Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas

Molecular techniques and virus concentration methods have shown that previously unknown viruses are shed by humans and animals, and may be transmitted by sewage-contaminated water. In the present study, river water from urban areas in Barcelona, Spain and Rio de Janeiro, Brazil, were analyzed to evaluate the dissemination of human viruses, while simultaneously optimizing and validating a low-cost concentration method for virus quantification in fresh water. The following three viral groups were analyzed. (i) Recently described viruses: klassevirus (KV), asfarvirus-like virus (ASFLV), and the polyomaviruses Merkel cell, KI and WU (MCPyV/KIPyV/WUPyV). (ii) Gastroenteritis agents: noroviruses (NoV) and rotaviruses (RV). (iii) Human fecal viral indicators in water: human adenoviruses (HAdV) and JC polyomaviruses (JCPyV). Virus detection was based on nested and quantitative PCR assays. Nested PCR assays were developed for KV and ASFLV. The method applied for virus concentration in water samples was a one-step procedure based on a skimmed milk flocculation procedure described previously for seawater. Using spiked river water samples, inter- and intra-laboratory assays showed a viral recovery rate of about 50% for HAdV, JCPyV, NoV and RV with a coefficient of variation ≤ 50%. HAdV and JCPyV were detected in 100% of the river samples from Barcelona and Rio de Janeiro. Moreover, NoV GGII was detected in 100% and MCPyV in 50% of the samples from Barcelona, whereas none of the other viruses analyzed were detected. NoV GGII was detected in 33%, KV in 33%, ASFLV in 17% and MCPyV in 50% of the samples from Rio de Janeiro, whereas KIPyV and WUPyV were not detected. RV were only tested for in Rio de Janeiro and resulted positive in 67% of the samples. The procedure applied here to river water represents a useful, straightforward and cost-effective method that could be applied in routine water quality testing.  The results of the assays expand our understanding of the global distribution of the viral pathogens studied here and their persistence in the environment. Fil: Calgua, B.. Universidad de Barcelona; España;Fil: Fumian, T.. Ministerio de Salud de Brasil. Fundacion Oswaldo Cruz; Brasil;Fil: Rusinol, M.. Universidad de Barcelona; España;Fil: Rodríguez Manzano, J.. Universidad de Barcelona; España;Fil: Mbayed, Viviana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Bofill Mas, S.. Universidad de Barcelona; España;Fil: Miagostovich, M.. Ministerio de Salud de Brasil. Fundacion Oswaldo Cruz; Brasil;Fil: Girones, R.. Universidad de Barcelona; España

Feline calicivirus virulent systemic disease: Clinical epidemiology, analysis of viral isolates and in vitro efficacy of novel antivirals in australian outbreaks

Feline calicivirus (FCV) causes upper respiratory tract disease (URTD) and sporadic outbreaks of virulent systemic disease (FCV-VSD). The basis for the increased pathogenicity of FCVVSD viruses is incompletely understood, and antivirals for FCV-VSD have yet to be developed. We investigated the clinicoepidemiology and viral features of three FCV-VSD outbreaks in Australia and evaluated the in vitro efficacy of nitazoxanide (NTZ), 2′-C-methylcytidine (2CMC) and NITD008 against FCV-VSD viruses. Overall mortality among 23 cases of FCV-VSD was 39%. Metagenomic sequencing identified five genetically distinct FCV lineages within the three outbreaks, all seemingly evolving in situ in Australia. Notably, no mutations that clearly distinguished FCVURTD from FCV-VSD phenotypes were identified. One FCV-URTD strain likely originated from a recombination event. Analysis of seven amino-acid residues from the hypervariable E region of the capsid in the cultured viruses did not support the contention that properties of these residues can reliably differentiate between the two pathotypes. On plaque reduction assays, dose–response inhibition of FCV-VSD was obtained with all antivirals at low micromolar concentrations; NTZ EC50, 0.4–0.6 µM, TI = 21; 2CMC EC50, 2.7–5.3 µM, TI > 18; NITD-008, 0.5 to 0.9 µM, TI > 111. Investigation of these antivirals for the treatment of FCV-VSD is warranted

Factors influencing terrestriality in primates of the Americas and Madagascar

Among mammals, the order Primates is exceptional in having a high taxonomic richness in which the taxa are arboreal, semiterrestrial, or terrestrial. Although habitual terrestriality is pervasive among the apes and African and Asian monkeys (catarrhines), it is largely absent among monkeys of the Americas (platyrrhines), as well as galagos, lemurs, and lorises (strepsirrhines), which are mostly arboreal. Numerous ecological drivers and species-specific factors are suggested to set the conditions for an evolutionary shift from arboreality to terrestriality, and current environmental conditions may provide analogous scenarios to those transitional periods. Therefore, we investigated predominantly arboreal, diurnal primate genera from the Americas and Madagascar that lack fully terrestrial taxa, to determine whether ecological drivers (habitat canopy cover, predation risk, maximum temperature, precipitation, primate species richness, human population density, and distance to roads) or species-specific traits (bodymass, group size, and degree of frugivory) associate with increased terrestriality. We collated 150,961 observation hours across 2,227 months from 47 species at 20 sites in Madagascar and 48 sites in the Americas. Multiple factors were associated with ground use in these otherwise arboreal species, including increased temperature, a decrease in canopy cover, a dietary shift away from frugivory, and larger group size. These factors mostly explain intraspecific differences in terrestriality. As humanity modifies habitats and causes climate change, our results suggest that species already inhabiting hot, sparsely canopied sites, and exhibiting more generalized diets, are more likely to shift toward greater ground use

Aetiology and incidence of diarrhoea requiring hospitalisation in children under 5 years of age in 28 low-income and middle-income countries: findings from the Global Pediatric Diarrhea Surveillance network.

Introduction: Diarrhoea remains a leading cause of child morbidity and mortality. Systematically collected and analysed data on the aetiology of hospitalised diarrhoea in low-income and middle-income countries are needed to prioritise interventions. Methods: We established the Global Pediatric Diarrhea Surveillance network, in which children under 5 years hospitalised with diarrhoea were enrolled at 33 sentinel surveillance hospitals in 28 low-income and middle-income countries. Randomly selected stool specimens were tested by quantitative PCR for 16 causes of diarrhoea. We estimated pathogen-specific attributable burdens of diarrhoeal hospitalisations and deaths. We incorporated country-level incidence to estimate the number of pathogen-specific deaths on a global scale. Results: During 2017–2018, 29 502 diarrhoea hospitalisations were enrolled, of which 5465 were randomly selected and tested. Rotavirus was the leading cause of diarrhoea requiring hospitalisation (attributable fraction (AF) 33.3%; 95% CI 27.7 to 40.3), followed by Shigella (9.7%; 95% CI 7.7 to 11.6), norovirus (6.5%; 95% CI 5.4 to 7.6) and adenovirus 40/41 (5.5%; 95% CI 4.4 to 6.7). Rotavirus was the leading cause of hospitalised diarrhoea in all regions except the Americas, where the leading aetiologies were Shigella (19.2%; 95% CI 11.4 to 28.1) and norovirus (22.2%; 95% CI 17.5 to 27.9) in Central and South America, respectively. The proportion of hospitalisations attributable to rotavirus was approximately 50% lower in sites that had introduced rotavirus vaccine (AF 20.8%; 95% CI 18.0 to 24.1) compared with sites that had not (42.1%; 95% CI 33.2 to 53.4). Globally, we estimated 208 009 annual rotavirus-attributable deaths (95% CI 169 561 to 259 216), 62 853 Shigella-attributable deaths (95% CI 48 656 to 78 805), 36 922 adenovirus 40/41-attributable deaths (95% CI 28 469 to 46 672) and 35 914 norovirus-attributable deaths (95% CI 27 258 to 46 516). Conclusions: Despite the substantial impact of rotavirus vaccine introduction, rotavirus remained the leading cause of paediatric diarrhoea hospitalisations. Improving the efficacy and coverage of rotavirus vaccination and prioritising interventions against Shigella, norovirus and adenovirus could further reduce diarrhoea morbidity and mortality

Factors influencing terrestriality in primates of the Americas and Madagascar

Among mammals, the order Primates is exceptional in having a high taxonomic richness in which the taxa are arboreal, semiterrestrial, or terrestrial. Although habitual terrestriality is pervasive among the apes and African and Asian monkeys (catarrhines), it is largely absent among monkeys of the Americas (platyrrhines), as well as galagos, lemurs, and lorises (strepsirrhines), which are mostly arboreal. Numerous ecological drivers and species-specific factors are suggested to set the conditions for an evolutionary shift from arboreality to terrestriality, and current environmental conditions may provide analogous scenarios to those transitional periods. Therefore, we investigated predominantly arboreal, diurnal primate genera from the Americas and Madagascar that lack fully terrestrial taxa, to determine whether ecological drivers (habitat canopy cover, predation risk, maximum temperature, precipitation, primate species richness, human population density, and distance to roads) or species-specific traits (body mass, group size, and degree of frugivory) associate with increased terrestriality. We collated 150,961 observation hours across 2,227 months from 47 species at 20 sites in Madagascar and 48 sites in the Americas. Multiple factors were associated with ground use in these otherwise arboreal species, including increased temperature, a decrease in canopy cover, a dietary shift away from frugivory, and larger group size. These factors mostly explain intraspecific differences in terrestriality. As humanity modifies habitats and causes climate change, our results suggest that species already inhabiting hot, sparsely canopied sites, and exhibiting more generalized diets, are more likely to shift toward greater ground use

The evolving epidemiology of rotavirus A infection in Brazil a decade after the introduction of universal vaccination with Rotarix®

Abstract Background Brazil introduced the monovalent rotavirus vaccine (Rotarix®) in 2006. This study aimed to assess the epidemiology and genotype distribution of species-A rotavirus (RVA) in Brazil, comparing the pre- and post-vaccination periods. Methods Laboratory-based RVA surveillance included 866 municipalities in 22 Brazilian states, over a 21-year period. A total of 16,185 children with diarrheal diseases (DD) aged up to 12 years between 1996 and 2005 (pre-vaccination period, n = 7030) and from 2006 to 2017 (post-vaccination period, n = 9155) were enrolled. RVA was detected using ELISA immune assay and/or polyacrylamide gel electrophoresis and genotyped using nested PCR and/or nucleotide sequencing. RVA-positivity and genotypes detection rates were compared in distinct periods and age groups and Rotarix vaccination status. Results RVA-positivity in pre- and post-vaccination periods was, respectively: 4–11 months bracket, 33.3% (668/2006) and 16.3% (415/2547) (p <  0.001); 12–24 months, 28.2% (607/2154) and 22.2% (680/3068) (p <  0.001); 25–48 months, 17.4% (215/1235) and 29.4% (505/1720) (p <  0.001). Genotypes distribution in the pre- and post-vaccination periods was, respectively: G1P [8]/G1P[Not Typed], 417/855 (48.8%) and 118/1835 (6.4%) (p <  0.001); G2P [4]/G2P[NT], 47/855 (5.5%) and 838/1835 (45.7%) (p <  0.001); G3P [8]/G3P[NT], 55/855 (6.4%) and 253/1835 (13.8%) (p <  0.001); G9P [8]/G9P[NT], 238/855 (27.8%) and 152/1835 (8.3%) (p <  0.001); G12P [8]/G129P[NT], 0/871 (0%) and 249/1835(13.6%) (p <  0.001). Concerning infants aged 4–11 months, RVA frequency in fully vaccinated and non-vaccinated individuals was 11.9% (125/1052) and 24.5% (58/237) (p <  0.001), respectively. In children aged 12–24 months, RVA detection rate was 18.1% (253/1395) and 29.6% (77/260) (p <  0.001), for the vaccinated and non-vaccinated individuals, respectively (p <  0.001). Conclusions RVA infection was significantly less frequent in children aged ≤2 years with DD after implementing vaccination, mainly among vaccinated children. It was also observed a decrease of P [8] circulation and emergence of G2P[4] in 2005, and afterwards in the post-vaccine era, with spreading of G12P[8] in 2014–2015 and of G3P[8] in 2017. Continuous RVA surveillance must be carried out in this scenario

Rotavirus seasonality in urban sewage from Argentina: Effect of meteorological variables on the viral load and the genetic diversity

In Argentina, the rotavirus disease exhibits seasonal variations, being most prevalent in the fall and winter months. To deepen the understanding of rotavirus seasonality in our community, the influence of meteorological factors on the rotavirus load and the genetic diversity in urban raw sewage from Córdoba city, Argentina were evaluated. Wastewater samples were collected monthly during a three-year study period and viral particles were concentrated by polyethylene glycol precipitation. RT-nested PCR was applied for rotavirus detection, and VP7/VP4 characterization and real-time PCR for rotavirus quantification. Both molecular techniques showed relatively similar sensitivity rates and revealed rotavirus presence in urban wastewater in cold and warm seasons, indicating its circulation in the local community all year round. However, a slight trend for rotavirus circulation was noted by real-time PCR in the fall and winter seasons, showing a significantly higher peak of rotavirus concentration at mean temperatures lower than 18. °C and also higher, although not statistically different during drier weather. VP7 and VP4 gene characterization showed that G1 and P[8] genotypes were dominant, and temporal variations in genotype distribution were not observed. Rotavirus spread is complex and our results point out that weather factors alone cannot explain the seasonal quantitative pattern of the rotavirus disease. Therefore, alternative transmission routes, changes in human behavior and susceptibility, and the stability and survivability of the virus might all together contribute to the seasonality of rotavirus. The results obtained here provide evidence regarding the dynamics of rotavirus circulation and maintenance in Argentina.Fil: Barril, Patricia Angelica. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Fumian, T. M.. Fundação Oswaldo Cruz. Instituto Oswaldo Cruz; BrasilFil: Prez, Verónica Emilse. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología "Dr. J. M. Vanella"; ArgentinaFil: Gil, Pedro Ignacio. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Martínez, L. C.. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Giordano, M.O.. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Masachessi, Gisela. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Isa, Maria Beatriz. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Ferreyra, Leonardo Jesús. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Ré, Viviana Elizabeth. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Miagostovich, M.. Fundação Oswaldo Cruz. Instituto Oswaldo Cruz; BrasilFil: Pavan, Jorge Victorio. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; ArgentinaFil: Nates, Silvia Viviana. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología ; Argentin