Rotavirus Isolates Research Articles

Novel Universal Recombinant Rotavirus A Vaccine Candidate: Evaluation of Immunological Properties.

Rotavirus infection is a leading cause of severe dehydrating gastroenteritis in children under 5 years of age. Although rotavirus-associated mortality has decreased considerably because of the introduction of the worldwide rotavirus vaccination, the global burden of rotavirus-associated gastroenteritis remains high. Current vaccines have a number of disadvantages; therefore, there is a need for innovative approaches in rotavirus vaccine development. In the current study, a universal recombinant rotavirus antigen (URRA) for a novel recombinant vaccine candidate against rotavirus A was obtained and characterised. This antigen included sequences of the VP8* subunit of rotavirus spike protein VP4. For the URRA, for the first time, two approaches were implemented simultaneously-the application of a highly conserved neutralising epitope and the use of the consensus of the extended protein's fragment. The recognition of URRA by antisera to patient-derived field rotavirus isolates was proven. Plant virus-based spherical particles (SPs), a novel, effective and safe adjuvant, considerably enhanced the immunogenicity of the URRA in a mouse model. Given these facts, a URRA + SPs vaccine candidate is regarded as a prospective basis for a universal vaccine against rotavirus.

Results and prospects of rotavirus immunisation in the Russian Federation

Scientific relevance. Rotavirus disease and its consequences remain a leading vaccine-preventable cause of mortality in young children. Russia has not yet included rotavirus immunisation in the national vaccination schedule, but paediatric rotavirus vaccines are provided under several regional immunisation programmes.Aim. The authors aimed to review literature covering Russian clinical evidence of the safety and effectiveness of rotavirus vaccines and to analyse the prospects for the development of the national immunisation programme for young children using the available rotavirus vaccines and considering promising pipeline ones.Discussion. Local epidemiological studies on a range of pathogens suggest that rotaviruses are the most common cause of acute intestinal infections, especially in children under 3 years of age. Since Russia’s first rotavirus vaccine approval in 2012, the healthcare system has acquired sufficient experience with rotavirus vaccines to evaluate the safety and effectiveness of rotavirus immunisation. Long-term monitoring of the rotavirus A genotypes circulating in the country shows that currently available rotavirus vaccines cover the majority of identified rotavirus isolates. Local observational studies confirm the favourable safety profile of rotavirus vaccines and demonstrate notable effectiveness of vaccination. In the regions with high immunisation coverage, the overall morbidity has declined dramatically in vaccinated children compared to unvaccinated children; some of these regions have reported only individual hospital visits or admissions for acute intestinal infections.Conclusion. This review demonstrates that rotavirus immunisation should be included in the Russian National Immunisation Schedule.

Rotavirus genotypes in children under five years hospitalized with diarrhea in low and middle-income countries: Results from the WHO-coordinated Global Rotavirus Surveillance Network.

Rotavirus is the most common pathogen causing pediatric diarrhea and an important cause of morbidity and mortality in low- and middle-income countries. Previous evidence suggests that the introduction of rotavirus vaccines in national immunization schedules resulted in dramatic declines in disease burden but may also be changing the rotavirus genetic landscape and driving the emergence of new genotypes. We report genotype data of more than 16,000 rotavirus isolates from 40 countries participating in the Global Rotavirus Surveillance Network. Data from a convenience sample of children under five years of age hospitalized with acute watery diarrhea who tested positive for rotavirus were included. Country results were weighted by their estimated rotavirus disease burden to estimate regional genotype distributions. Globally, the most frequent genotypes identified after weighting were G1P[8] (31%), G1P[6] (8%) and G3P[8] (8%). Genotypes varied across WHO Regions and between countries that had and had not introduced rotavirus vaccine. G1P[8] was less frequent among African (36 vs 20%) and European (33 vs 8%) countries that had introduced rotavirus vaccines as compared to countries that had not introduced. Our results describe differences in the distribution of the most common rotavirus genotypes in children with diarrhea in low- and middle-income countries. G1P[8] was less frequent in countries that had introduced the rotavirus vaccine while different strains are emerging or re-emerging in different regions.

Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates


 Background:
 Viruses are being used as alternative and complementary tools for treating cancers. Oncolytic viruses exhibit tumor tropism, ability to enhance anti-tumor immunity and ability to be used in combination with conventional chemotherapy and radiotherapy. We have recently selected some rotavirus isolates which are adapted to efficiently infect and kill tumor cell lines.
 
 
 Aim:
 We tested five tumor cell-adapted rotavirus isolates for their ability to infect the human adenocarcinoma cell line MCF-7.
 
 
 Methods:
 Cell surface membrane-associated proteins mediating virus particle attachment were characterized using ELISA, immunoprecipitation, FACS analysis, and antibody blocking.
 
 
 Results:
 It was found that heat shock proteins (HSPs) such as Hsp90, Hsp70, Hsp60, and Hsp40 are expressed on the cell surface forming complexes with protein disulfide isomerase (PDI), integrin β3, and heat shock cognate protein 70 (Hsc70) in lipid raft microdomains. Interaction of rotavirus isolates with these cellular proteins was further confirmed by a competition assay and an inhibition assay involving the HSPs tested.
 
 
 Conclusion:
 Our findings suggest that the tumor cell-adapted rotavirus isolates studied here offer a promising tool for killing tumor cells, thus encouraging further research into this topic, including animal models.

Genome characterization of a Turkish bovine rotavirus field isolate by shotgun metagenomics.

A bovine rotavirus (BRV) isolate from Kirsehir was isolated from feces of a neonatal calf with diarrhea, identified, and sequenced by shotgun sequencing. Its genotype constellation is G10-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The structural genes and the non-structural genes NSP1, NSP3, and NSP4 of the Kirsehir isolate were similar in sequence to those of BRVs identified in Turkey. However, VP2, NSP2, NSP4, and NSP5/6 showed similarity to those of rotaviruses from different animal hosts. These findings not only expand our current understanding of the diversity of rotaviruses but also contribute to our understanding of the evolution of rotaviruses at both the national and global levels and reinforce the significance of conducting further research on rotaviruses in Turkey.

Circulation of Rotavirus Genotypes and their Distribution among Vaccinated and Non-vaccinated Children in Abuja, Nigeria

Introduction: Nigeria had planned to introduce the rotavirus vaccine in the National Immunisation Programme in 2014 but this has yet to be done. Nigeria has the continent’s highest mortality due to diarrhoeal diseases with little information on specific prevalent genotypes. The main objectives of the study were to identify the predominant rotavirus genotypes and to examine the effects of existing local vaccination programmes on prevailing rotavirus genotypes and on preventing rotavirus diarrhoea.
 Methodology: A one-year prospective descriptive study of children under 5 with acute diarrhoea was conducted from September 2012 to August 2013. Children with acute diarrhoea attending three government hospitals and one private hospital were recruited. Children without diarrhoea were also recruited as a control group. Rotavirus ELISA and RNA extraction were done with commercially available kits and positive samples were subjected to RT-PCR and electrophoresis to determine VP7 (G) and VP4 (P) genotypes.
 Results: Stool samples were collected from 1240 (93.3%) participants, of whom 957 (77.0%) were ambulatory, 123 (9.9%) hospitalised and 160 (12.8%) controls without diarrhoea. Rotavirus-ELISA was positive among 123 (11.4%) children with diarrhoea. The predominant VP7 genotypes were G2 (n=33, 26.4%) followed by G9 (n=24, 19.2). The main VP4 (P) genotypes included P [4] (n=45, 36.0%) followed by P [6] (n=40, 32.0%). The predominant genotype combinations found were G2 P [4] (n=21, 16.8%), G3 P [6] and G1 P [6] (each n=16, 12.8%), and G12 P [8] (n=15, 12.0%). Very few mixed infections were found in only one government hospital 4 (6.4%). Among 94 unvaccinated children with rotavirus isolates that were genotyped, G2 P [4] (n=19, 20.2%) and G1 P [6] (n=16, 17.0%) were predominant. Among 12 vaccinated children, 2 isolates each (16.6%) were found of G3 P [6], G9 P [4], G12 P [8] and G2 P [NT] with no G1 isolates.
 Conclusion: The emergence of new genotypes such as G 12 P [8] found in this study emphasizes the need for continued prospective monitoring of rotavirus at the molecular level to detect new threats to vaccine programmes in future.

Characterization of bovine rotavirus isolates from diarrheic calves in Türkiye.

Neonatal calf diarrhea, which is the most common cause in calf deaths, leads to significant economic losses in dairy farming around the world. Diarrhea develops due to infectious and non-infectious reasons. Group A Rotaviruses (RVA) are the leading and predisposing factor for acute neonatal gastroenteritis. In this study, 20 diarrheic fecal samples were collected from one farm in Balıkesir province of Turkey. During virus isolation, a total of 2 stool samples were detected to produce cytopathogenic effects in MA-104 cell line. The two samples (RV-36, RV-38) were tested positive with antigen ELISA kits detecting RVA antigens. In order to detect the presence of rotavirus viral nucleic acid in cell supernatants, VP6 gene region-specific RT-PCR test was performed and the samples RV-36 and RV-38 were positive for RVA viral nucleic acid. By RT-PCR using genotype specific primers, both the isolates RV-36 and RV-38 formed amplicons compatible with G10 and P[11] genotypes of RVA. RVA nucleic acids segments were also visualized by poliacrilamide gel electrophoresis (PAGE) method. The phylogenetic tree constructed according to the VP6 gene region showed that these isolates were in the Rotavirus A group and in the I2 cluster sameas other bovine and some human RVA isolates. Succesful isolation of RVA G10P[11] was echieved inthe cattle farm. As rotaviruses play the most important role in the etiology of diarrhea in newborn calves respected genotype G10P[11] should be considered in selection of the vaccines applied to the dams. Those isolates can be further evaluated as vaccine candidate.

Isolation and Characterization of Distinct Rotavirus A in Bat and Rodent Hosts.

Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.

Isolation, Propagation and Genotyping of Human Rotaviruses Circulating among Children with Gastroenteritis in Two Egyptian University Hospitals.

Simple SummaryGroup A rotaviruses are the most common cause of acute gastroenteritis affecting Egyptian children under the age of five, with symptoms ranging from asymptomatic infection to severe dehydration or death. In the present work, diarrheal samples from Egyptian children admitted to gastrointestinal pediatric wards of two main governmental hospitals were collected and molecularly analyzed for Group A rotavirus. Our findings revealed that rotaviruses accounted for more than one-sixth of all cases under study, peaking in the winter. G1P[8] was the most prevalent rotavirus genotype in this study. The two cell lines used in our work coherently isolated and propagated rotavirus strains. Continuous rotavirus detection and genome sequencing of the successfully isolated strains will be recommended in the future in order to support the control of such viruses, and tackle the problem in Egypt.The most prevalent cause of infectious neonatal diarrhea is Group A rotavirus (RVA). Unfortunately, there is a dearth of data on the incidence of rotavirus-associated infections among Egyptian children. The present study aimed to isolate, propagate, and genotype human rotaviruses circulating among Egyptian children with acute gastroenteritis admitted to two main university pediatric hospitals, Abo El-Reesh and El-Demerdash, over two consecutive winters, 2018–2020. Diarrheal samples (n = 230) were screened for Group A rotavirus RNA using RT-PCR assay. In positive samples (n = 34), multiplex semi-nested PCR was utilized to determine G and P genotypes. Thirty-four (14.8%) of the collected samples tested positive. The genotype distribution revealed that G1P[8] was the predominant rotavirus genotype throughout the current study. All rotavirus-positive fecal samples were passaged twice on human colorectal adenocarcinoma cell line (Caco-2) and rhesus monkey kidney epithelial cell line (MA104). Both cell lines could successfully isolate 14.7% (n = 5 out of 34) of the identified strains; however, Caco-2 cell line was shown to be more efficient than MA104 in promoting the propagation of human rotaviruses identified in Egyptian children’s feces.

Prevalence of rotavirus isolates in the stools of under-5 children presenting with diarrhoea at University Maiduguri Teaching Hospital, Borno State.

Diarrhoea is a major cause of childhood morbidity and mortality in developing countries including Nigeria. Rotavirus is a leading cause of acute watery diarrhoea in children under 5 years of age. The aim of the study is to determine the prevalence of rotavirus diarrhoea in children less than 5 years old presenting with watery diarrhoea at the University of Maiduguri Teaching Hospital. The cross-sectional study was carried out at University of Maiduguri Teaching Hospital (UMTH), a referral tertiary centre for northeast Nigeria and neighbouring Cameroon, Chad, and Niger Republic. Study population were children under five presenting to UMTH with acute diarrhoea. Freshly passed stool was collected from each participant in a universal sterile container and transported to the department of medical microbiology laboratory UMTH, Rotavirus antigen was detected using Rota - dipstick an immunochromatographic test. The positive samples were subjected to RT-PCR to detect the VP 7 gene of the dsRNA. SPSS Version 25. The prevalence was found to be 14.5% in the population studied and was highest among children below 1 year of age. This study has confirmed that rotavirus is an important cause of childhood diarrhoea. The burden of childhood diarrhoea can be reduced by introduction of vaccines, and children of 1 year old and younger will benefit from this vaccine as most study participants have not been vaccinated. Creating awareness on prevention and control of this infection with mass vaccination will go a long way in reducing the prevalence and mortality rate of rotavirus diarrhoea.

Molecular Variation between RT-PCR Detected Rotavirus Infection of Naturally Diarrheic Neonatal Calves and Rotavirus Strains of Commercial Vaccines

Neonatal diarrhea is the main cause of morbidity and mortality in calves, and Rotavirus is the main viral etiology. Rotavirus vaccines are one of the main important methods for control of diarrhea in neonates’ calves. In the current study, Deoxyribonucleic acid (DNA) sequencing and phylogenetic analysis of Bovine Rotavirus Group a (BRVA) were performed in our study. 1 Calf guard® vaccine genotype (G6P1) and 5 different field genotypes (2 G6P5, 1 G10P5, G10P? and 1 G10P11) were subjected to DNA sequencing. We observed that at the nucleotide level, G10P5 and G10P? Sequences were 100 % identical with each other, two G6P5 sequences were 100% identical with each other and there was no significant similarity between sequences of G10P11 with sequences of G6P5, G10P5, and G10P? The phylogenetic analysis of G10P5 and G10P? Isolates showed a close cluster with G10 isolates of Sharkia governorate, Egypt, phylogenetic analysis of two G6P5 and one G10P11 isolate showed a close cluster with the VP4 gene of Rotavirus isolates of Dakahlia Governorate, Egypt. Molecular comparison between detected and typed Rotaviruses’ genotypes with other genotypes of common vaccines indicated that there were genetically close or distance between field and vaccine Rotavirus strains. Our results can be concluded as the following, Molecular comparison between detected and typed Rotaviruses’ genotypes with other genotypes of common vaccines indicated that there was genetically close or distance between field and vaccinal Rotavirus strains. Also, we suggest that Rotavac vaccine containing G6P5 Rotavirus strain and Scour guard vaccine containing can be used in Assiut governorate due to circulating of G6P5 and G10P11 strains of Rotavirus in Assiut.

Detection of adenovirus, rotavirus, and hepatitis E virus in meat cuts marketed in Uruguaiana, Rio Grande do Sul, Brazil

The aim of this study was to investigate the presence of adenovirus (AdV), rotavirus (RV), and hepatitis E virus (HEV) in beef, pork, and chicken meat cuts in retail trade in the city of Uruguaiana, RS, Brazil. A total of 131 meat products were collected (beef, n = 55; chicken, n = 47; pork, n = 29) from 18 commercial establishments (supermarkets, n = 7; butchers, n = 7; markets/grocery stores, n = 4). All samples were evaluated for AdV, RV, and HEV. The genomes of RV and AdV were identified in 29% (n = 38) and 5.34% (n = 7) of the samples, respectively. HEV was not identified in any of the samples. Chicken cuts had a higher frequency of AdV and RV isolates compared to beef and pork (P < 0.05). Among the categories of commercial establishments evaluated, all revealed at least one positive sample for AdV and RV; however, supermarkets showed a higher frequency of RV than others (P < 0.05). The genetic material of AdV and RV was identified simultaneously in 2.29% (n = 3) of samples from supermarkets (n = 2) and grocery stores (n = 1). This is the first report on detection of enteric viruses in meat cuts in the western region of the state of Rio Grande do Sul, Brazil, and the presence of AdV and RV in these products may indicate flaws during the process of handling these foods, especially in places where commercialization provides important public health issues.

Clinical Profile and Characterization of Rotavirus Strains in Under-Five Children with Diarrhea: A Hospital-Based Study from Eastern Odisha, India

Abstract Objective Diarrheal diseases are one of the most common causes of hospitalization in children under five. Rotavirus is the most common cause of acute diarrhea in younger children, and the prevalence decreases rapidly with increasing age. The objective of the study was to estimate the burden of rotavirus infection in acute gastroenteritis among under-five children admitted to a tertiary care hospital in eastern Odisha, for the clinical profile and identity of the prevalent strains. Methods This was a prospective observational study linked to the National Rotavirus Surveillance Network (NRSN), where 720 under-five children with diarrhea were enrolled. In total, 675 stool samples of eligible candidates were sent for rotavirus isolation, and identification of strains was done by identifying VP7 (G-type) and VP4 (P-type) genes by reverse transcription polymerase chain reaction. Results Categorical variables were presented as frequency and percentage, and continuous variables were expressed as mean ± standard deviation. Rotavirus was detected in 256 (37.92%) samples. Males outnumbered females. The most common affected age group was 7 to 12 months, followed by 13 to 18 months. G3P[8] was the most prevalent strain in this study. Conclusion Children between the age of 7 and 18 months were most vulnerable to rotavirus infection. The most prevalent strain varies from one region to another and continuous surveillance is needed.

Replication of SARS-CoV-2 in cell lines used in public health surveillance programmes with special emphasis on biosafety.

Background & objectives:Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS-CoV-2 used in these programmes.Methods:Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin–Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription–polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control.Results:SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per µl. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively.Interpretation & conclusions:Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories.

ISOLATION OF ROTAVIRUS FROM A SICK CALF AND OPTIMIZATION OF ITS CULTIVATION PARAMETERS

The article is devoted to the isolation of rotavirus from a sick calf and optimization of its cultivation parameters in the transplanted lines of homologous and heterologous cell cultures. Rotavirus isolate was isolated in MDBK cell culture, which was confirmed in polymerase chain reaction and neutralization reaction, from the faeces of a sick calf. A method of culturing the isolated isolate in homologous (MDBK) and heterologous (SPEV) cell culture has been worked out. The cultivation of rotavirus in an alkaline medium and its treatment with trypsin at a concentration of 20 mcg/ml and the introduction of trypsin into a supportive medium (15 mcg/ml) led to an increase in the titer of the virus to 8.0–8.25 lg TCD50/ml. It was found that the accumulation of isolated rotavirus in the rollers led to an increase in the yield of the virus and an increase in its titer to 8.5 lg TCD50/ml. The isolated rotavirus isolate has antigenic properties and leads to the formation of an immune response when administered intramuscularly to rabbits.

Rotavirus Genotypes in Abuja, Nigeria

Introduction: Nigeria had planned to introduce the rotavirus vaccine in the National Immunisation Programme in 2014, but this has yet to be done. Nigeria has the continent's highest mortality due to diarrhoeal diseases with little information on specific, prevalent genotypes.&#x0D; Aim: The study's main objectives were to identify the predominant rotavirus genotypes and examine the effects of existing local vaccination programs on prevailing rotavirus genotypes and on preventing rotavirus diarrhoea.&#x0D; Methodology: A one-year prospective descriptive study of children under 5 with acute diarrhoea was conducted from June 2018 to May 2019. Children with acute diarrhoea attending Asokoro District Hospital, Abuja. Children without diarrhoea were also recruited as a control group. Rotavirus ELISA and RNA extraction were done with commercially available kits, and positive samples were subjected to RT-PCR and electrophoresis to determine VP7 (G) and VP4 (P) genotypes.&#x0D; Results: Rotavirus-ELISA was positive among 231 (17.8%) children with diarrhoea and 29 (2.2%) of controls, with November, December. The predominant VP7 genotypes was G1 (n=116, 50.2%) followed by G9 (n=66, 28.5%). Viral Protein, VP4 (P) was mostly P [8] (n=143, 74.8%) followed by P [4] (n=21, 10.9%). The predominant genotype combinations found were G1 P [8] (n=108, 46.7%), G9 P [8] (n=62, 26.8%), and G2 P [4] (n=18, 7.7%). Very few mixed infections were found in the study, 2 (0.8%). Among 94 unvaccinated children with rotavirus isolates that were genotyped, G1 P [8] (n=88, 49.4%) and G9 P [8] (n=43, 24.1%) were predominant. Among 32 vaccinated children, G1 P [8] (n=13, 40.2%) and G9 P [8] (n=9, 28.1%) were predominant.&#x0D; Conclusion: The emergence of new genotypes such as G 12 P [4] found in this study emphasize the need for continued prospective monitoring of rotavirus at the molecular level to detect new threats to vaccine programs in future.

Perspectives for the optimization and utility of the rotavirus reverse genetics system

Following Kobayashi and colleagues’ seminal paper in 20171, in the past four years the rotavirus (RV) field has witnessed a burst in research and publications based on the use of a fully plasmid-based RV reverse genetics systems and subsequent modifications2,3. However, in most cases, the rotaviral strain under interrogation has been the prototypic simian RV SA11-L2 strain (G3P[2]). Of note, a variety of other weakly-to-modestly replication-competent animal and human RV strains, bioluminescent and fluorescent reporter viruses, and clinical isolates of human RVs have proved hard or impossible to rescue using the original reverse genetics system2,4, highlighting a critical need to further enhance the recovery efficiency and expand the rescue tool kit. A number of further modifications of the initial reverse genetics system have enabled the rescue of other RV strains such as the human RV KU and CDC-9 strains, and a murine RV D6/2-like strain4,5. Here, we discuss future possible modifications of existing RV reverse genetics systems to further increase efficiency based on past experience with the improvement of influenza A virus recovery. The development of RV to accommodate the insertion and expression of heterologous sequences has substantial potential in the design of next-generation RV vaccine candidates and enteric viral vectors.

Cell surface heat shock protein-mediated entry of tumor cell-adapted rotavirus into U-937 cells.

Rotaviruses infect cells by binding to specific cell surface molecules including gangliosides, heat shock protein cognate protein 70 (Hsc70), and some integrins. The characterization of cell surface receptors defining viral tropism is crucial for inhibiting entry into the normal cells or the cancer cells. In the present work, several tumor cell-adapted rotavirus isolates were tested for their interaction with some heat shock proteins (HSPs) present in the U-937 cells, derived from a human pleural effusion (histiocytic lymphoma monocyte). This interaction was examined by virus overlay protein-binding (VOPB), immunochemistry, immuno-dot blot assays, and flow cytometry. The results indicated that the rotavirus isolates studied were able to infect U937 cells by interacting with Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, protein disulfide isomerase (PDI), and integrin β3, which are implicated in cellular proliferation, differentiation, and cancer development. Interestingly, these cellular proteins were found to be associated in lipid microdomains (rafts), facilitating in this way eventual sequential interactions of the rotavirus particles with the cell surface receptors. The rotavirus tropism for U937 cells through the use of these cell surface proteins made this rotavirus isolates an attractive target for the development of oncolytic strategies in the context of alternative and complementary treatment of cancer.

Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates.

Background: Viruses are being used as alternative and complementary tools for treating cancers. Oncolytic viruses exhibit tumor tropism, ability to enhance anti-tumor immunity and ability to be used in combination with conventional chemotherapy and radiotherapy. We have recently selected some rotavirus isolates which are adapted to efficiently infect and kill tumor cell lines. Aim: We tested five tumor cell-adapted rotavirus isolates for their ability to infect the human adenocarcinoma cell line MCF-7. Methods: Cell surface membrane-associated proteins mediating virus particle attachment were characterized using ELISA, immunoprecipitation, FACS analysis, and antibody blocking. Results: It was found that heat shock proteins (HSPs) such as Hsp90, Hsp70, Hsp60, and Hsp40 are expressed on the cell surface forming complexes with protein disulfide isomerase (PDI), integrin β3, and heat shock cognate protein 70 (Hsc70) in lipid raft microdomains. Interaction of rotavirus isolates with these cellular proteins was further confirmed by a competition assay and an inhibition assay involving the HSPs tested. Conclusion: Our findings suggest that the tumor cell-adapted rotavirus isolates studied here offer a promising tool for killing tumor cells, thus encouraging further research into this topic, including animal models.

Rotavirus Gastroenteritis in Eastern Uttar Pradesh, India

To identify cases of rotavirus diarrhea in Uttar Pradesh among children less than 5 y of age presenting with acute gastroenteritis, to study the clinical profile of the cases and to identify the rotavirus genotypes. Any child who is 0-59 mo of age who is admitted to the study facility with diarrhea as the presenting complaint were enrolled into the surveillance after obtaining informed consent. Clinical history and sociodemographic information was recorded for all the patients. Patients with dehydration were treated as per standard protocol. Stool samples were collected for isolation of rotavirus and identification of its genotype. Of the 418 stool specimens collected, rotavirus was isolated in 47 (11.33%) samples. Rotavirus gastroenteritis was most common below the age of two years. A significant difference in the clinical characteristics between children who were positive for rotavirus and those who tested negative were not observed in this study. But patients with rotavirus diarrhea had longer duration of hospital stay (OR 3.31; CI 1.24-8.87). The outcome was similar in the two groups (OR 2.64; CI 0.27-25.89). G3P[8] was the most common type genotype isolated in 13 (28.89%) patients followed by G2P[4] in 7 (15.56%) patients. Thirteen patients (28.89%) had mixed genotype. Rotavirus diarrhea was most common below two years of age. G3P[8] was the most common genotype identified.