The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

CACO-2 cells: A continuous cell line with sensitive and broad-spectrum utility for respiratory virus culture.

BACKGROUND: Primary rhesus monkey kidney cells (RhMK) can be used for the detection of respiratory viruses, including influenza and parainfluenza. The human colon adeno-carcinoma cell line, CACO-2, has been previously used for the growth of multiple influenza viruses, including seasonal, novel and avian lineages. OBJECTIVE: We compared CACO-2, Madin-Darby Canine Kidney (MDCK), and RhMK cells for the isolation of viruses from patients presenting with influenza like-illness (ILI). STUDY DESIGN: Nasopharyngeal specimens from patients with ILI in primary care settings were processed for conventional viral culture in MDCK, RhMK, and CACO-2. Cells were examined microscopically for cytopathic effect (CPE) and confirmatory testing included immunofluorescent antigen (IFA) detection and real-time RT-PCR. Additionally, 16 specimens positive for respiratory syncytial virus (RSV) by PCR were inoculated on CACO-2 cells. Statistical analysis was done using Chi-square test with IBM Statistical Program. RESULTS: Of 1031 respiratory specimens inoculated, viruses were isolated and confirmed from 331 (32.1 %) in MDCK cells, 304 (29.5 %) in RhMk cells, and 433 (42.0 %) in CACO-2 cells. These included influenza A/(H1N1)pdm09, influenza A(H3N2), influenza B, parainfluenza virus (PIV) types 1, 2, and 3, human coronavirus 229E (CoV-229E), human adenovirus (HAdV), herpes simplex virus 1 (HSV 1), and enterovirus (EV). Influenza A viruses grew best in the CACO-2 cell line. Time to observation of CPE was similar for all three cell types but unlike RhMK and MDCK cells, virus-specific morphological changes were indistinguishable in CACO-2 cells. None of the 16 specimens positive for RSV by PCR grew on CACO-2 cells. CONCLUSIONS: The overall respiratory virus culture isolation rate in CACO-2 cells was significantly higher than that in RhMK or MDCK cells (p < 0.05). CACO-2 cells also supported the growth of some viruses that did not grow in either RhMK or MDCK cells. Except for RSV, CACO-2 cells provide a worthwhile addition to culture algorithms for respiratory specimens.

Detection of Human Adenovirus, Rotavirus, and Enterovirus in Tap Water and Their Association with the Overall Quality of Water in Karachi, Pakistan.

Drinking water supplies in the developing world often serve as a biosphere for various organisms. Viral gastroenteritis is a neglected area of research in Pakistan, there are no data for the prevalence of enteric viruses in drinking water of the largest city of Karachi. The present study aimed to provide a survey of the existence of enteric viruses: human adenovirus (HAdV), human enteroviruses (hEV), and genotype A rotavirus (GARV) in tap water. Using a simple PCR approach, we detected 20%, 43%, and 23% of HAdV, hEV, and GARV in tap water samples, respectively. We have also shown an overall quality deficit of tap water at the pumping station and consumer tap. We have found no sample free from bacterial contaminations. The ranges for a total number of the heterotrophic plate count and coliform were found 8.7 × 102-4.5 × 106 CFU/mL and 210 to uncountable coliforms/100 mL, respectively. Moreover, we assessed the efficiency of small-scale water treatment methods for the removal of viruses.

Differential Splicing of Human Adenovirus 5 E1A RNA Expressed in cis versus in trans.

Human adenovirus (HAdV) is used extensively as a vector for gene delivery for a variety of purposes, including gene therapy and vaccine development. Most adenoviral vectors used for these approaches have a deletion of early region 1 (E1), which is complemented by the cell line. Most commonly, these are 293 cells for HAdV serotype 2 or 5. The 293 cells have the left end of HAdV5 integrated into chromosome 19 and express the E1 genes and protein IX. We observed that viruses with the E1 region deleted often grow less well on 293 cells than E1 wild-type viruses. Therefore, we investigated whether this poor growth is caused by splicing differences between the E1A RNA provided by the cell line (in trans) and the E1A RNA provided by the infecting viral genome (in cis). We observed that E1A RNA that was expressed from the genomes of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus than E1A RNA from the same cells infected with dl309 or wt300. Importantly, 293 cells were not able to fully complement the late E1A transcripts, specifically 11S, 10S, and 9S RNA, which express the E1A217R, E1A171R, and E1A55R isoforms, respectively. We observed that these splicing differences likely arise due to different subnuclear localizations of E1A RNA. E1A RNA expressed from the viral genome was localized to viral replication centers, while E1A RNA expressed from the cell's genome was not. This loss of the late E1A mRNAs and their associated proteins impacts viral growth, gene expression, and protein levels. Complementation of the late E1A mRNAs in 293 cells restored some of the growth defect observed with dl312 and resulted in higher virus growth.IMPORTANCE Human adenovirus has become an important tool for medicine and research, and 293 cells and various similar cell lines are used extensively for virus production in situations where high viral yields are important. Such complementing cell lines are used for the production of viral vectors and vaccines, which often have deletions and replacements in various viral genes. Deletions in essential genes, such as E1, are often complemented by the cell line that is used for virus propagation in trans Here, we show that even complete genetic complementation of a viral gene does not result in full protein complementation, a defect that compromises virus growth. This is particularly important when high viral yields are crucial, as in virus production for vaccine development or gene therapy.

Generation and characterization of an Il2rg knockout Syrian hamster model for XSCID and HAdV-C6 infection in immunocompromised patients.

Model animals are indispensable for the study of human diseases, and in general, of complex biological processes. The Syrian hamster is an important model animal for infectious diseases, behavioral science and metabolic science, for which more experimental tools are becoming available. Here, we describe the generation and characterization of an interleukin-2 receptor subunit gamma (Il2rg) knockout (KO) Syrian hamster strain. In humans, mutations in IL2RG can result in a total failure of T and natural killer (NK) lymphocyte development and nonfunctional B lymphocytes (X-linked severe combined immunodeficiency; XSCID). Therefore, we sought to develop a non-murine model to study XSCID and the infectious diseases associated with IL2RG deficiency. We demonstrated that the Il2rg KO hamsters have a lymphoid compartment that is greatly reduced in size and diversity, and is impaired in function. As a result of the defective adaptive immune response, Il2rg KO hamsters developed a more severe human adenovirus infection and cleared virus less efficiently than immune competent wild-type hamsters. Because of this enhanced virus replication, Il2rg KO hamsters developed more severe adenovirus-induced liver pathology than wild-type hamsters. This novel hamster strain will provide researchers with a new tool to investigate human XSCID and its related infections.

Quantitative Detection of Human Adenovirus and Human Rotavirus Group A in Wastewater and El-Rahawy Drainage Canal Influencing River Nile in the North of Giza, Egypt.

Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this study, the prevalence of human adenovirus (HAdV), rotavirus group A (RVA) was determined in urban sewage, activated sludge, drainage water, drainage sediment, Nile water, and Nile sediment, using quantitative polymerase chain reaction (qPCR) analysis. HAdV was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 genome copies/liter (GC/L), 33% of activated sludge with viral concentrations ranging from 103 to 107 GC/kilogram (GC/kg), 95% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 75% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, 50% of Nile water with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile sediment with viral concentrations ranging from 103 to 107 GC/kg. RVA was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 GC/L, 75% of activated sludge with viral concentrations ranging from 103 to 107 GC/L, 58% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 50% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile water with viral concentrations ranging from 103 to 107 GC/kg. In conclusion, Abu-Rawash WWTP acts as a source of HAdV and RVA, releasing them into El-Rahawy drain then to the River Nile Rosetta branch.

Histone Deacetylase Inhibitors Promote Latent Adenovirus Reactivation from Tonsillectomy Specimens.

Adenovirus (HAdV) infection is a common cause of illness among young children, immunocompromised patients, and transplant recipients. The majority of HAdV infections are self-limited, but recurring infection is frequently encountered in young children and may require hospitalization. In this study, we surveyed the presence of HAdV in tonsillectomy samples and investigated epigenetic conditions that contributed to HAdV reactivation. HAdV DNA was detected from 86.7% donors. The lymphocytes isolated from the samples failed to produce infectious HAdV after incubation, suggesting the viruses remained in a latent status. To determine whether epigenetic factors played a role in HAdV reactivation, isolated lymphocytes were treated with a small compound library. Viral DNA replication and infectious HAdV production were assayed by PCR and by a secondary infection assay. We identified several compounds, mainly pan- and selective histone deacetylase (HDAC) inhibitors, which showed activity to reactivate HAdV from latency. The viruses were isolated and were determined as species C HAdV. Using a model of HAdV lytic infection, we showed that the compounds promoted histone-3 acetylation and association with viral early gene promoters. In addition to demonstrate the palatine tonsils as a reservoir of latent HAdV, this study uncovers a critical role of histone acetylation in HAdV reactivation, linking HAdV latency to recurrent HAdV infection.IMPORTANCE Respiratory tract infection by adenoviruses is among the most common diseases in children, attributing to approximately 20% of hospitalizations of children with acute respiratory infection (ARI). Adenovirus transmits by direct contact, but recurrent infection is common. Ever since its isolation, adenovirus has been known to have the ability to establish persistent or latent infection. We found 87.7% tonsillectomy specimens contained detectable amounts of adenoviral DNA. Isolated lymphocytes did not produce infectious adenoviruses without stimulation. By screening an epigenetic informer compound library, we identified several histone deacetylase inhibitors that promoted adenovirus reactivation that was evidenced by increased viral DNA replication and production of infectious viruses. The human tonsils are covered with bacterial pathogens that may utilize pathogen-associated pattern molecules or metabolites to cause epigenetic activation and proinflammatory gene transcription, which may lead to viral reactivation from latency. The study shows that recurrent adenovirus infection could arise from reactivation of residing virus from previous infections.

Gastroenteric Viruses Detection in a Drinking Water Distribution-to-Consumption System in a Low-Income Community in Rio de Janeiro.

The availability of drinking water is one of the main determinants of quality of life, disease prevention and the promotion of health. Viruses are important agents of waterborne diseases and have been described as important markers of human faecal contamination. This study aimed to investigate viruses' presence as an indicator of drinking water quality in low-income communities in the Manguinhos area, Rio de Janeiro, Brazil. Three hundred and four drinking water samples (2L/each) were collected along the drinking water distribution-to-consumption pathway in households, as well as healthcare and school units. Water samples were collected both directly from the water supply prior to distribution and after storage in tanks and filtration units. Using qPCR, viruses were detected 50 times in 45 water samples (15%), 19 of these being human adenovirus, 17 rotavirus A and 14 norovirus GII. Viral loads recovered ranged from 5E+10 to 8.7E+106 genome copies/Liter. Co-detection was observed in five household water samples and there was no difference regarding virus detection across sampling sites. Precarious and inadequate environmental conditions characterized by the lack of local infrastructure regarding basic sanitation and waste collection in the territory, as well as negligent hygiene habits, could explain viral detection in drinking water in regions with a water supply system.

Mineral Waste Containing High Levels of Iron from an Environmental Disaster (Bento Rodrigues, Mariana, Brazil) is Associated with Higher Titers of Enteric Viruses.

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60 million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fundão), reaching the Gualaxo do Norte River (sites evaluated in this study), the "Rio Doce" River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (< 20 µg/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil.

Inactivation of Adenovirus in Water by Natural and Synthetic Compounds.

Millions of people use contaminated water sources for direct consumption. Chlorine is the most widely disinfection product but can produce toxic by-products. In this context, natural and synthetic compounds can be an alternative to water disinfection. Therefore, the aim of this study was to assess the inactivation of human adenovirus by N-chlorotaurine (NCT), bromamine-T (BAT) and Grape seed extract (GSE) in water. Distilled water artificially contaminated with recombinant human adenovirus type 5 (rAdV-GFP) was treated with different concentrations of each compound for up to 120 min, and viral infectivity was assessed by fluorescence microscopy. The decrease in activity of the compounds in the presence of organic matter was evaluated in water supplemented with peptone. As results, NCT and GSE inactivated approximately 2.5 log10 of adenovirus after 120 min. With BAT, more than 4.0 log10 decrease was observed within 10 min. The oxidative activity of 1% BAT decreased by 50% in 0.5% peptone within a few minutes, while the reduction was only 30% for 1% NCT in 5% peptone after 60 min. Organic matter had no effect on the activity of GSE. Moreover, the minimal concentration of BAT and GSE to kill viruses was lower than that known to kill human cells. It was concluded that the three compounds have potential to be used for water disinfection for drinking or reuse purposes.

Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA.

Human adenovirus (HAdV) encodes a multifunctional DNA-binding protein pVII, which is involved in virus DNA packaging and extracellular immune signaling regulation. Although the pVII is an essential viral protein, its exact role in the virus life cycle and interplay with cellular proteins have remained to a large extent unclear. We have recently identified the cellular zinc finger protein 622 (ZNF622) as a potential pVII-interacting protein. In this study, we describe the functional consequences of the ZNF622-pVII interplay and the role of ZNF622 in the HAdV life cycle. ZNF622 protein expression increased, and it accumulated similarly to the pVII protein in the nuclei of virus-infected cells. The lack of the ZNF622 protein specifically increased pVII binding to viral DNA in the infected cells and elevated the pVII protein levels in the purified virions. In addition, ZNF622 knockout cells showed an increased cell lysis and enhanced accumulation of the infectious virus particles. Protein interaction studies revealed that ZNF622 forms a trimeric complex with the pVII protein and the cellular histone chaperon protein nucleophosmin 1 (NPM1). The integrity of this complex is important since ZNF622 mutations and NPM1 deficiency changed pVII ability to bind viral DNA. Collectively, our results implicate that ZNF622 may act as a cellular antiviral protein hindering lytic HAdV growth and limiting pVII protein binding to viral DNA.IMPORTANCE Human adenoviruses (HAdVs) are common human pathogens causing a wide range of acute infections. To counteract viral pathogenicity, cells encode a variety of antiviral proteins and noncoding RNAs to block virus growth. In this study, we show that the cellular zinc finger protein 622 (ZNF622) interacts with an essential HAdV protein known as pVII. This mutual interaction limits pVII binding to viral DNA. Further, ZNF622 has a role in HAdV life cycle since the lack of ZNF622 correlates with increased lysis of the infected cells and accumulation of the infectious virions. Together, our study reveals a novel cellular antiviral protein ZNF622, which may impede lytic HAdV growth.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection.

Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5' to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

Assessment of the Virological Quality of Marine and Running Surface Waters in NW Greece: A Case Study.

The virological quality of surface marine and running water samples collected from Igoumenitsa gulf and Kalamas river (NW Greece) was assessed from October 2012 to September 2013. Sampling sites were exposed to different land and/or anthropogenic effects. Seawater samples were collected monthly from five sampling stations (new harbor, old harbor, wastewater treatment plant outlet, protected Natura area, Drepano beach). Viral targets included human adenoviruses (hAdVs), as index human viruses, while noroviruses (NoVs) and hepatitis A virus (HAV) were also studied. Kalamas river samples were collected seasonally, from three sampling stations (Soulopoulo, Dam, Sagiada-estuaries), while viral targets included also porcine adenoviruses (pAdVs) and bovine polyoma viruses (bPyVs), as additional index viruses. All water samples were analyzed for standard bacterial indicators, as well. Physicochemical and meteorological data were also collected. Based on the standard bacterial indices, both sea and river water samples did not exceed the limits set according to Directive 2006/7/EU. However, positive samples for hAdVs were found occasionally in all sampling sites in Igoumenitsa gulf (23.3%, 14/60) showing fecal contamination of human origin. Moreover, HAV was detected once, in the sampling site of the old port (at 510 GC/L). Most of the Kalamas water samples were found positive for hAdVs (58.3%, 7/12), while human noroviruses GI (NoVGI) (8.3%, 1/12) and GII (NoVGII) (16.7%, 2/12) were also detected. HAV, pAdVs, and bovine polyomaviruses (bPyVs) were not detected in any of the analyzed samples. No statistically significant correlations were found between classic bacterial indicators and viral targets, nor between viruses and meteorological data. Overall, the present study contributed to the collection of useful data for the biomonitoring of the region, and the assessment of the overall impact of anthropogenic activities. It provided also valuable information for the evaluation of the risk of waterborne viral infections and the protection of public health. It was the first virological study in the area and one of the few in Greece.

Evaluation of Bacterial Contamination as an Indicator of Viral Contamination in a Sedimentary Aquifer in Uruguay.

In Uruguay, groundwater is frequently used for agricultural activities, as well as for human consumption in urban and rural areas. As in many countries worldwide, drinking water microbiological quality is evaluated only according to bacteriological standards and virological analyses are not mentioned in the legislation. In this work, the incidence of human viral (Rotavirus A, Norovirus GII, and human Adenovirus) and bacterial (total and thermotolerant coliform and Pseudomonas aeruginosa) contamination in groundwater in the Salto district, Uruguay, as well as the possible correlation between these groups of microorganisms, was studied. From a total of 134 groundwater samples, 42 (32.1%) were positive for Rotavirus, only 1 (0.7%) for both Rotavirus and Adenovirus, and 96 (72.6%) samples were positive for bacterial indicators. Results also show that Rotavirus presence was not associated with changes in chemical composition of the aquifer water. Bacteriological indicators were not adequate to predict the presence of viruses in individual groundwater samples (well scale), but a deeper spatial-temporal analysis showed that they are promising candidates to assess the viral contamination degree at aquifer scale, since from the number of wells with bacterial contamination the number of wells with viral contamination could be estimated.

Broad-spectrum antiviral activity of the deubiquitinase inhibitor HBX against human adenoviruses

BACKGROUND: Human adenoviral (HAdV) infections are usually mild and self-limited, however, some infections from species A, B, C, D and E, can cause severe illnesses, which have raised public health concerns over the past few years. Current available antiviral therapies have limited efficacy and severe toxicity; therefore, finding new targets for specific anti-adenoviral drug design is urgently needed. Our previous work showed that the small molecule compound, HBX, inhibits HAdV type 5 (species C, HAdV-C5) replication and oncogenic transformation through inhibition of the cellular pro-viral factor ubiquitin-specific protease 7 (USP7). Here, we have tested the ability of HBX to inhibit other HAdV species, as well as different clinical isolates that are the cause of severe infections. METHODS: We treated HAdV-infected A549 cells with different concentrations of HBX and analysed the antiviral efficacy of the drug by determining the half maximal inhibitory concentration (IC50) necessary to decrease both viral genome copies and virus progeny production at different time points after infection. RESULTS: In addition to its effect on HAdV-C5, HBX was able to significantly inhibit virus genome replication and progeny release of all adenovirus types tested, with the exception of types 12 and 31, from species A. Of note, clinical isolates were more sensitive to HBX treatment than their prototype strains. CONCLUSIONS: These results point to HBX as a promising broad-spectrum anti-adenoviral drug, opening new opportunities to prevent severe adenoviral infections and to improve their treatment.

Whole-genome analyses of human adenovirus type 55 emerged in Tibet, Sichuan and Yunnan in China, in 2016.

Three outbreaks of acute respiratory disease occurred at military camps in 2016 at Tibet, Sichuan and Yunnan province, China. The pathogen induced these three outbreaks were all confirmed as HAdV-55 by genotype-specific PCR. The outbreak in Tibet was the first report that HAdV-55 occurred in the high altitude (HA, above sea level 3658 m). This study aims to determine the gene variation and evolution characteristics of these viral strains. Three strains of adenoviruses, LS89/Tibet/2016 (GenBank accession no. KY002683), SF04/SC/2016 (GenBank accession no. KY002684) and KM03/YN/2016 (GenBank accession no. KY002685) were obtained and confirmed by wholegenome sequencing. No multi-gene fragments recombination were found in these isolated HAdV-55 virus compared with previous reported HAdV-55 strains in China. The outbreaks in Tibet and in Sichuan continuously occurred. Virus isolated from Tibet (LS89/Tibet/2016) and Sichuan (SF04/SC/2016) had a similar mutation pattern and had a closer genetic evolutionary distance than KM03/YN/2016 strain, which indicates that the pathogens causing these two outbreaks may be of the same origin. Moreover, we found that heating was an effective way to inactive these viruses, which provide valuable information for the development of HAdV-55 vaccines. Our data provide new information for genetic evolution of HAdV-55, and contribute to the prevention and control of HAdV-55 infection in the future.

Temporal characterization of the non-structural Adenovirus type 2 proteome and phosphoproteome using high-resolving mass spectrometry.

The proteome and phosphoproteome of non-structural proteins of Adenovirus type 2 (Ad2) were time resolved using a developed mass spectrometry approach. These proteins are expressed by the viral genome and important for the infection process, but not part of the virus particle. We unambiguously confirm the existence of 95% of the viral proteins predicted to be encoded by the viral genome. Most non-structural proteins peaked in expression at late time post infection. We identified 27 non-redundant sites of phosphorylation on seven different non-structural proteins. The most heavily phosphorylated protein was the DNA binding protein (DBP) with 15 different sites. The phosphorylation occupancy rate could be calculated and monitored with time post infection for 15 phosphorylated sites on various proteins. In the DBP, phosphorylations with time-dependent relation were observed. The findings show the complexity of the Ad2 non-structural proteins and opens up a discussion for potential new drug targets.

Human adenovirus in tissues of freshwater snails living in contaminated waters.

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.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.