OBJECTIVES: Direct genotyping of adenovirus or enterovirus from clinical material using polymerase chain reaction (PCR) followed by Sanger sequencing is often difficult due to the presence of multiple virus types in a sample, or due to varying efficacy of PCR amplifying the capsid gene on the background of foreign nucleic acids. Here we present a simple protocol for virus genotyping using massive parallel amplicon sequencing. METHODS: The protocol utilized a set of 16 tailed degenerate primers flanking the seventh hypervariable region of the adenovirus hexon gene and 9 tailed degenerate primers targeted to the proximal portion of the enterovirus VP1 gene. Subsequent addition of dual indices enabled simultaneous sequencing of 384 different samples on an Illumina MiSeq instrument. Downstream bioinformatic analysis was based on remapping to a set of references representative of the presently known repertoire of virus types. RESULTS: After validation with known virus types, the sequencing method was applied on 301 adenovirus-positive samples and 350 enterovirus-positive samples from a longitudinally collected series of stools from 83 children aged 3 to 36 months. We detected 7 different adenovirus types and 27 different enterovirus types. There were 37 (6.2%) samples containing more than one genotype of the same viral genus. At least one dual infection was experienced by 23 of 83 (28%) of the children observed over the 3 years' observation period. CONCLUSIONS: Amplicon sequencing with a multiplex set of degenerate primers seems to be a rapid and reliable technical solution for genotyping of large collections of samples where simultaneous infections with multiple strains can be expected.

• MeSH
• Adenoviridae klasifikace   genetika   izolace a purifikace   MeSH
• adenovirové infekce virologie   MeSH
• DNA primery genetika   MeSH
• enterovirové infekce virologie   MeSH
• Enterovirus klasifikace   genetika   izolace a purifikace   MeSH
• genotyp * MeSH
• genotypizační techniky metody   MeSH
• kojenec MeSH
• lidé MeSH
• longitudinální studie MeSH
• předškolní dítě MeSH
• sekvenční analýza DNA metody   MeSH
• výpočetní biologie MeSH
• zvířata MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Norsko MeSH

Adenoviruses are a widespread cause of diverse human infections with recently confirmed zoonotic roots in African great apes. We focused on savanna-dwelling chimpanzees in the Issa Valley (Tanzania), which differ from those from forested sites in many aspects of behavior and ecology. PCR targeting the DNA polymerase gene detected AdV in 36.7% (69/188) of fecal samples. We detected five groups of strains belonging to the species Human mastadenovirus E and two distinct groups within the species Human mastadenovirus C based on partial hexon sequence. All detected AdVs from the Issa Valley are related to those from nearby Mahale and Gombe National Parks, suggesting chimpanzee movements and pathogen transmission.

• MeSH
• Adenoviridae genetika   izolace a purifikace   MeSH
• adenovirové infekce epidemiologie   veterinární   virologie   MeSH
• DNA-dependentní DNA-polymerasy genetika   MeSH
• feces virologie   MeSH
• fylogeneze MeSH
• nemoci lidoopů epidemiologie   virologie   MeSH
• Pan troglodytes virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Tanzanie epidemiologie   MeSH

The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus.

• MeSH
• Adenoviridae klasifikace   genetika   imunologie   MeSH
• antigeny virové genetika   imunologie   MeSH
• ELISA MeSH
• exprese genu * MeSH
• krocani MeSH
• protilátky virové krev   imunologie   MeSH
• rekombinantní proteiny genetika   imunologie   izolace a purifikace   MeSH
• specificita protilátek imunologie   MeSH
• virové plášťové proteiny genetika   imunologie   izolace a purifikace   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Both, common gene variants and human adenovirus 36 (Adv36) are involved in the pathogenesis of obesity. The potential relationship between these two pathogenic factors has not yet been investigated. The aim of our study was to examine the association of obesity susceptibility loci with Adv36 status. Genotyping of ten gene variants (in/near TMEM18, SH2B1, KCTD15, PCSK1, BDNF, SEC16B, MC4R, FTO) and analysis of Adv36 antibodies was performed in 1,027 Czech adolescents aged 13.0-17.9 years. Variants of two genes (PCSK1 and BDNF) were associated with Adv36 seropositivity. A higher prevalence of Adv36 antibody positivity was observed in obesity risk allele carriers of PCSK1 rs6232, rs6235 and BDNF rs4923461 vs. non-carriers (chi(2)=6.59, p=0.010; chi(2)=7.56, p=0.023 and chi(2)=6.84, p=0.033, respectively). The increased risk of Adv36 positivity was also found in PCSK1 variants: rs6232 (OR=1.67, 95 % CI 1.11-2.49, p=0.016) and rs6235 (OR=1.34, 95 % CI 1.08-1.67, p=0.010). PCSK1 rs6232 and BDNF rs925946 variants were closely associated with Adv36 status in boys and girls, respectively (chi(2)=5.09, p=0.024; chi(2)=7.29, p=0.026). Furthermore, PCSK1 rs6235 risk allele was related to Adv36 seropositivity (chi(2)=6.85, p=0.033) in overweight/obese subgroup. In conclusion, our results suggest that obesity risk variants of PCSK1 and BDNF genes may be related to Adv36 infection.

• MeSH
• Adenoviridae genetika   MeSH
• adenovirové infekce lidí epidemiologie   genetika   MeSH
• genetická variace genetika   MeSH
• genetické asociační studie metody   MeSH
• lidé MeSH
• mladiství MeSH
• mozkový neurotrofický faktor genetika   MeSH
• obezita epidemiologie   genetika   MeSH
• proproteinkonvertasa 1 genetika   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Efficient intravenous delivery is the greatest single hurdle, with most nanotherapeutics frequently found to be unstable in the harsh conditions of the bloodstream. In the case of nanotherapeutics for gene delivery, viral vectors are often avidly recognized by both the innate and the adaptive immune systems. So, most modern delivery systems have benefited from being coated with hydrophilic polymers. Self-assembling delivery systems can achieve both steric and lateral stabilization following surface coating, endowing them with much improved systemic circulation properties and better access to disseminated targets; similarly, gene delivery viral vectors can be 'stealthed' and their physical properties modulated by surface coating. Polymers that start degrading under acidic conditions are increasingly investigated as a pathway to trigger the release of drugs or genes once the carrier reaches a slightly acidic tumor environment or after the carrier has been taken up by cells, resulting in the localization of the polymer in acidic endosomes and lysosomes. Advances in the design of acid-degradable drug and gene delivery systems have been focused and discussed in this article with stress placed on HPMA-based copolymers. We designed a system that is able to "throw away" the polymer coat after successful transport of the vector into a target cell. Initial biological studies were performed and it was demonstrated that this principle is applicable for real adenoviral vectors. It was shown that the transfection ability of coated virus at pH 7.4 is 75 times lower then transfection at pH 5.4.

• MeSH
• Adenoviridae genetika   MeSH
• chemie farmaceutická * MeSH
• endozomy MeSH
• genetické vektory MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• lyzozomy MeSH
• mikrosféry MeSH
• nanomedicína metody   MeSH
• polymery MeSH
• technika přenosu genů * MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: PFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis. METHODS: Paired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n=10) (b) from children with obstructive sleep apnoea syndrome as controls (n=10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR. RESULTS: The PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls. CONCLUSIONS: Our findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome.

• MeSH
• Adenoviridae genetika   izolace a purifikace   MeSH
• aftózní stomatitida komplikace   imunologie   chirurgie   MeSH
• antigeny CD279 biosyntéza   MeSH
• B-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• chemokin CCL19 biosyntéza   MeSH
• chemokin CXCL10 biosyntéza   MeSH
• chemokin CXCL9 biosyntéza   MeSH
• dítě MeSH
• faryngitida komplikace   imunologie   chirurgie   MeSH
• horečka neznámého původu komplikace   imunologie   chirurgie   MeSH
• kojenec MeSH
• krční mandle cytologie   imunologie   chirurgie   MeSH
• lidé MeSH
• lidský herpesvirus 6 genetika   izolace a purifikace   MeSH
• lymfadenitida komplikace   imunologie   chirurgie   MeSH
• obstrukční spánková apnoe imunologie   chirurgie   MeSH
• počet lymfocytů MeSH
• předškolní dítě MeSH
• receptory antigenů T-buněk genetika   MeSH
• T-lymfocyty - podskupiny imunologie   MeSH
• tonzilektomie MeSH
• virus Epsteinův-Barrové genetika   izolace a purifikace   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nanomedicines have provided fresh impetus in the fight against cancer due to their selectivity and power. However, these agents are limited when delivered intravenously due to their rapid clearance from the bloodstream and poor passage from the bloodstream into target tumours. Here we describe a novel stealthing strategy which addresses both these limitations and thereby demonstrate that both the passive and mechanically-mediated tumour accumulation of the model nanomedicine adenovirus (Ad) can be substantially enhanced. In our strategy gold nanoparticles were thoroughly modified with 2kDa polyethyleneglycol (PEG) and then linked to Ad via a single reduction-cleavable 5kDa PEG. The resulting Ad-gold-PEG construct was compared to non-modified Ad or conventionally stealthed Ad-poly[N-(2-hydroxypropyl)methacrylamide] (Ad-PHPMA). Notably, although Ad-gold-PEG was of similar size and surface charge to Ad-PHPMA the increase in density, resulting from the inclusion of the gold nanoparticles, provided a substantial enhancement of ultrasound-mediated transport. In an in vitro tumour mimicking phantom, the level and distance of Ad-gold-PEG transport was shown to be substantially greater than achieved with Ad-PHPMA. In in vivo studies 0.1% of an unmodified Ad dose was shown to accumulate in tumours, whereas over 12% of the injected dose was recovered from the tumours of mice treated with Ad-gold-PEG and ultrasound. Ultimately, a significant increase in anti-tumour efficacy resulted from this strategy. This stealthing and density-increasing technology could ultimately enhance clinical utility of intravenously delivered nanoscale medicines including viruses, liposomes and antibodies.

• MeSH
• Adenoviridae genetika   MeSH
• játra metabolismus   MeSH
• kovové nanočástice * aplikace a dávkování   chemie   MeSH
• kyseliny polymethakrylové aplikace a dávkování   chemie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   terapie   MeSH
• nanomedicína MeSH
• onkolytická viroterapie MeSH
• polyethylenglykoly * aplikace a dávkování   chemie   MeSH
• ultrazvuk MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zlato * aplikace a dávkování   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human adenoviruses (HAdV; species HAdV-A to -G) are highly prevalent in the human population, and represent an important cause of morbidity and, to a lesser extent, mortality. Recent studies have identified close relatives of these viruses in African great apes, suggesting that some HAdV may be of zoonotic origin. We analyzed more than 800 fecal samples from wild African great apes and humans to further investigate the evolutionary history and zoonotic potential of hominine HAdV. HAdV-B and -E were frequently detected in wild gorillas (55%) and chimpanzees (25%), respectively. Bayesian ancestral host reconstruction under discrete diffusion models supported a gorilla and chimpanzee origin for these viral species. Host switches were relatively rare along HAdV evolution, with about ten events recorded in 4.5 My. Despite presumably rare direct contact between sympatric populations of the two species, transmission events from gorillas to chimpanzees were observed, suggesting that habitat and dietary overlap may lead to fecal-oral cross-hominine transmission of HAdV. Finally, we determined that two independent HAdV-B transmission events to humans occurred more than 100,000 years ago. We conclude that HAdV-B circulating in humans are of zoonotic origin and have probably affected global human health for most of our species lifetime.

• MeSH
• Adenoviridae * genetika   patogenita   MeSH
• adenovirové infekce * genetika   přenos   MeSH
• druhová specificita MeSH
• Hominidae virologie   MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• zoonózy genetika   přenos   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

V sezóně 2013/14 byly dominujícím respiračním patogenem adenoviry (hAdV). Adenoviry jsou poměrně bohatá skupina virů obsahujících dle současné klasifikace 60 sérotypů. Většina z nich je spojována s mnoha různými mírnými onemocněními od respiračních, přes gastroenteritidy až po encefalitidy. Pouze některé sérotypy vyvolávají závažné infekce s tendencí k lokálním epidemiím. Pro majoritní výskyt jsme přistoupili k charakterizaci hAdV v sentinelových materiálech. Namísto klasické sérotypizace byla použita metoda parciálního sekvenování úseku hexonového genu o velikosti cca 800 párů bazí. Na základě BLAST (Basic Local Alignment Search Tool) analýzy bylo provedeno systematické zařazení u celkem 27 materiálů z celkového počtu 52 vzorků. V rámci klasifikace bylo nalezeno pouze 5 sérotypů (3B, 2C, 1C, 7B, 4E). Naprosto převažujícím sérotypem byl hAdV 3B, který byl prokázán v 63 % sekvenovaných materiálů. V souvislosti s adenovirovým onemocněním udávali téměř všichni nemocní rýmu, horečku s teplotou nad 38 °C a kašel. Zhruba polovina nemocných trpělo bolestí hlavy a malátností. Nebyly prokázány žádné sérotypy dávané do souvislosti se závažným onemocněním, což odpovídá vybrané kohortě pacientů. Nejméně u poloviny materiálů byl sérotyp určen přímo z klinického materiálu bez předchozí izolace. Lze tedy shrnout, že sérotypizace /genotypizace provedená na základě parciálního sekvenování hexonového genu s použitím „nested“ (vnořených) primerů nevyžaduje izolaci viru na tkáňové kultuře. Metoda je dostupná a efektivní při charakterizaci adenovirů.

In the season 2013/14, the predominant respiratory pathogen was adenovirus (hAdV). Adenoviruses are a relatively large family of DNA viruses consisting of 60 serotypes classified into seven groups. Most of them are associated with many various mild diseases from respiratory infections through gastroenteritis to encephalitis. Some serotypes only cause severe infection with a tendency towards local outbreaks. We have implemented the classification of hAdV in sentinel samples. Instead of conventional serotyping, partial sequencing of the approximately 800 pb segments of the hexon gene was used. Based on the BLAST (Basic Local Alignment Search Tool) analysis, 27 of 52 samples were fitted into the current classification system. Only five serotypes were found (3B, 2C, 1C, 7B, 4E). The clearly prevailing hAdV serotype was 3B, detected in 63 % of the samples sequenced. Nearly all patients with adenovirus infection reported runny nose, temperature above 38°C, and cough. About half of the patients complained of headache and malaise. No severe disease-associated serotype was detected, as expected given the selected cohort of patients. In at least half of the samples, the serotype was determined without the need for virus isolation on cell culture. It can be concluded that serotyping based on partial hexon gene sequencing in combination with nested PCR does not require virus isolation on tissue culture. The method is time saving and effective in the characterization of adenoviruses.

• Klíčová slova
• genotypizace,
• MeSH
• Adenoviridae genetika   MeSH
• adenovirové infekce lidí * diagnóza   genetika   MeSH
• DNA primery MeSH
• genotypizační techniky MeSH
• infekce dýchací soustavy * diagnóza   etiologie   MeSH
• lidé MeSH
• sekvenční analýza DNA MeSH
• sérotypizace * metody   statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH

• MeSH
• Adenoviridae genetika   izolace a purifikace   MeSH
• Bacteroides genetika   izolace a purifikace   MeSH
• DNA analýza   genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   normy   MeSH
• Legionella pneumophila genetika   izolace a purifikace   MeSH
• mikrobiologie životního prostředí normy   MeSH
• společenstvo * MeSH
• výzkumný projekt MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH