Alejandro A. Castello

Molecular Epidemiology of Group A Rotavirus Diarrhea among Children in Buenos Aires, Argentina, from 1999 to 2003 and Emergence of the Infrequent Genotype G12

ABSTRACT To examine the epidemiology of rotaviruses in Buenos Aires, Argentina, we screened 1,212 stool samples from children with diarrhea in the southern district of Buenos Aires from 1999 to 2003. We identified 187 samples (15.4%) that were positive for group A rotavirus by use of antigen enzyme-linked immunosorbent assay. Among these specimens, 112 were available for typing: 93 (83.0%) were single-type infections, 9 (8.0%) were mixed-type infections with more than one G or P type, and 10 (8.9%) were G and/or P nontypeable. In contrast to the findings in our last study, from 1996 to 1998, genotype P[4], G2 strains were almost completely absent and P[8], G1 and P[8], G4 strains were dominant, representing more than 80% of the G and P types found. Genotypes G2 and G9 were detected in few samples, and type G3 was completely absent. We identified several uncommon genotype G12 strains, representing the first detections outside of Asia and the United States, by sequencing. Using a genotype G12-specific reverse transcription-PCR, we identified eight (6.7%) positive samples for the 1999 to 2003 period. The high degree of sequence identity between recent G12 isolates from Argentina, the United States, and Asian countries suggests a relatively recent introduction(s) of these strains into humans from a common progenitor. The Argentinean G12 strains belonged to genotype P[9], similar to most of the recently described Asian G12 strains. The finding of G12 strains in several other regions of the world raises the possibility that G12 may be emerging globally and suggests that surveillance for this strain should be conducted routinely.

Rotavirus strain surveillance in Latin America: a review of the last nine years.

Background: Latin America will likely be the first area in the developing world where rotavirus vaccine will be introduced into the routine childhood immunization schedule. In anticipation of that goal, we reviewed the distribution of group A rotavirus genotypes in Latin America to understand the diversity of strains to be targeted by vaccines and to identify novel strains that may pose challenges for vaccines. Methods: We reviewed studies characterizing rotavirus strains in Latin America (published in English since 1995) that used molecular methods to type genes encoding the G and P outer capsid proteins, VP7 and VP4, and that reported data on >50 specimens. Results: Fifteen studies from 5 countries met our criteria. In total, 1989 samples were characterized; 12% (233) were mixed rotavirus infections with more than 1 strain, and 20% (402) were not fully typable. Of the remaining 1354 samples that were fully typed, 83% represented the 4 common strains: P[8],G1 (40%); P[4],G2 (30%); P[8],G3 (6%); P[8],G4 (7%). The unusual strains provide interesting insights into virus evolution: some strains (G5) were regionally common; the emerging G9 strains were widely distributed; many animal-human reassortants were present; and some common serotypes (G3 and G4) were of animal origin. Also an unusual G12 serotype was recently detected in Argentina. Conclusions: The common rotavirus serotypes should remain the prime targets for vaccine development. However, the changing profile of rare strains, animal-human reassortants and nontypable strains suggest that rotavirus is constantly evolving. Laboratory surveillance is needed to monitor rotavirus strains now in circulation and to detect those that might escape the immunity induced by vaccines or represent vaccine strains entering the environment.

Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration

Group A rotaviruses (RV-A) are the most common agents of viral gastroenteritis in children worldwide. The goal of this study was to compare two different methods to concentrate RV-A from sewage samples and to improve the detection and quantification of RV-A using a multiplex quantitative PCR assay with an internal control. Both RV-A and the internal control virus, bacteriophage PP7, were seeded into wastewater and then concentrated using either an ultrafiltration-based adsorption-elution protocol or an ultracentrifugation-based protocol. Real time multiplex quantitative PCR was used to quantify the purified RV-A and PP7, and the results of the multiplex assay were compared with the results of the monoplex assays. The ultracentrifugation-based method had a mean recovery rate of 47% (range: 34-60%), while the ultrafiltration-based adsorption-elution method had a mean recovery rate of 3.5% (range: 1.5-5.5%). These results demonstrate that ultracentrifugation is a more appropriate method for recovering RV-A from wastewater. This method together with the multiplex qPCR assay may be suitable for routine laboratory use.

Characterization of genotype P[9]G12 rotavirus strains from Argentina: high similarity with Japanese and Korean G12 strains.

The circulation of the unusual P[9]G12 strains was previously reported in suburban Buenos Aires, Argentina and in Far Eastern Asian countries. To examine genetic relationships of these strains the genes coding VP7, VP4, and NSP1 from two Argentine, one Japanese and one Korean P[9]G12 isolates were sequenced and their overall genome relatedness was determined by liquid hybridization. In addition, liquid hybridization was used to compare this group of strains to the previous G12 isolates L26 and Se585, and prototype Wa, DS‐1, and AU‐1 strains. The genomes of the Argentinean, Japanese and Korean strains were virtually indistinguishable by hybridization assays, suggesting very high sequence relatedness for all 11 segments. Hybridization assays also demonstrated that these four strains belong to the AU‐1 genogroup and that their genetic relationship with rotaviruses L26 and Se585 is limited to the VP7 gene. The VP7, VP4, and NSP1 genes of the Argentinean, Japanese and Korean strains were highly homologous to each other and to Thai strain T152 (∼99% identity). These results together with the report of a similar strain detected during 2003 in Brazil are consistent with a recent importation and dissemination of the G12 strains from Far Eastern countries into South America. Increasing reports from several regions of the world demonstrating a variety of different G12 reassortant strains suggests that routine surveillance for this serotype should be conducted to determine its potential for global emergence. J. Med. Virol. 81:371–381, 2009.

Characterization of human group C rotavirus in Argentina

A survey was conducted for identification of human group C rotaviruses in stool specimens taken from children suffering diarrhea in suburban Buenos Aires regions. Among 90 true negative group A samples as defined by ELISA, RT‐PCR and PAGE, five were positive by group C specific RT‐PCR (VP7 and VP6 genes) and three of these samples exhibited the characteristic 4‐3‐2‐2 dsRNA pattern of group C rotavirus. These results were further confirmed by electron microscopy and by ELISA for detection of group C VP6 specific antigens. Sequence analysis of the VP7 gene from one of these isolates revealed a 97.3–98.6% nucleotide identity and up to 99.1% protein homology with human group C rotavirus strains found scattered throughout the last ten years in other countries. Conversely, similar analysis performed with porcine strains showed a much lower homology degree both at the nucleotide (75.5% nucleotide identity) and amino acid level (85.5% protein homology). Detection of group C rotavirus in children with acute diarrhea in Argentina extends the identification range of this agent in the region and is consistent with previous reported data that demonstrate a global distribution of this virus. J. Med. Virol. 62:199–207, 2000.

A rapid method to produce high yields of purified rotavirus particles

A rapid purification method of rotavirus particles to high yield retaining the double shelled structure of infectious virus is described. Group A rotavirus (UK strain) was concentrated through a cushion of colloidal silica (rho=1.10 g/cm(3)) or by precipitating with polyethylene glycol 8000. After concentration, infectious rotavirus was cleared from host cell proteins by density equilibrium centrifugation in gradients of colloidal silica using near vertical rotors. Characterisation of purified virus assessed by electron microscopy and poliacrylamide gel electrophoresis (PAGE) revealed the typical wheel shape structure of rotavirus particles and the presence of the 11 segments of dsRNA arranged in the 4-2-3-2 pattern. Presence of rotavirus structural proteins including VP6, VP4 and VP7 from the outer shell, was demonstrated by SDS-PAGE and Western blot using polyclonal and VP6-specific monoclonal antibodies. This method achieved a approximately 1500 fold purification, which retained approximately 80% infectivity depending on the concentration protocol used, while yielding 160 microg of viral protein per each litre of infected cell culture medium. The time required for the isopycnic centrifugation was only 25 min and the entire completion of the method required 3.5 h. The method is simple technically and applicable to the purification of large as well as minute amounts of virus.

HSV-1 amplicon vectors launch the production of heterologous Rotavirus-like particles and induce Rotavirus-specific immune responses in mice

Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines.

Measles Virus-Specific Antibody Levels in Individuals in Argentina Who Received a One-Dose Vaccine

ABSTRACT In spite of active measles virus (MV) vaccination strategies, reemergence continues to occur, impairing global eradication programs. The immune status against measles was evaluated in 350 vaccinated healthy Argentine children and teenagers who received a single dose of the MV Schwarz strain Lirugen vaccine (Aventis Pasteur). Sera were assessed for immunoglobulin G (IgG) antibodies by a commercial enzyme immunoassay (EIA) (Enzygnost; Behring), an in-house EIA, and neutralization EIA. Results obtained with these methods showed a marked decline in IgG level with increasing age. At 1 to 4 years of age, 84% of children had IgG antibodies above 200 mIU/ml, conventionally accepted as protective levels, whereas only 32% of older children and teenagers had antibody levels exceeding 200 mIU/ml. Moreover, the MV IgG content in the teenage group was significantly lower than the IgG antibody level of the group of younger children (P < 0.0001). In contrast, screening for IgG antibody levels to inactivated tetanus vaccine showed that, on average, 80% of this population was fully protected and that this high level of protection remained through the teenage years. This study suggests that within this population a considerable proportion of individuals had low measles antibody levels that may be insufficient to protect against reinfections or clinical disease.

Surveillance of group A Rotavirus in Buenos Aires 2008–2011, long lasting circulation of G2P[4] strains possibly linked to massive monovalent vaccination in the region

BACKGROUND Group A rotaviruses (RVA) are the most frequent single etiological agents of severe diarrhea in infants. Since 2006 RVA vaccines have been introduced in national schedules of middle and high income countries with substantial declines in rotavirus associated disease burden. However, surveillance must be maintained to, eventually, detect emerging types or variants selected by the new pressure imposed by vaccination. OBJECTIVES To analyze the molecular epidemiology of group A rotavirus after vaccine introduction in the region in the context of data from more than 15 years of continuous surveillance in Buenos Aires. STUDY DESIGN RVA positive diarrhea samples collected in Buenos Aires from 2008 to 2011 were genotyped by RT-PCR. Selected samples were sequenced to gain insight on evolution of common and globally emerging human RVA strains. RESULTS Lineage III G12P[8] strain emerged in 2008 in Buenos Aires and shared co-dominancy with G3 strains during 2009. An atypical long lasting circulation of G2P[4] strains since 2004 reached rates around 80% in 2011 in Buenos Aires. Sequencing of the VP7 and VP4 genes of representative G2P[4] isolates suggests Brazil as the origin of the 2010-2011 strains. CONCLUSIONS Globally emergent G12 lineage III strains could be established as dominant strains in a very populated area in two years since emergence. In this work it was also shown that the persistence of G2P[4] strains during 8 years could be related to massive immunization with the monovalent vaccine in the region.

Prevalence of VP4 and VP7 genotypes of human rotavirus in Ecuadorian children with acute diarrhea.

The objective of the present study was to determine rotavirus etiology and prevalence of the different rotavirus serotypes in Ecuadorian children younger than 5 years of age with gastroenteritis. Children (729) less than 5 years of age with acute diarrhea from either public or private primary health care centers in 10 different provinces of Ecuador, between March 2006 and August 2006 were included in the study. Rotavirus infection was diagnosed using a commercial immunoenzymatic test. Rotavirus isolated from stool samples was genotyped. Rotavirus was detected in the feces of 269 of the 729 children (37%) with diarrhea. The most prevalent G genotypes were G9 (46.1%) and G2 (27.2%), while the predominant P genotypes were P[8] (57%) and P[4] (29.5%). Among the single infections, the predominant P/G combinations were: P[8]G9 (56.9%) and P[4]G2 (32.6%). The present countrywide survey is one of the major studies for one single season in Latin America and the first in its class in Ecuador. The value of expanding laboratory capability throughout Latin America in order to monitor rotavirus strains over time, with special attention directed at those strains obtained from children who experience vaccine failure, is critical. Only continuous monitoring of rotavirus disease burden and genotype surveillance will provide this information. J. Med. Virol. 80:1106–1111, 2008.