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The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps. Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein. Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Martin Radrizzani & Mariano Grasselli has patent pending to Universidad Nacional de San Martin/Universidad Nacional de Quilmes., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The COVID-19 pandemic highlighted testing inequities in developing countries. Lack of lateral flow test (LFT) manufacturing capacity was a major COVID-19 response bottleneck in low- and middle-income regions. Here we report the development of an open-access LFT for SARS-CoV-2 detection comparable to commercial tests that requires only locally available supplies. The main critical resource is a locally developed horse polyclonal antibody (pAb) whose sensitivity and selectivity are greatly enhanced by affinity purification. We demonstrate that these Abs can perform similarly to commercial monoclonal antibodies (mAbs), as well as mAbs and other pAbs developed against the same antigen. We report a workflow for test optimization using nasopharyngeal swabs collected for RT-qPCR, spiked with the inactivated virus to determine analytical performance characteristics as the limit of detection, among others. Our final prototype showed a performance similar to available tests (sensitivity of 83.3% compared to RT-qPCR, and 90.9% compared to commercial antigen tests). Finally, we discuss the possibility and the challenges of utilizing affinity-purified pAbs as an alternative for the local development of antigen tests in an outbreak context and as a tool to address inequalities in access to rapid tests.

Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Background: SARS-CoV-2 infection is associated with a wide range of clinical manifestations and severity. Pediatric cases represent <10% of total cases, with a mortality rate below 1%. Data of correlation between SARS-CoV-2 viral load in respiratory samples and severity of disease in pediatric patients is scarce. The cycle threshold (CT) value for the detection of SARS-CoV-2 could be used as an indirect indicator of viral load in analyzed respiratory samples., Objective: The aim of this study was to describe CT values and their correlation with clinical manifestations, epidemiology and laboratory parameters in pediatric patients with confirmed COVID-19., Methods: In this observational, retrospective, analytic and single-center study we included patients under 15 years with confirmed COVID-19 by RT-PCR SARS-CoV-2 admitted to the Isidoro Iriarte Hospital (Argentina) between March 1st 2020 and April 30th 2021., Results: 485 patients were included, the distribution according to disease severity was: 84% (408 patients) presented mild disease, 12% (59 patients) moderate disease and 4% (18 patients) severe disease. Patients with moderate and severe illness had an increased hospitalization rate, prolonged hospitalization, higher frequency of comorbidities and oxygen and antibiotics use. CT values, that could be used as an indirect measure of viral load, was associated with severity of clinical manifestations and age under 12 months. No patient required admission to PICU nor mechanical ventilation. No deaths were registered., Conclusions: In this study, the viral load of SARS-CoV-2 in respiratory samples, determined by the cycle threshold, was significantly correlated with moderate to severe cases and with age., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brizuela, Goñi, Cardama, Zinni, Castello, Sommese and Farina.)

Group A rotaviruses (RVA) are the most frequent etiological agents causing severe diarrhea in infants and surveillance of genotype, and genetic characteristics of circulating strains are necessary in order to evaluate vaccine programs. The objectives of this work were to describe G and P genotype from 2012 through 2014 in Buenos Aires, Argentina completing an overview of 19 years of genotype surveillance in our region and to characterize an emerging G1P[8] strain associated with severe cases and five fatalities in 2014. We performed genotyping by RT-PCR. The sequencing of several genes, phylogenetic analyses, and comparative epidemiological data were used to know the origin and phylogenetic relationships of the emerging G1P[8] strain. Along with this report, 19 years of continuous RVA genotype surveillance in Argentina in the pre-vaccine era was covered. During the last year of this surveillance, 2014, a significantly increased incidence of RVA associated gastroenteritis was related to the reemergence of G1P[8] strains, being these ones detected in low frequency in the last nine years. Interestingly, the patients affected were significantly older when compared with those from the last six seasons. Additionally, phylogenetic analysis of several genes infer that these G1P[8] strains were closely related to Asian strains circulating during 2012 and 2013. In addition to this, the suggested extra continental origin for the 2014 G1P[8] strains and the very low circulation of G1 type during nine years probably explain the increased incidence and severity in the gastroenteritis cases and the particular epidemiologic characteristics. In conclusion, this work gives us a whole panorama of the pre-vaccine era of the RVA molecular epidemiology in the most populated region of Argentina. In this way, this work inspires us to continue with this type of studies in the post-vaccination era., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery., Competing Interests: The authors have declared that no competing interests exist.

Group C Rotavirus (RVC) has been associated globally with sporadic outbreaks of gastroenteritis in children and adults. RVC also infects animals, and interspecies transmission has been reported as well as its zoonotic potential. Considering its genetic diversity and the absence of effective vaccines, it is important and necessary to develop new generation vaccines against RVC for both humans and animals. The aim of the present study was to develop and characterize an HSV-1-based amplicon vector expressing a human RVC-VP6 protein and evaluate the humoral immune response induced after immunizing BALB/c mice. Local fecal samples positive for RVC were used for isolation and sequencing of the vp6 gene, which phylogenetically belongs to the I2 genotype. We show here that cells infected with the HSV[VP6C] amplicon vector efficiently express the VP6 protein, and induced specific anti-RVC antibodies in mice immunized with HSV[VP6C], in a prime-boost schedule. This work highlights that amplicon vectors are an attractive platform for the generation of safe genetic immunogens against RVC, without the addition of external adjuvants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

DCs very potently activate CD8(+) T cells specific for viral peptides bound to MHC class I molecules. However, many viruses have evolved immune evasion mechanisms, which inactivate infected DCs and might reduce priming of T cells. Then MHC class I cross-presentation of exogenous viral Ag by non-infected DCs may become crucial to assure CD8(+) T cell responses. Although many vital functions of infected DCs are inhibited in vitro by many different viruses, the contributions of cross-presentation to T cell immunity when confronted with viral immune inactivation in vivo has not been demonstrated up to now, and remains controversial. Here we show that priming of Herpes Simplex Virus (HSV)-, but not murine cytomegalovirus (mCMV)-specific CD8(+) T cells was severely reduced in mice with a DC-specific cross-presentation deficiency. In contrast, while CD8(+) T cell responses to mutant HSV, which lacks crucial inhibitory genes, also depended on CD8α(+) DCs, they were independent of cross-presentation. Therefore HSV-specific CTL-responses entirely depend on the CD8α(+) DC subset, which present via direct or cross-presentation mechanisms depending on the immune evasion equipment of virus. Our data establish the contribution of cross-presentation to counteract viral immune evasion mechanisms in some, but not all viruses.

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.

The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Routine rotavirus A (RV-A) surveillance is based on clinical cases, so only symptomatic infections are reported. The objective of this study was to determine whether the RV-A genotypes and cold seasonal pattern described in patients with diarrhea is reflected by sewage surveillance, which could be representative of the RV-A genotypes circulating in the population. The genotype distribution of RV-A in effluent samples from a local sewage treatment plant was compared to those from local clinical cases. A total of 52 sewage samples and 70 stool specimens from children with acute non-bacterial diarrhea were collected from January to December 2006. The effluent specimens were concentrated and RNA extracts from concentrated sewage and clinical samples were genotyped for the rotavirus VP7 gene. The proportional distribution of the RV-A G-genotypes in sewage and clinical samples during the cold season was similar: G1 accounted for 26.6% of the typed sewage isolates and 28.8% of the clinical infections; G3 type accounted for 21.9% and 25.8%; G2 type 15.6% and 10.6%; G4 type 17.2% and 21.2%; G8 type 1.6% and 0%; and the G9 type 17.2% and 13.6%, respectively. A similar picture of RV-A genotype detection was obtained in sewage samples collected during the cold and warm seasons. The results indicate that there is a correlation between genotypes of RV-A isolates from human diarrheic patients and of those from sewage samples. In addition, sewage monitoring highlighted the uniform all-year RV-A circulation, which was in contrast to the peak incidence of RV-A infection in the community., ((c) 2010 Wiley-Liss, Inc.)

Detection and characterization of group A rotavirus in Buenos Aires, Argentina, was conducted on 710 fecal samples from children 0-15 years old collected between 2004 and 2007. Rotavirus was detected in 140 (19.7%) samples with G9P[8] (30.0%) and G2P[4] (21.4%) as the most common genotypes. Mixed (G and/or P) infections accounted for 17.9% of the samples and the emerging G12 strain was detected during 2004 (3.5%) and 2007 (2.5%). Genotype G2 was the most prevalent during 2004 (43.9%) and 2007 (57.5%) and G9 during 2005 (58.0%) and 2006 (61.5%). Analysis of genotype prevalences from studies performed since 1996 in the same area showed striking natural fluctuations in G and P genotype frequencies. In particular, G2P[4] strains disappeared after 1999 and reemerged in 2004 to become the predominant strain by 2007 with a concomitant major decrease in G1P[8] prevalence. The VP7 genes from Argentinian G9 and G2 strains were sequenced and phylogenetic analysis was conducted in order to compare with sequences from strains isolated in regional countries reported previously. Several changes in the deduced amino acid sequence in antigenic regions of the VP7 protein from Argentinian and Brazilian strains were identified compared to vaccine strains. Overall, this study revealed relationships in the circulation of rotavirus strains in South American countries and major replacements in dominant genotypes, including the virtual disappearance of G1P[8] strains in a non-vaccinated population. High numbers of mixed infections speeding up evolution, circulation of rare serotypes, and antigenic drift could, eventually, become challenges for new vaccines., ((c) 2010 Wiley-Liss, Inc.)

The role of group C rotaviruses as a cause of diarrhea was examined among children <17 years of age admitted to a Hospital in a suburban area of Buenos Aires, Argentina between 1997 and 2003. A total of 1,579 fecal samples were screened for group A (RVA) and C (RVC) rotaviruses by two in-house ELISA methods at Quilmes University (UNQ-ELISA). Samples positive, doubtful and negative by RVC specific UNQ-ELISA (n = 246) were examined further for RVC by another in-house ELISA (CDC-ELISA), electron microscopy, RT-PCR, nested PCR, and Southern hybridization. Sensitivity, specificity, and predictive values for each test were determined. While the sensitivity was comparable for the nested PCR and CDC-ELISA methods (82.5%), the molecular methods were slightly more specific. Poorly preserved particles were often seen in fecal samples, suggesting that degradation of RNA could be a factor influencing the performance of molecular methods. The incidence of RVC was estimated to be 3% without apparent differences among seasons. RVC infected patients had a significantly (P < 0.001) higher median age (6 years vs. 1 year) than those with RVA infection. Sequence of the RVC VP7 gene from six Argentinean strains and sequences reported previously in different countries showed high nucleotide (94.4-99.9%) sequence identities, indicating a high degree of conservation for human RVC VP7 genes among strains collected on five continents over a period of 17 years. These findings indicate that RVC is a significant cause of diarrhea and it is necessary to develop simple and sensitive serological methods for its detection.

A previous rotavirus epidemiological survey in Córdoba, Argentina, revealed an unusually high frequency of mixed G-type infections (41.5%). The genotype distribution of those mixed infections showed that the most prevalent G-type combinations were G1+G4 (65.0%), G1+G2 (12.5%), G2+G4 (3.1%) and G1+G9 (2.5%). In the present study we analyzed the competitive growth in CaCo-2 cell cultures of strains from the most frequent rotavirus G-type coinfections in order to explain some aspect of the dynamic of G-type replacement along the time. Our results indicated that G1-type was preferentially selected compared with G2 and G9-genotypes, meanwhile, G1-G4 coinfections showed an efficient co-amplification of both types. Interestingly, this mirrored the high detection rates of both genotypes as single and mixed infections (G1+G4, 65.0%) in our region. On the other hand, G2-type revealed a better amplification rate with respect to G4-type. Fluctuant rates in the prevalence of different genotypes usually observed along the time could, in part, be explained by successive replacement of strains with different growth characteristics. We hypothesized that one aspect of these different fitnesses can be measured as differential growth in culture of the strains contained in the sample of a mixed infection. Our findings here provide the first data supporting the validity of the competitive replication in vitro to better understand rotavirus G-type circulation patterns.

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 ( approximately 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., ((c) 2008 Wiley-Liss, Inc.)

The incidence of human rotavirus G types was determined over a 25-year period (1979-2003) by using reverse transcription-PCR (RT-PCR) to examine 519 stool specimens found to be positive for rotavirus by enzyme linked immunosorbent assay (ELISA) or polyacrylamide gel electrophoresis (PAGE). These stool samples were obtained from children under 3 years old who had been treated for acute diarrhea at public hospitals in Córdoba, Argentina. The present study describes the continued circulation of the common human G types G1 (53.8%), G2 (10.2%), G3 (4.4%), and G4 (27%), and also the detection of the unusual types G8 (0.5%) and G9 (4.2%). Genotype G9 was detected during the 1980-1988 and 1997-2003 periods at relatively low rates. Rotavirus G types distribution was independent of age (1-18 months), gender or out-patient or in-patient status. Unexpectedly, 44.6% of mixed infections were detected, involving common and unusual genotypes. Overall, 95.4% of the typed strains belonged to the most prevalent human serotypes (G1-G4) but the detection of G9 infection throughout this study period highlights the importance of this serotype as a human pathogen., (2006 Wiley-Liss, Inc.)

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.

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.

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.

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.

The incidence of human group C rotavirus infections among children and adults in Buenos Aires was evaluated by enzyme linked immunosorbent assays (ELISA) based on recombinant group C VP6 protein (Cowden strain). A total of 976 stool samples taken from patients (ages 6 months to 15 years) with acute diarrhea were tested for the presence of group C rotavirus. Among these, only 10 (1.02%) were group C rotavirus positive by enzyme-linked immunosorbent assay (ELISA) confirmed by absorption with group C VP6 antibodies and by RT-PCR for both VP6 and VP7 genes. The average age (5.86 years) was significantly superior to that in group A-infected patients (1.63 years). Previous exposure to this virus was assessed by detecting specific IgG in sera taken from healthy individuals grouped by age. Of 844 sera tested, 425 (50.3%) were group C IgG positive by ELISA, confirmed by Western blot analysis. The rates of IgG positivity for group A and C rotaviruses during the first years of life indicated that infections with group C are frequent in older children (3-5 years), whereas group A infections are prevalent in infants and young children (6-18 months). This study shows that group C rotavirus infections in Argentine children occur later in life than group A and are relatively common in spite of the low detection rate of this virus., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2000 Wiley-Liss, Inc.)