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Vaccination has proven to be a valuable tool to combat SARS-CoV-2. However, reports of rare adverse reactions such as thrombosis/thrombocytopenia syndrome after ChAdOx1 nCoV-19 vaccination have caused scientific, public and media concern. ChAdOx1 was vectorised from the Y25 chimpanzee adenovirus, which was selected due to low human seroprevalence to circumvent pre-existing immunity. In this study, we aimed to explore patterns of T-cell activation after SARS-CoV-2 COVID-19 vaccine exposure in vitro using PBMCs collected from pre-pandemic ChAdOx1 nCoV-19 naïve healthy donors (HDs), and ChAdOx1 nCoV-19 and Pfizer vaccinated controls. PBMCs were assessed for T-cell proliferation using the lymphocyte transformation test (LTT) following exposure to SARS-CoV-2 COVID-19 vaccines. Cytokine analysis was performed via intracellular cytokine staining, ELISpot assay and LEGENDplex immunoassays. T-cell assays performed in pre-pandemic vaccine naïve HDs, revealed widespread lymphocyte stimulation after exposure to ChAdOx1 nCoV-19 (95%), ChAdOx-spike (90%) and the Ad26.COV2. S vaccine, but not on exposure to the BNT162b2 vaccine. ICS analysis demonstrated that CD4+ CD45RO+ memory T-cells are activated by ChAdOx1 nCoV-19 in vaccine naïve HDs. Cytometric immunoassays showed ChAdOx1 nCoV-19 exposure was associated with the release of proinflammatory and cytotoxic molecules, such as IFN-γ, IL-6, perforin, granzyme B and FasL. These studies demonstrate a ubiquitous T-cell response to ChAdOx1 nCoV-19 and Ad26.COV2. S in HDs recruited prior to the SARS-CoV-2 pandemic, with T-cell stimulation also identified in vaccinated controls. This may be due to underlying T-cell cross-reactivity with prevalent human adenoviruses and further study will be needed to identify T-cell epitopes involved. [ABSTRACT FROM AUTHOR]

Human adenoviruses (HAdVs) usually cause asymptomatic or mild infection, but infrequently, they are responsible for various severe syndromes including neurological disorders. Various research studies have investigated the association of HAdVs with acute flaccid paralysis (AFP). The purpose of this study was to investigate the genetic diversity of HAdVs and their association with AFP. Stool samples from patients ≤ 12 years of age with suspected AFP were collected from all over Pakistan within the framework of poliovirus surveillance. Poliovirus- and enterovirus-negative samples were screened for HAdVs. For virus isolation, the human epithelial cell line HEp-2c was used, culture-positive samples were screened by nested PCR assay, and partial hexon gene sequences were used for genotype identification. Out of 172 samples, 94 were positive by virus isolation, 89 were positive by PCR, and 32 isolates were genotyped successfully. Phylogenetic analysis showed that the HAdVs belonged to species A (HAdV-A12 and A31), B (HAdV-B3 and B7), C (HAdV-C1 and C6), D (HAdV-D19 and D93), and F (HAdV-F41), showing 99-100% nucleotide sequence identity and 98.3-100% amino acid sequence identity). Most of these genotypes have been reported previously in AFP cases, but this is the first report of the detection of HAdV-D93 in stool samples from AFP cases. The detection of a significant fraction of the HAdVs genotypes indicates that these genetically distinct genotypes are circulating in Pakistan and suggests their possible role in the pathogenesis of AFP. [ABSTRACT FROM AUTHOR]

Human adenoviruses (HAdVs) are highly contagious pathogens of clinical importance, especially among the pediatric population. Studies on comparative viral genomic analysis of cases associated with severe and mild infections due to HAdV are limited. Using whole-genome sequencing (WGS), we investigated whether there were any differences between circulating HAdV strains associated with severe infections (meningitis, sepsis, convulsion, sudden infant death syndrome, death, and hospitalization) and mild clinical presentations in pediatric patients hospitalized between the years 1998 and 2017 in a tertiary care hospital group in Bern, Switzerland covering a population base of approx. 2 million inhabitants. The HAdV species implicated in causing severe infections in this study included HAdV species C genotypes (HAdV1, HAdV2, and HAdV5). Clustering of the HAdV whole-genome sequences of the severe and mild cases did not show any differences except for one sample (isolated from a patient presenting with sepsis, meningitis, and hospitalization) that formed its own cluster with HAdV species C genotypes. This isolate showed intertypic recombination events involving four genotypes, had the highest homology to HAdV89 at complete genome level, but possessed the fiber gene of HAdV1, thereby representing a novel genotype of HAdV species C. The incidence of potential recombination events was higher in severe cases than in mild cases. Our findings confirm that recombination among HAdVs is important for molecular evolution and emergence of new strains. Therefore, further research on HAdVs, particularly among susceptible groups, is needed and continuous surveillance is required for public health preparedness including outbreak investigations. [ABSTRACT FROM AUTHOR]

Human Adenoviruses (HAdVs) are a family of clinically and therapeutically relevant viruses. A precise understanding of their host cell attachment and entry mechanisms can be applied in inhibitor design and the construction of targeted gene delivery vectors. In this article, structural data on adenovirus attachment and entry are reviewed. HAdVs engage two types of receptors: first, an attachment receptor that is bound by the fibre knob protein protruding from the icosahedral capsid, and next, an integrin entry receptor bound by the pentameric penton base at the capsid vertices. Adenoviruses use remarkably diverse attachment receptors, five of which have been studied structurally in the context of HAdV binding: Coxsackie and Adenovirus Receptor, CD46, the glycans GD1a and polysialic acid, and desmoglein-2. Together with the integrin entry receptors, they display both symmetrical and asymmetrical modes of binding to the virus as demonstrated by the structural analyses reviewed here. The diversity of HAdV receptors contributes to the broad tropism of these viruses, and structural studies are thus an important source of information on HAdV-host cell interactions. The imbalance in structural data between the more and less extensively studied receptors remains to be addressed by future research. [ABSTRACT FROM AUTHOR]

Codon usage is biased in most species, and the pattern of codon usage bias is specific to each species or group of closely related species. Although viruses use the host translational machinery for synthesis of their proteins, their codon usage patterns do not match those of their host. Viral codon usage is determined by a complex interplay of mutational bias, genome composition constraints, translational adaptation to the host, and host cellular innate defense. The codon usage of parvoviruses was previously shown not to be strongly biased and selective pressure was found to be a dominating factor driving codon usage. The family Parvoviridae includes the genus Dependoparvovirus, some of the members of which require a helper virus to complete their replication cycle, whereas the rest of the family can replicate without the need for helper viruses. Here, we show that difference in the replication strategy of these viruses may be an important factor determining viral codon usage. Hierarchical clustering and principal component analysis revealed that the codon usage pattern of adeno-associated viruses (AAVs) of the genus Dependoparvovirus is distinct from that of members of the other genera of vertebrate parvoviruses, and even from that of independent viruses of the genus Dependoparvovirus. Furthermore, the codon usage of human AAVs was found to be similar to that of some human adenoviruses in hierarchical clustering and principal component analysis. This suggests that the codon usage of AAVs is different from that of other parvoviruses because of their distinctive replication strategy and that their codon usage is probably driven by forces similar to those that shaped the codon usage pattern of their helper viruses. [ABSTRACT FROM AUTHOR]

The 2015 rupture of the Fundão dam near the district of Bento Rodrigues in Mariana, Minas Gerais, Brazil, released around 50 million m3 of iron ore tailings. The first tributary of the Rio Doceto receive this waste was the Gualaxo do Norte River. Many groups in Brazil and from around the world have studied the environmental and social impacts of this disaster. However, relationships between the introduction of mining waste, the geological complexity of the area, and the presence of pathogenic organisms have not yet been investigated. The present study aimed to measure the concentrations of enteric pathogens along the Gualaxo do Norte River after the environmental disaster and to correlate their abundance with the presence of metals and semimetals coming from both mining tailings and geological sources. For this purpose, we collected water samples from 27 stations along the entire basin during a hydrological year. The concentrations of metals and semimetals measured in this study were generally within limits established by national and international legislation, except for those of iron and manganese. Positive correlations between the human adenovirus (HAdV) and arsenic, barium, iron, lead, manganese, and nickel were confirmed, allowing us to observe that there is an abundance of the potentially infectious virus present in the studied sites containing metal/semimetal concentrations. These studies indicate the importance of investigations that consider viral enteric pathogens complexed with metals and may favor the stability and prolongation of the infectivity of such pathogens in water destined for human and animal usage. [ABSTRACT FROM AUTHOR]

A variety of viruses can cause acute flaccid paralysis (AFP). However, the causative agent, sometimes, remains undetermined. Metagenomics helps in identifying viruses not diagnosed by conventional methods. Stool samples from AFP (n = 104) and non-AFP (n = 114) cases that tested enterovirus-negative by WHO standard methods were investigated. A metagenomics approach, first used on five pools of four samples each, revealed the presence of adenovirus sequences. Amplification in A549 cells and full-genome sequencing were used for complete virus identification and for designing a PCR assay to screen individual related samples. Metagenomic analysis showed that adenovirus sequences that were closely to the A31 and A61 genotypes were the most abundant. Two out of the corresponding 20 individual samples were found positive by PCR, and isolates were obtained in cell culture. Phylogenetic analysis based on complete genome sequences showed that the viruses belong to HAdV-A31 genotype (98-100% nucleotide sequence identity). PCR analysis of stool samples from all AFP and non-AFP cases revealed that a larger proportion of the positive samples were from AFP cases (17.3%) than from non-AFP cases (2.4%). These results open the way to studies aiming to test a possible role of HAdV-A31 in the pathogenesis of AFP. [ABSTRACT FROM AUTHOR]

Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics. [ABSTRACT FROM AUTHOR]

Immunocompromised patients are susceptible to multiple viral infections. Relevant interactions between co-infecting viruses might result from viral regulatory genes which trans-activate or repress the expression of host cell genes as well as the genes of any co-infecting virus. The aim of the current study was to show that the replication of human adenovirus 5 is enhanced by co-infection with BK polyomavirus and is associated with increased expression of proteins including early region 4 open reading frame 1 and both the large tumor antigen and small tumor antigen. Clinical samples of whole blood and urine from 156 hematopoietic stem cell transplant recipients were tested. We also inoculated adenocarcinomic human alveolar basal epithelial cells with both human adenovirus 5 and BK polyomavirus to evaluate if co-infection of viruses affected their replication. Data showed that adenovirus load was significantly higher in the plasma (mean 7.5 x 103 ± 8.5 x 102 copies/ml) and urine (mean 1.9 x 103 ± 8.0 x 102 copies/ml) of samples from patients with co-infections, in comparison to samples from patients with isolated adenovirus infection. In vitro co-infection led to an increased (8.6 times) expression of the adenovirus early region 4 open reading frame gene 48 hours post-inoculation. The expression of the early region 4 open reading frame gene positively correlated with the expression of BK polyomavirus large tumor antigen (r = 0.90, p < 0.0001) and small tumor antigen (r = 0.83, p < 0.001) genes. The enhanced expression of the early region 4 open reading frame gene due to co-infection with BK polyomavirus was associated with enhanced adenovirus, but not BK polyomavirus, replication. The current study provides evidence that co-infection of adenovirus and BK polyomavirus contributes to enhanced adenovirus replication. Data obtained from this study may have significant importance in the clinical setting. [ABSTRACT FROM AUTHOR]

Sewage treatment may be insufficient for the complete removal of enteric viruses, such as human adenoviruses (HAdV) and group A rotavirus (RVA). The differences in the efficiency of the treatment methodologies used may interfere with the detection of these viruses. The objective of this study was to optimize a skimmed-milk flocculation technique for the recovery of HAdV and RVA in the samples of treated effluent. The treated effluent collected at the wastewater treatment plant (WWTP) was processed via four protocols including modifications in the initial centrifugation step and the final concentration of skimmed-milk. The viral load and recovery rate were determined by quantitative PCR TaqMan® System. The highest recovery rates of HAdV, RVA, and bacteriophage PP7 (internal control process) were obtained when the concentration of skimmed-milk was doubled and no centrifugation step was used for the sample clarification. The optimized protocol was assessed in a field study conducted with 24 treated effluent samples collected bi-monthly during 2015. HAdV and RVA were detected in 50.0% (12/24) and 33.3% (08/24) of the samples tested, respectively, throughout the year, without seasonal variation (p > 0.05). This study corroborates the use of the organic flocculation method for virus recovery in environmental samples with the adaptation of the protocols to different aquatic matrices. [ABSTRACT FROM AUTHOR]

Hospital wastewater (HWW) represents a major source of the diffusion of many antibiotics and some toxic pathogenic microorganisms in the aquatic environment. Sanitation services play a critical role in controlling transmission of numerous waterborne pathogens, especially enteric human adenoviruses (HAdVs) that can cause acute gastroenteritis. This study intended to evaluate the human adenoviruses (HAdVs) detection rates, to determine the genotype of these viruses and to assess the efficiency of HAdVs removal in hospital pilot wastewater treatment plant (PWWTP) in Tunis City, Tunisia. Therefore, hospital wastewater samples (n = 102) were collected during the study year from the two biological wastewater treatment techniques: natural oxidizing ponds and the rotating biological disks or biodisks. Nested polymerase chain reaction (Nested PCR) was used to evaluate the HAdVs detection rates. The genotype of HAdVs positive samples was achieved by the sequencing of the PCR products. HAdVs were detected in 64% (65/102) of positive wastewater samples. A substantial increase in the frequencies of HAdVs was observed at the exit of the two wastewater treatment techniques studied. The typing of HAdVs species F showed the occurrence of only HAdVs type 41. This data acquired for the first time in Tunisia showed high persistence and survival of HAdVs in the two biological wastewater treatment techniques experienced, and mainly highlighted the poor virological quality of the treated wastewater intended for recycling, agriculture reuse, and discharges into the natural receiving environments. Consequently, tertiary wastewater treatment appeared necessary in this case to decrease the load of enteric viruses flowing in the water environment. [ABSTRACT FROM AUTHOR]

Large-scale wastewater schemes rely on multi-barrier approach for the production of safe and sustainable recycled water. In multi-barrier wastewater reclamation systems, conventional activated sludge process (ASP) often constitutes a major initial treatment step. The main aim of this research was to determine most appropriate sampling approach to establish pathogen removal efficacy of ASP. The results suggest that ASP is capable of reducing human adenovirus (HAdV) and polyomavirus (HPyV) by up to 3 log. The virus removal data suggests that HAdV removal is comparable to somatic bacteriophage belonging to Microviridae family. Due to the high removal of Escherichia coli (>3 log) and very poor correlation with the enteric virus, it is not recommended that E. coli be used as a surrogate for enteric virus removal. The results also demonstrated no statistically significant differences ( t test, P > 0.05) in calculated log removal values (LRVs) for HAdV, HPyV, and Microviridae from samples collected on hydraulic retention time (HRT) or simultaneous paired samples collected for influent and effluent. This indicates that a more practical approach of simultaneous sampling for influent and effluent could be used to determine pathogen removal efficiency of ASP. The results also suggest that a minimum of 10, preferably 20 samples, are required to fully capture variability in the removal of virus. In order to cover for the potential seasonal prevalence of viruses such as norovirus and rotavirus, sampling should be spread across all seasons. [ABSTRACT FROM AUTHOR]

Human adenoviruses (HAdVs) are DNA viruses found in recreational water, such as water parks and swimming pools. Human adenovirus 41 (HAdV-41) is the most common serotype detected and is a leading cause of acute diarrheal disease. The focus of this study is to determine the prevalence of HAdVs in hot springs. Of 57 samples collected from four different geological sites, 16 samples have shown evidence of HAdVs (28.1%). HAdV-41 and porcine adenovirus 5 (PAdV-5) were the two types isolated, with a greater frequency of HAdV-41, which in other settings has been associated with acute diarrhea. The highest occurrence was found in private hot tubs/Yuya (37.5%), followed by an outlet of hot springs (30.8%); public pools and foot pools shared the same detection rate of 21.4% (3/14). However, there was no evidence supporting a link between water quality indicators and HAdV detection rate. From a phylogenic analysis and BLAST against the NCBI database, it was concluded that HAdV-41 obtained from hot spring areas are closely related to global environmental genotypes. [ABSTRACT FROM AUTHOR]

Human adenovirus (HAdV) is resistant to environment and can be used as a marker to detect fecal contamination. Considering the importance of freshwater snails in the aquatic environment, their use as concentrators for HAdV is a complementary tool for viral analysis of water. The goal of the study was to detect HAdV in snails and surface water collected from wetlands of the Sinos River (Rio Grande do Sul, Brazil) basin and to compare rates and viral loads found in both samples. HAdV was detected through real-time PCR. Total and fecal coliforms were detected by Colilert kit, and viral infectivity of positive samples of the DNA genome was performed in A549 human cell line. All wetlands presented bacterial and viral contamination, but no viral particle was considered viable. The wetland that showed lower fecal coliform mean was Campo Bom, and São Leopoldo (both cities in Rio Grande do Sul) was representative of the highest mean. HAdV was detected in water samples (53%), gastropods' hemolymph (31%) and tissues (16%). Wetlands proved to be environments already altered by human action. Water samples exhibited a higher frequency of HAdV detection; however, in some instances, the target viral genomes were only found in gastropod biological samples. This was a pioneer study in the use of freshwater snails for human enteric viral assessment thus demonstrating that the human organism can retain fecal contamination, complementing and assisting in microbiological water analyzes. [ABSTRACT FROM AUTHOR]

Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are difficult to concentrate from environmental waters due to low numbers and small sizes. Rapid enumeration of viruses by quantitative polymerase chain reaction (qPCR) has the potential to improve water quality analysis and risk assessment. However, capturing and recovering these viruses from environmental water remain formidable barriers to routine use. Here, we compared the recovery efficiencies of human adenoviruses (HAdVs) and human polyomaviruses (HPyVs) from 10-L river water samples seeded with raw human wastewater (100 and 10 mL) using hollow-fiber ultrafiltration (HFUF) and glass wool filter (GWF) methods. The mean recovery efficiencies of HAdVs in river water samples through HFUF were 36 and 86 % for 100 and 10 mL of seeded human wastewater, respectively. In contrast, the estimated mean recovery efficiencies of HAdVs in river water samples through GWF were 1.3 and 3 % for 100 and 10 mL seeded raw human wastewater, respectively. Similar trends were also observed for HPyVs. Recovery efficiencies of HFUF method were significantly higher ( P < 0.05) than GWF for both HAdVs and HPyVs. Our results clearly suggest that HFUF would be a preferred method for concentrating HAdVs and HPyVs from river water followed by subsequent detection and quantification with PCR/qPCR assays. [ABSTRACT FROM AUTHOR]

The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17-4.59) and 1.65 (0.15-3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24-2.93), 0.64 (0.12-1.97), 0.45 (0.04-2.54) and 0.72 (0.22-2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water. [ABSTRACT FROM AUTHOR]

In this study, the seasonal difference and the observable presence/absence of human adenovirus (HAdV) in the Puzih River basin in Taiwan was investigated. A total of 288 water samples were collected from 24 sites from March 2014 to February 2015. Human AdV analysis of sample was subjected to viral concentration using a GN-6 Metricel® filter, followed by DNA extraction, nested-PCR, and qPCR. Human AdV was detected in 34.3 % (99/288) of the entire river water sample. A higher percentage of HAdV (76.4 %) was obtained during the winter. The HAdV median concentration was relatively high in fall (1.4 × 10 copies/L) and winter (2.8 × 10 copies/L). Significant difference and correlation were found between the seasonal variation of HAdV and water quality parameters, including heterotrophic plate count, total coliform, water temperature, and turbidity. The most frequently identified HAdV (subgenus F) serotype was 41. Human AdV-41 is the main cause of gastroenteritis and should be considered for associated human health risk potential in the Puzih River basin. [ABSTRACT FROM AUTHOR]

Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses. [ABSTRACT FROM AUTHOR]

This study was designed to assess the presence of human adenovirus (HAdV), rotavirus-A (RVA), hepatitis A virus (HAV), and porcine circovirus-2 (PCV2) in groundwater from deep wells, and recreational and network waters. The water samples were collected and concentrated and the virus genomes were assessed and quantified by quantitative PCR (qPCR). Infectious HAdV was evaluated in groundwater and network water samples by integrated cell culture using transcribed messenger RNA (mRNA) (ICC-RT-qPCR). In recreational water samples, HAdV was detected in 100 % (6/6), HAV in 66.6 % (4/6), and RVA in 66.6 % (4/6). In network water, HAdV was detected in 100 % (6/6) of the samples (these 83 % contained infectious HAdV), although HAV and RVA were not detected and PCV2 was not evaluated. In groundwater from deep wells, during rainy period, HAdV and RVA were detected in 80 % (4/5) of the samples, and HAV and PCV2 were not detected; however, during dry period, HAdV and RVA were detected in 60 % (3/5), HAV in only one sample, and PCV2 in 60 % (4/5). In groundwater, all samples contained infectious HAdV. PCV2 presence in groundwater is indicative of contamination caused by swine manure in Concórdia, Santa Catarina, Brazil. The disinfection of human and animal wastes is urgent, since they can contaminate surface and groundwater, being a potential threat for public and animal health. [ABSTRACT FROM AUTHOR]

Enteric viruses enter surface waters through discharge of sewage treatment plants. They have a high environmental resistance and persistence and have low infectious doses. The aim of this study was to investigate the efficiency of polishing pond in the removal of viruses and bacteria. The samples were taken approximately once a week at the influent of secondary treatment ( n = 39), effluent of secondary treatment ( n = 39), and polishing pond (tertiary treatment, n = 29). Human adenoviruses (HAdV) were detected in 82-100 % of wastewater samples, whereas 62-79 % of the samples were positive for human polyomavirus (HPyV). The median concentrations ranged from 6.8 × 10 genome equivalents/l (HAdV) to 6.0 × 10 genome equivalents/l (HPyV). The concentration of HAdV and HPyV did not change significantly during the wastewater treatment. For somatic coliphages and bacteria an overall reduction of 1.84-2.65 log has been detected. Based on the data collected, this type of tertiary treatment achieved a significant reduction in bacteria and phages, but not for viruses. [ABSTRACT FROM AUTHOR]

This study aims to establish a straightforward and original workflow for high-throughput typing of human adenoviruses (HAdVs) in environmental samples. Occurrence of HAdVs in water is well documented worldwide, but data on diversity of HAdV types circulating in water are scarcely available. Here, the characterisation of viral particles was performed by determination of amplicon sequences using a next-generation sequencing (NGS) approach. Adenoviral DNA was either directly isolated from wastewater or river water concentrates or after a cell culture passage. Genome amplification targeted a hyper variable region of the hexon gene, allowing the discrimination of the 54 human adenoviral types described until now. After read generation on the benchtop MiSeq platform (Illumina), data were analysed using the Mothur software for identification of all HAdV species and types simultaneously present in a unique sample. NGS results showed a relatively wide HAdV diversity of up to six types in one sample, whereas Sanger sequencing always only retrieved the dominant one. Detected types included HAdV-1, HAdV-2, HAdV-3, HAdV-6, HAdV-12, HAdV-31, HAdV-40 and HAdV-41, HAdV-41 being the most abundant in tested samples. In addition, the influence of the cell line (A549 vs 293A cells) on the infectious HAdV typing results was clearly determined. The 293A appeared to be the most suitable cell line allowing the detection of a larger diversity of infectious HAdVs and reflecting a more realistic initial species distribution than using the A549 cells. These findings demonstrated the feasibility of amplicon sequencing NGS approach to identify viruses in complex environmental water samples. [ABSTRACT FROM AUTHOR]

The prevalence of human adenoviruses (HAdV) in river waters was investigated in this study. Water samples were collected from 13 rivers in Taiwan, concentrated, and assessed for the presence of HAdVs using nested polymerase chain reaction (PCR). Human AdV positive samples were then subjected to real-time PCR (qPCR) to quantify the viral genomes and further subjected to primer-based genotyping to identify the various serotypes present. For each water sample, several water quality parameters were evaluated, including heterotrophic plate count, total coliform, Escherichia coli, water temperature, pH, conductivity, and dissolved oxygen. Among the 13 rivers examined, four rivers (30.8 %) were found to contain HAdVs. The major genotype was F species HAdV serotype 41. The mean HAdVs concentrations ranged from 6.10 × 10 to 8.51 × 10 copies/L. No significant differences were observed between the presence of HAdVs, and all of the water quality parameters evaluated (heterotrophic plate count, total coliform, E. coli, water temperature, pH, conductivity, and dissolved oxygen). Given the potential health risks posed by the presence of enteric viruses in environmental waters, further assessment is desirable with respect to possible sources, virus transport, and survival of viruses in the aquatic environment. [ABSTRACT FROM AUTHOR]

The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×10 TCID was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level. [ABSTRACT FROM AUTHOR]

Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: [ABSTRACT FROM AUTHOR]

Human adenoviruses (HAdVs) are highly contagious pathogens causing acute respiratory disease (ARD), among other illnesses. Of the ARD genotypes, HAdV-7 presents with more severe morbidity and higher mortality than the others. We report the isolation and identification of a genome type HAdV-7d (DG01_2011) from a recent outbreak in Southern China. Genome sequencing, phylogenetic analysis, and restriction endonuclease analysis (REA) comparisons with past pathogens indicate HAdV-7d has re-emerged in Southern China after an absence of twenty-one years. Recombination analysis reveals this genome differs from the 1950s-era prototype and vaccine strains by a lateral gene transfer, substituting the coding region for the L1 52/55 kDa DNA packaging protein from HAdV-16. DG01_2011 descends from both a strain circulating in Southwestern China (2010) and a strain from Shaanxi causing a fatality and outbreak (Northwestern China; 2009). Due to the higher morbidity and mortality rates associated with HAdV-7, the surveillance, identification, and characterization of these strains in population-dense China by REA and/or whole genome sequencing are strongly indicated. With these accurate identifications of specific HAdV types and an epidemiological database of regional HAdV pathogens, along with the HAdV genome stability noted across time and space, the development, availability, and deployment of appropriate vaccines are needed. [ABSTRACT FROM AUTHOR]

Human adenovirus 36 (Ad36) is positively associated with obesity in humans and animals. Ad36 infection is characterized by increased adiposity and inflammation. To investigate the possibility that a prophylactic vaccine candidate might protect against Ad36-induced obesity and inflammation, we purified Ad36 and ultraviolet-irradiated virus to obtain a vaccine candidate. After immunizing the mice with the vaccine candidate (vaccinated group), live Ad36 was injected into mice as a challenge test. Unvaccinated mice (control group) were immunized with phosphate-buffered saline and then challenged with live Ad36. Fourteen weeks after challenge, we compared adiposity and inflammation in vaccinated and control mice. The control group showed 17% greater body weight and 20% more epididymal fats compared with the vaccinated group. In addition, the vaccinated group had decreased serum levels of pro-inflammatory cytokines, and infiltrated immune cells, especially M1 macrophages, in fat tissue. Therefore, the vaccine candidate for Ad36 was able to protect against Ad36-increased body weight and fat as well as inflammatory states after challenge. These results provide proof-of-concept for prophylactic vaccination against virus-induced adiposity. [ABSTRACT FROM AUTHOR]

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge. [ABSTRACT FROM AUTHOR]

Viral infections caused by human adenovirus (HAdV) or CMV remain life-threatening complications in immunocompromised patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive immunotherapy with virus-specific T cells showed impressive clinical results without or with only mild GvHD. However, because of high costs and high regulatory barriers, these protocols are accessible to only a few centers. The infusion of unmanipulated donor lymphocytes (DLIs) that contain virus-specific T cells is not feasible because of the risk of GvHD. Reports about three patients treated with irradiated granulocytes or DLIs that potentially comprised virus-specific T cells discussed an active role of virus-specific lymphocytes despite irradiation, but real evidence could not be provided. Therefore, we tested the effect of irradiation on HAdV-specific T cells, which had been expanded in vitro, by stimulating PBMCs with HAdV-peptide pools and IL-15 for 12 days. Cells were then irradiated with 30 Gy, as performed for normal granulocyte concentrates. Cell viability and polyfunctional activity were determined by flow cytometry. Even 48 h after irradiation, 15.6% of expanded HAdV-specific T cells were apparently viable and cytolytically active. Although the in vivo antiviral activity was not tested, these data support earlier assumptions about the potential role of irradiated cells in patients. [ABSTRACT FROM AUTHOR]

In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture-PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses. [ABSTRACT FROM AUTHOR]

Buffalo River is an important water resource in the Eastern Cape Province of South Africa. Over a 1-year period (August 2010-July 2011), we assessed the prevalence of human adenoviruses (HAdVs) at a total of 6 sites on the river and three dams along its course. HAdVs were detected by real-time quantitative PCR in about 35 % of the samples with concentrations ranging from 1.2 × 10 genome copies (GC)/l to 4.71 × 10 GC/l. HAdVs were detected at 5 of the 6 sampling sites with the detection rate ranging from 8.3 % at Rooikrantz Dam to 92 % at Parkside. The HAdV concentrations across the sampling sites were as follows: Parkside (3.25 × 10-4.71 × 10 GC/); King William's Town (1.02 × 10-4.56 × 10 GC/l); and Eluxolzweni (1.17 × 10-3.97 × 10 GC/l). Significantly ( P < 0.05) higher concentrations were detected at the non-dam sites compared to the dam sites. A very low mean concentration of 1.86 × 10 HAdV GC/l was observed at Bridle Drift Dam. While HAdVs were detected only once at Rooikrantz Dam (1.74 × 10 GC/l), no HAdV was detected at Maden Dam. Epidemiologically important serotypes, Ad40/41, constituted 83.3 %, while Ad21 made up 16.7 % of the all HAdVs detected and were characterized by qualitative PCR. The Buffalo River presents a public health risk heightened by the presence of Ad 40/41 and Ad21. Our results make imperative the need for assessing water sources for viral contamination in the interest of public health. This work is a significant contribution to the molecular epidemiology of adenoviruses and to the best of our knowledge this is the first report on detection of enteric virus from surface waters in the Eastern Cape. [ABSTRACT FROM AUTHOR]

One of the major limitations of cancer gene therapy using recombinant human adenovirus (Ad) is rapid Ad inactivation from systemic delivery. To eliminate this, biotin-coated ultrasound contrast agents, or microbubbles (MBs), were streptavidin-coupled with biotinylated antibodies to three distinct tumor vasculature-associated receptors (αVβ3 integrin, P-selectin and vascular endothelial growth factor receptor-2) for systemic targeting of a previously generated vector Ad5/3-Id1-SEAP-Id1-mCherry. This cancer-specific, dual-reporter vector was loaded in the targeted MBs and confirmed by confocal microscopy. MB loading capacity was estimated by functional assays as 4.72±0.2 plaque forming unit (PFU) per MB. Non-loaded (free) Ad particles were effectively inactivated by treatment with human complement. The Ad-loaded, targeted-MBs were injected systemically in mice bearing MDA-MB-231 tumors (Grp 1) and compared with two control groups: Ad-loaded, non-targeted MBs (Grp 2) and free Ad (Grp 3) administered under the same conditions. Two days after administration the blood levels of secreted embryonic alkaline phosphatase (SEAP) reporter in Grp 1 mice (16.1 ng ml-1±2.5) were significantly higher (P<0.05) than those in Grp 2 (9.75 ng ml-1±1.5) or Grp 3 (4.26 ng ml-1±2.5) animals. The targeted Ad delivery was also confirmed by fluorescence imaging. Thus, Ad delivery by targeted MBs holds potential as a safe and effective system for systemic Ad delivery for the purpose of cancer screening. [ABSTRACT FROM AUTHOR]

The major goal of this study is to gain a perspective on the prevalence of DNA enteric virus genomes in mesophilic anaerobic-digested (MAD) sewage sludge and manure by comparing their quantitative PCR (qPCR) concentrations and removals with traditional fecal indicators ( Escherichia coli, enterococci, and Bacteroidetes). In addition, relationships between qPCR and culture measurements of fecal indicators (FIs) were determined. There was no significant difference between the qPCR concentrations of human adenovirus and E. coli/enterococci in MAD sewage sludge; however, the qPCR concentrations of bovine adenovirus were significantly lower than FIs and bovine polyomavirus (BPyV) in MAD manure. The qPCR concentrations of human polyomavirus were slightly lower than E. coli and enterococci ( p ≤ 0.05), but no significant difference was observed between the qPCR concentrations of BPyV and FIs. The digestion treatment achieved higher genome removal of bovine DNA enteric viruses than FIs ( p ≤ 0.05). Significant correlations were observed between qPCR and culture measurements of FIs, but the concentrations and removals of FIs determined by qPCR assays were still significantly different than those determined by culture assays. Overall, we determined that the prevalence of DNA enteric virus genomes in MAD biological wastes was high due to their comparable in qPCR concentrations to FIs, indicating that mesophilic anaerobic digestion treatment alone may not be effective enough to remove DNA viral pathogens in biological wastes. [ABSTRACT FROM AUTHOR]

A new DNA reporter construct has been designed for protein quantification by immuno-PCR. It has been shown that the amplification efficiency of a reporter that contains a fragment of human adenovirus 2 flanked by homoprimer sequences is much higher than standard PCR format based on two different primers. The further application of the new construct and its homoprimer-based detection could represent new ways to increase the sensitivity of the immuno-PCR and the efficiency of single-molecule PCR. [ABSTRACT FROM AUTHOR]