THE BRAZILIAN JOURNAL OF INFECTIOUS DISEASES

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1 269 ISSN Volume 1 Number 6 December 1997 THE BRAZILIAN JOURNAL OF INFECTIOUS DISEASES An Official Publication of the Brazilian Society of Infectious Diseases EDITOR-IN-CHIEF Roberto Badaró EDITOR Thomas C. Jones PUBLISHED BY CONTEXTO December 1997 Printed in Brazil

2 Rotavirus Infection in Brazil: Epidemiology, Immunity, and Potential Vaccination Alexandre C. Linhares Serviço de Virologia Geral, Instituto Evandro Chagas, Fundação Nacional de Saúde Worldwide, it is estimated that rotaviruses cause more than 125 million diarrheal episodes and nearly 1 million deaths per year among infants and young children. In Brazil, these agents have been associated with 13%-40% of cases of acute diarrhea affecting pediatric inpatients and outpatients. Longitudinal community-based studies involving children followed from birth to the age of three years, yielded an average of 0.3 rotavirus-related episodes of diarrhea per child per year. While seasonality of rotavirus infections is not evident in the more northern tropical areas, a peak incidence during the driest months (May to September) has been noted in Brazil s central and southern states. All four epidemiologically important rotavirus serotypes have been identified in Brazil, with G1 and G2 accounting for about two-thirds of typed strains. The recent characterization of G- and P- genotypes has demonstrated that predominant strains are essentially the same as those most commonly identified worldwide: taken together, P[8]G1 and P[4]G2 rotaviruses account for over 50% of genotyped strains. In addition, the occurrence of unusual types, such as P[8]G5, and mixed infections, have been reported in about 10% and 20% of diarrheal cases, respectively. Outbreaks of rotavirus diarrhea have been reported in both urban and remote communities, G2 type being reported to cause severe gastroenteritis among adults and children. Among Indian populations the clinical attack rate has approached 90%. Rotavirus group C has been associated with outbreaks of diarrhea in day-care centers, yielding clinical attack rates of nearly 50%. Seroprevalence studies in several regions in Brazil indicate that at least 70% of children aged 4-5 years have antibodies to rotavirus. It has also been demonstrated in longitudinal studies that heterotypic immune response in a primary infection may be an intrinsic property of the rotavirus strain. A tetravalent rhesus-human reassortant rotavirus vaccine (4 x 10,000 pfu/dose) has been evaluated in northern Brazil with the following main results: a) the vaccine was safe with only low-grade fever on days 3-5 in 2%-3% of vaccinees after the first dose; b) an IgA antibody response occurred in 58% of vaccinees and 33% of placebo recipients; and c) the overall vaccine efficacy was 35% (p = 0.03) against any rotavirus diarrhea for the 2-year study period, but reached 57% (p = 0.008) during the first year of follow-up. In view of findings indicating strain diversity throughout Brazil, a country-wild surveillance system is urgently needed to monitor circulating rotavirus strains. Key Words: rotavirus, vaccination, epidemiology, immunity. Received in 7 December 1997; revised 21 December Address reprint requests to: Alexandre C. Linhares, M.D., Serviço de Virologia Geral, Instituto Evandro Chagas, Av. Almirante Barroso, 492, , Belém, Pará, Brasil. The Brazilian Journal of Infectious Diseases 1997;1(6): by The Brazilian Journal of Infectious Diseases. All rights reserved

3 Rotavirus Infection in Brazil 285 Epidemologic Overview Wolrdwide rotaviruses are associated with more than 125 million cases of infantile gastroenteritis and nearly one million deaths per year, mainly in tropical regions [1]. The similar incidence of the illness in both industrialized and developing countries supports the view that control of rotavirus diarrhea would more likely be achieved by the development of an effective rotavirus vaccine than by improvement of water supply, sanitation or hygiene practices [2, 3]. In Brazil, rotavirus particles were first demonstrated in 1976, by electron microscopy of fecal material from children admitted to a public hospital in Belém with acute gastroenteritis (Figure 1) [4, 5]. Several other studies subsequently assessed the importance of these agents in the etiology of acute infantile diarrhea throughout the country. Rotaviruses have been detected in 13%-40% of diarrheic infants and young children who were either hospitalized or attending public health units and private clinics in Brazil as outpatients [6-17]. Most of these studies indicate that approximately 70% of symptomatic rotavirus infections occur among children aged 6 months to 2 years [11, 17, 18]. In addition, rotaviruses have been associated with nearly 40% of cases of nosocomial infantile diarrhea in northern Brazil [19]. These studies have also shown that rotavirus strains which circulate in the hospital environment reflect those infecting the general community. Very few community-based longitudinal studies have been conducted in Brazil. In northern Brazil, for instance, approximately 3.0 episodes of diarrhea per child annually were recorded during a 3-year prospective study carried out from December, 1982, to March, 1986, involving children aged 0 to 3 years [20]. As rotaviruses occurred in about 10% of the cases of gastroenteritis, the average number of rotavirus diarrhea per child/annually was 0.3. In this study, the overall age-specific attack-rate of diarrhea was higher in children over 6 months (mainly up to 2 years) than in those between birth and 6 months of age. In a more recent longitudinal study in the same area in , rates of 5.9 and 0.2 episodes of diarrhea per child/annually were noted for all cases and the rotavirus-related ones, respectively [21]. A prospective village surveillance conducted among low-income families in northeastern Brazil revealed diarrhea attack-rates of more than 7 episodes per child/annually among children aged 6 months to 11 months [22]. In this investigation, rotaviruses accounted for 20% of acute diarrhea cases. All of these studies indicate that the clinical picture in children with rotavirusrelated diarrhea has been more severe than in those suffering from acute diarrhea due to other agents. In addition, cases of successive rotavirus infection were detected during these prospective communitybased investigations. Most of these first suffered an infection with rotavirus G type 1 (within the first year of life), and then a subsequent diarrheal episode associated with G type 2. The seasonal distribution of rotavirus gastroenteritis in children less than 5 years of age throughout the country was assessed during an eight state laboratory surveillance, carried out between 1984 and 1990 [23]. There was a peak incidence of rotavirus diarrhea in May-September (the dry season) in Brazil s central and southern states, but no seasonality was evident in the more tropical northern areas (Figure 2). Data on the epidemiology of rotavirus serotypes in Brazil are still scarce. During a longitudinal community-based study carried out in Belém over 3 years ( ), G serotypes 1, 2, 3 and 4 were found in 50%, 30%, 3.3% and 16.7% of the serotyped samples, respectively [20, 24]. In a further (1988) hospital-based study conducted in the same area, only G serotype 3 could be identified among infants and young children (1 month-24 months) with diarrhea [11]. In a similar investigation carried out during approximately the same period in São Luís, Maranhão (northern Brazil), G type 3 was also found to be predominant and accounted for 41% of the cases of infantile diarrhea [25]. In the course of a field trial with a

4 Rotavirus Infection in Brazil Figure 1. First rotavirus particles detected in Brazil, from a child with diarrhea in Belém, Brazil Reproduced from Linhares, et al., 1977 (x100,000)

5 Rotavirus Infection in Brazil 287 Figure 2. Surveillance for rotavirus gastroenteritis in Brazil from 1981 to 1983 BRAZIL Adapted from Pereira, et al., 1993.

6 Rotavirus Infection in Brazil Figure 3. Occurrence of P and G genotypes of rotavirus strains from children with diarrhea in Brazil Adapted from Leite, et al

7 Rotavirus Infection in Brazil 289 rotavirus candidate vaccine in Belém, Brazil, from 1990 to 1992, G type 1 was found to be more prevalent, and accounted for 68% of serotyped strains [21]. In a hospital-based study conducted in the same area during November May 1994, however, rotavirus G serotype 2 prevailed over the other types, accounting for 80% of isolates from nosocomial diarrheal episodes [19]. The use of molecular approaches has enabled several investigators, mostly in southeastern Brazil, to identify human rotavirus G and P types in stool specimens. During 1986 to 1992, a study involving children under 4 years of age revealed frequencies of the four major G types as follows: G1, 17% of the specimens; G2, 6%; G3, 22%; and G4, 5% [26]. A predominance of P1 type (21%) was also observed, consistent with the association with G1, G3 and G4 specificities. Other investigations have also identified rotavirus strains belonging to G types 1 to 4 having a P3 genotype (M37-like VP4) among children with symptomatic and asymptomatic infections [27]. An extensive survey to determine the rotavirus P and G genotypes was recently carried out in 9 States and the Federal District of Brazil involving children under 5 years of age [28]. The predominant strains were essentially the same as those most commonly found worldwide, as follows: P[8]G1, 43%; P[4]G2, 12%; P[8]G3, 6%; and P[8]G4, 6%. It is of interest that unusual types (e.g., P[8]G5) and mixed infections were associated with 12% and 21% of the cases, respectively (Figure 3). In addition, genotype G5 strains were identified in specimens obtained from all 9 study areas, supporting previous observations made in São Paulo, Brazil [29]. Although limited, the available data in Brazil suggest that G and P types are equally virulent, and that inapparent infections may occur with all types. In addition to serotypes, the diversity of electrophoretic profiles has been investigated by several authors. As observed worldwide, most studies in Brazil indicate that the long electropherotype predominates (at least 70% of isolates) over the short pattern; the former genomic profile, on the other hand, shows a greater degree of heterogeneity than the latter one [10, 30, 31]. Outbreaks of diarrhea due to rotavirus have been reported in both urban and remote communities in Brazil, and data suggests that some populations may be at greater risk for illness than others. An extensive outbreak of gastroenteritis associated with both rotavirus and a virulent strain of Shigella sonnei occurred in Rio de Janeiro in May 1980, affecting approximately 75% of both students and staff of a private school [32]. Rotavirus serotype 1 has been associated with an outbreak of diarrhea in a day-care nursery, also in Rio de Janeiro, affecting 60% of infants and nearly 7% of the staff; no enteropathogens other than rotaviruses could be identified in stool samples from patients with diarrhea [33]. Day-care was also found to present a potential risk-factor for rotavirus infection among infants attending an emergency room in São Paulo, Brazil [34]. Rotavirus G type 2 was associated with an outbreak of severe gastroenteritis affecting children and adults in a small city in southeastern Brazil, where contamination of the main water supply was the most likely source [35]. During July and August, 1977, an explosive water-borne outbreak of acute diarrhea due to rotavirus serotype 1 occurred in the isolated, nonimmune Tiriyó Indian tribe in North Pará. All agegroups were affected with an overall clinical attackrate that approached 90%. The proportion of symptomatic cases was greatest in young children [36]. The occasional contact of Indians with urban populations may accounts for the spread of virus in these remote settings, but studies are needed in order to establish whether or not there are other sources of infection, such as wild animals [37]. To date, a limited number of atypical group A rotavirus strains has been detected throughout Brazil. During a community-based longitudinal study in northern Brazil, 5 naturally occurring serotype 2/subgroup II rotavirus reassortants were identified [38]. An unusual avian-like rotavirus strain was detected among three hospitalized children in Belém, Brazil, reflecting a genomic rearrangement [39].

8 Rotavirus Infection in Brazil Group C rotaviruses have been detected among children with diarrhea in Rio de Janeiro [40] and Belém, Brazil [41]. In Belém, they were found to be the etiological agents of an outbreak in a daycare center in which a 38% attack-rate was reported (unpublished data). Subsequent studies on direct sequencing of PCR-amplified cdna corresponding to the VP6 gene of the Belém isolates confirmed the presence of Group C rotaviruses [42]. Clinical Features of the Disease Clinical studies conducted in Brazil have consistently demonstrated the greater severity of rotavirus diarrheal episodes (very often leading to moderate/severe dehydration) compared with those due to other agents [8, 20, 21]. In this context, the routine use of glucose-electrolyte rehydration therapy, recommended by the World Health Organization (WHO), is widely accepted and efficacious. Diarrheic (and dehydrated) children given oral treatment in northeastern Brazil showed a greater improvement in their condition than did those receiving intravenous rehydration [13]. Immune Response to Rotavirus Limited information is available concerning the immune response to rotavirus infection in Brazil. Findings from seroprevalence studies in northern [43], central [44] and southeastern [45, 46] regions indicate that at least 70% of children aged 4-5 years have been infected with rotavirus. In a longitudinal, community-based study carried out in Belém [47], it has been shown that passively transferred maternal antibody may last about 3 months; subsequently, a low level of rotavirus antibody appears at 6-7 months, reaching a peak at 11 months of age. From 1 year on, seropositivity in children gradually increases, reaching its highest value (about 80%) at age 34 months. In a recent study involving children from Recife, Pernambuco, northeastern Brazil [48], a high (80%) seropositivity rate was found among 0 to 2 month old infants, suggesting that future immunization against rotavirus should be started at 3 to 5 months of age when both seronegativity and partial immunity are maximal. Seroepidemiological studies involving several isolated tribes in the Amazon region yielded prevalence-rates of rotavirus antibody that ranged from 18% to 90% [49,50]. In most of these remote communities, the youngest (0-10 years of age) were consistently positive, suggesting that the virus may be endemic. To our knowledge, only two studies dealing with serotype-specific immune response have been carried out in Brazil. The potential for crossreaction between rotavirus serotypes 1 and 3 was noted during an outbreak of diarrhea among Indians inhabiting the north of Pará State, where infection by serotype 1 boosted pre-existing type 3 antibodies [36]. An evaluation of the immune response to rotavirus infection in 25 children who participated in a longitudinal study carried out in Belém yielded the following information: During primary infection, the serotype G1 generally induced a homotypic response while, in addition, serotypes G2 and G4 viruses induced a heterotypic response directed primarily to serotype G1 [51]. These results indicate that heterotypic immune response in a primary rotavirus infection may be an intrinsic property of the virus strain. No protection (p = 0.21) conferred by maternal milk against clinical rotavirus disease was noted in the Belém longitudinal study [20]. In a recent hospital-based survey conducted in the same area (unpublished data), however, when mothers relied on breastfeeding exclusively, there was a significant (p < 0.01) protection against rotavirus diarrhea in the infants. In Rio de Janeiro, experiments using a murine model indicated that a high proportion of CD8+ and/or CD4+CD8+ T cells in the intestines of suckling-mice may be associated with a greater susceptibility to rotavirus infections [52].

9 Rotavirus Infection in Brazil 291 Vaccine Use Against Rotavirus On the basis of the incidence data for rotaviral illness outlined above, Belém, North Brazil, was selected by the WHO as a suitable site to conduct a field trial with a rotavirus candidate vaccine. A tetravalent rhesus-human reassortant rotavirus (RRV-TV) vaccine (4 x 10,000 plaque-forming units (pfu)/dose) - manufactured at Wyeth-Ayerst Laboratories - was evaluated for safety, immunogenicity and efficacy in a prospective, randomized, double-blind, placebo-controlled trial involving 540 infants. The vaccine was well tolerated and only low grade fever seemed to be the most likely side effect on days 3, 4 and 5 after the first dose. An IgA antibody response to RRV- TV occurred in 60% of vaccinees and 33% of placebo recipients. On the other hand, neutralizing antibody responses to individual serotypes did not exceed 20% when measured by fluorescent focus reduction assay. The overall vaccine efficacy against any rotavirus diarrhea was 35% (Table 1). Protection during the first year of follow-up, when serotype G 1 predominated, was nearly 60%, but fell to 12% in the second year. It was also noted that vaccine efficacy may be somewhat greater for preventing severe illness than for all diarrheal episodes [53]. The results of the trial in Belém should encourage further evaluations of the RRV-TV vaccine in Brazil using increasing doses to at least 4 x 100,000 pfu, in an effort to achieve enhanced and prolonged protection. Protection against this severe, potentially life-threatening illness must be emphasized. In order to support the future introduction of these vaccines into routine use for immunization of children in Brazil (ideally if combined with oral polio vaccine), however, additional studies are needed on the molecular epidemiology of rotavirus infections throughout the country. This should include better information on the distribution of antigenic types of rotaviruses in Brazil and laboratory surveillance for the emergence of unusual strains (particularly genotype G5) as well as novel reassortants that may evolve from children with mixed infections [23, 28]. Table 1. Protective efficacy of RRV-tetravalent vaccine against all- and- pure* rotavirus diarrheal cases among children from Belém, Brazil. Adapted from Linhares et al., 1996 [53] RV diarrhea episodes, Placebo group (N) Vaccine group (N) % protection p** by year of follow-up Two years All cases Pure RV cases First year All cases Pure RV cases Second year All cases Pure RV cases * Cases in which rotavirus was the only enteropathogen found ** By analysis of variance N = number.

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