Aptamer-quantum dots platform for SARS-CoV-2 viral particle detection by fluorescence microscopy.

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.

Correlation between rotavirus A genotypes detected in hospitalized children and sewage samples in 2006, Córdoba, Argentina.

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.

Antigenicity and immunogenicity of rotavirus VP6 protein expressed on the surface of Lactococcus lactis.

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.

Rotavirus VP7-gene selection during coinfections in CaCo-2 cells.

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.

Detection of group a human rotavirus G9 genotype circulating in Córdoba, Argentina, as early as 1980.

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.

Molecular epidemiology of group A rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to 2003 and emergence of the infrequent genotype G12.

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.

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.