV infection were detected in 10 (35.7%) of the 28 fecal samples, confirming the large NoV outbreak occurring during the same period⁴. Mixed NoV and RV infections were not observed.

In State of São Paulo surveillance data available on the CVE website (http://www.cve.saude.sp.gov.br/htm/hidrica/hidri_estat.html), there were no RV outbreak reports in the metropolitan area of Santos during the summer periods. In addition, no RV cases from Santos were identified in the Enteric Diseases Laboratory records during the rainy seasons. Taken together, these findings suggest that a minor RV outbreak occurred in Guarujá alongside the major NoV outbreak. RV outbreaks during the summer season are very uncommon⁸. The seasonal pattern of RV diarrhea cases may be a consequence of unmeasured factors apart from climate factors, including human activity and environmental factors other than the weather⁹.

The RT-PCR assay identified the G2P[4] genotype in 4 samples. Recently, a high prevalence of G2P[4] has been reported in Brazil and linked with the inclusion of the G1P[8] live oral RV vaccine in the Brazilian vaccination program¹⁰, suggesting that this monovalent vaccine possibly created conditions in which G2P[4] could acquire selective advantage over P[8] genotypes. Alternatively, a temporal periodicity, within an approximately 10-year cyclic pattern of G2P[4] have been observed in Brazil¹¹, and should be considered as an alternative explanation to the increased detection of this genotype since 2006.

The genetic relationships between the VP7 sequences of 4 G2 RV strains: R2047 (HQ844990), R2050 (HQ844988), R2053 (HQ844989), and R2057 (HQ844987), and representative strains of the G2 genotype: DS1 (AB118023), Thailand (EF199723), Australia (HRU73955), India (GQ229046), Italy (DQ172854), KO-2 (AF401754), and Brazil (FJ492764), are shown as a distance tree in Figure 1. The SP G2 RV sequences showed 93%-97.5% similarity when compared to the representative strains, and 100% similarity between them. The analysis of the VP7 gene showed a genetic relationship with the RV strain from India, and also allowed classification of these strains into lineage II using the Page and Steele¹² classification. In a previous study conducted in the State of Pará, Brazil¹³, the phylogenetic analysis performed in G2 RV strains also grouped to lineage II.

FIGURE 1
Dendrogram of the VP7 gene nucleotide sequences of human rotavirus G2 strains (SP R2047, SP R2057, SP R2050, and SP R2053) from Guarujá, São Paulo, Brazil. The tree was constructed using the neighbor joining method with the molecular evolutionary genetics analysis (MEGA) 4.0 software package. The numbers on each branch indicate the bootstrap values.

The genetic relationships between the VP4 sequences of 3 P[4] RV strains: R2047 (HQ844984), R2050 (HQ844985), and R2053 (HQ844986), and representative strains of the P[4] genotype: Brazil (DQ857927), Italy (DQ172842), DS1 (AB118025), Brazil (FJ492780), Bangladesh (EU839950), and Russia (GU356610), are shown as a distance tree in Figure 2. The SP P[4] RV sequences showed 94.6%-99.6% similarity when compared to the representative strains, and 99.9%-100% similarity between them. The SP P[4] RV strains analyzed in this study showed a genetic relationship with the RV strain from Russia.

FIGURE 2
Dendrogram of the VP4 gene nucleotide sequences of human rotavirus P[4] strains (SP R2047, SP R2050, and SP R2053) from Guarujá, São Paulo, Brazil. The tree was constructed using the neighbor joining method with the molecular evolutionary genetics analysis (MEGA) 4.0 software package. The numbers on each branch indicate the bootstrap values.

A P-mixed unusual infection G2P[4]+P[6] was detected in 1 sample from a 10-year-old unvaccinated male. The dual infection was confirmed by RT-PCR using specific set of primers; however, although several attempts were made to obtain the VP4 sequences from this sample, they were unsuccessful. Thus, no data was obtained that could identify the potential origin of this strain.

The isolation of a Brazilian G2 RV strain exhibiting double P[4] and P[6] specificity was not unexpected, suggesting that this was not the same strain circulating during this period and that there was not a common source of RV contamination. In Nigeria, a G2 RV strain displaying dual P[4] and P[6] specificity was identified in 2000¹², and in Guinea-Bissau this unusual strain was the most frequent cause of RV infections in hospitalized children in 2002¹⁴. These findings may reflect environmental characteristics related to tropical areas, as Brazil, Nigeria, and Guinea-Bissau share the hot and humid tropical climate characteristic of the region between the tropics of Cancer and Capricorn.

The inclusion of alternative P types in serotype G2 strain gene constellations may provide a means of escaping protective immunity and generating increased RV diversity¹². Uncommon associations could also be related to reassortment between co-circulating RV strains¹⁵.

The outbreak investigation also attempted to investigate the bacterial and parasitological etiology. These enteropathogenic investigations were conducted through the Adolfo Lutz Institute online Integrated Hospital Management system (Sistema Integrado de Gestão Hospitalar-SIGH), and the findings are summarized in Table 1. Overall, this study recognized pathogenic agents in 75% of the examined stool samples, and this elevated rate of isolation could be an indicative of a high rate of oral-fecal exposure in this population.

TABLE 1
- Detection of enteric pathogens in stool samples from patients with acute gastroenteritis during a norovirus outbreak in the summer of 2010, Guarujá, São Paulo, Brazil

Enteric pathogens	Positive
	Number	Percentage
RV	4	14.3
NoV	8	28.6
RV + E. coli (EPEC)	1	3.6
NoV + E. coli (EPEC)	2	7.1
E. coli (ETEC)	1	3.6
E. coli (EHEC)	1	3.6
E. coli (EPEC) + Ascaris lumbricoides	1	3.6
Giardia lamblia	2	7.1
Giardia lamblia + Entamoeba coli	1	3.6
Negative	7	24.9
Total	28	100.0

• RV: rotavirus, NoV: norovirus, EPEC: enteropathogenic Escherichia coli, ETEC: enterotoxigenic Escherichia coli, EHEC: enterohemorrhagic Escherichi.

In conclusion, RV was recognized as the etiological agent of a minor gastroenteritis outbreak, affecting children and adults, in the city of Guarujá during the summer of 2010. These results could improve the understanding of RV transmission in tropics, paying close attention to human and climatological variables, and its relationship to other viral gastroenteritis pathogens. A major finding in the present study was the detection of the G2 P[4]+P[6] genotype, reflecting the importance of continued monitoring of RV strains circulating throughout the world.

Ethical considerations

Ethics Committee approval was granted by the Adolfo Lutz Institute, São Paulo, Brazil (Ref. 14/2005). This was an anonymous, unlinked study and informed consent was not required according to the resolution 196/96 concerning research involving human beings (Conselho Nacional de Saúde/Ministério da Saúde, Brasília, 1996).

REFERENCES

• ¹
  Turcios RM, Curns AT, Holman RC, Pandya-Smith I, LaMonte A, Bresee JS, et al. Temporal and geographic trends of rotavirus activity in the United States, 1997-2004. Pediatr Infect Dis J 2006; 25:451-454.
• ²
  Greenberg HB, Estes MK. Rotaviruses: from pathogenesis to vaccination. Gastroenterology 2009; 136:1939-1951.
• ³
  Pereira HG, Linhares AC, Candeias JAN, Glass RI. National laboratory surveillance of viral agents of gastroenteritis in Brazil. Bull Pan Am Health Organ 1993; 27:224-233.
• ⁴
  Morillo SG, Luchs A, Cilli A, Ribeiro CD, Calux SJ, Carmona RCC, et al. Large gastroenteritis outbreak due to Norovirus GII in São Paulo, Brazil, summer 2010. Rev Inst Med Trop Sao Paulo 2011; 53:119-120.
• ⁵
  Eduardo MBP, Suzuki E, Fred J, Marques D, Lima LMA, Silva CMB, et al. Investigação de surto de diarréia por norovírus no município de Guarujá, SP, Brasil, Dezembro de 2009 a Janeiro de 2010. Poster presented at: EPI CVE 2010. Conferência Internacional em Epidemiologia; 2010 Nov 29-30; São Paulo, SP, Brazil. [cited 2011 May 28]. Available from: http://www.cve.saude.sp.gov.br/htm/hidrica/2010/Poster10_Surto_Guaruja.pdf/
  » http://www.cve.saude.sp.gov.br/htm/hidrica/2010/Poster10_Surto_Guaruja.pdf/
• ⁶
  Timenetsky MCST, Gouvêa V, Santos N, Carmona RCC, Hoshino Y. A novel human rotavirus serotype with dual G5-G11 specificity. J Genl Virol 1997; 78:1373-1378.
• ⁷
  Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24(8):1596-1599.
• ⁸
  Marin-Casanova P, Fernández-Fatou B, García-Martós P, Martín-Hernández C, Agudo-Pérez E. Atypical epidemic outbreak of gastroenteritis caused by rotavirus. Rev Sanid Hig Publica (Madr) 1989; 63:39-45.
• ⁹
  Hashizume M, Armstrong B, Wagatsuma Y, Faruque AS, Hayashi T, Sack DA. Rotavirus infections and climate variability in Dhaka, Bangladesh: a time-series analysis. Epidemiol Infect 2008; 136:1281-1289.
• ¹⁰
  Nakagomi T, Cuervas LE, Gurger RG, Elrokhsi SH, Belkhir YA, Abugalia M, et al. Apparent extinction of non-G2 rotavirus strains from circulation in Recife, Brazil, after introduction of rotavirus vaccine. Arch Virol 2008; 153:591-593.
• ¹¹
  Carvalho-Costa FA, Assis RM, Fialho AM, Bóia MN, Alves DP, Martins CM, et al. Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004. Mem Inst Oswaldo Cruz 2006; 101:291-294.
• ¹²
  Page NA, Steele AD. Antigenic and genetic characterization of serotype G2 human rotavirus strains from the African continent. J Clin Microbiol 2004; 42:595-600.
• ¹³
  Mascarenhas JD, Lima CS, Oliveira DS, Guerra SF, Maestri RP, Gabbay YB, et al. Identification of two sublineages of genotype G2 rotavirus among diarrheic children in Parauapebas, Southern Pará State, Brazil. J Med Virol 2010; 82:712-719.
• ¹⁴
  Nielsen NM, Eugen-Olsen J, Aaby P, Mølbak K, Rodrigues A, Fischer TK. Characterization of rotavirus strains among hospitalized and non-hospitalized children in Guinea-Bissau, 2002 A high frequency of mixed infections with serotype G8. J Clin Virol 2005; 34:13-21.
• ¹⁵
  Iturriza-Gómara M, Isherwood B, Desselberger U, Gray J. Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999. J Virol 2001; 75:3696-3705.

Publication Dates

• Publication in this collection
  Mar-Apr 2013

History

• Received
  28 May 2011
• Accepted
  8 Aug 2011