Publications by authors named "Michele Gastaldelli"

18 Publications

  • Page 1 of 1

Molecular Differentiation of Outbreaks: A Last Decade Study on Italian Farms Using GTS and MLST.

Vaccines (Basel) 2020 Nov 9;8(4). Epub 2020 Nov 9.

Mycoplasma Unit-SCT1, Istituto Zooprofilattico Sperimentale delle Venezie, Via San Giacomo 5, 37135 Verona, Italy.

(MG) infects many avian species and leads to significant economic losses in the poultry industry. Transmission of this pathogen occurs both horizontally and vertically, and strategies to avoid the spread of MG rely on vaccination and the application of biosecurity measures to maintain breeder groups as pathogen-free. Two live attenuated MG vaccine strains are licensed in Italy: 6/85 and ts-11. After their introduction, the implementation of adequate genotyping tools became necessary to distinguish between field and vaccine strains and to guarantee proper infection monitoring activity. In this study, 40 Italian MG isolates collected between 2010-2019 from both vaccinated and unvaccinated farms were genotyped using gene-targeted sequencing (GTS) of the cythadesin gene and multilocus sequence typing (MLST) based on six housekeeping genes. The discriminatory power of GTS typing ensures 6/85-like strain identification, but the technique does not allow the identification ts-11 strains; conversely, MLST differentiates both vaccine strains, describing more detailed interrelation structures. Our study describes MG genetic scenario within a mixed farming context. In conclusion, the use of adequate typing methods is essential to understand the evolutionary dynamics of MG strains in a particular area and to conduct epidemiological investigations in the avian population.
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http://dx.doi.org/10.3390/vaccines8040665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712042PMC
November 2020

VHSV Single Amino Acid Polymorphisms (SAPs) Associated With Virulence in Rainbow Trout.

Front Microbiol 2020 27;11:1984. Epub 2020 Aug 27.

Unit for Fish and Shellfish Diseases, EURL for Fish and Crustacean Diseases, National Institute of Aquatic Resources, Technical University of Denmark (DTU), Kongens Lyngby, Denmark.

The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.
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http://dx.doi.org/10.3389/fmicb.2020.01984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493562PMC
August 2020

The pathogen Shows High Minimum Inhibitory Concentrations for Antimicrobials Commonly Used for Bovine Respiratory Disease.

Antibiotics (Basel) 2020 Jul 29;9(8). Epub 2020 Jul 29.

Istituto Zooprofilattico Sperimentale delle Venezie, SCT1-Verona, 37135 Verona, Italy.

is an overlooked pathogen often involved in bovine respiratory disease (BRD), which affects cattle around the world. BRD results in lost production and high treatment and prevention costs. Additionally, chronic therapies with multiple antimicrobials may lead to antimicrobial resistance. Data on antimicrobial susceptibility to is limited so minimum inhibitory concentrations (MIC) of a range of antimicrobials routinely used in BRD were evaluated using a broth microdilution technique for 41 isolates collected in Italy between 2011-2019. While all isolates had low MIC values for florfenicol (<1 μg/mL), many showed high MIC values for erythromycin (MIC90 ≥8 μg/mL). Tilmicosin MIC values were higher (MIC50 = 32 μg/mL) than those for tylosin (MIC50 = 0.25 μg/mL). Seven isolates had high MIC values for lincomycin, tilmicosin and tylosin (≥32 μg/mL). More, alarmingly, results showed more than half the strains had high MICs for enrofloxacin, a member of the fluoroquinolone class considered critically important in human health. A time-dependent progressive drift of enrofloxacin MICs towards high-concentration values was observed, indicative of an on-going selection process among the isolates.
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http://dx.doi.org/10.3390/antibiotics9080460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459706PMC
July 2020

Infection Dynamics of and Other Respiratory Mycoplasmas in Newly Imported Bulls on Italian Fattening Farms.

Pathogens 2020 Jul 4;9(7). Epub 2020 Jul 4.

The Oaks, Nutshell Lane, Farnham, Surrey GU9 0HG, UK.

Italian beef production is mainly based on a feedlot system where calves are housed with mixed aged cattle often in conditions favourable to bovine respiratory disease (BRD). In Veneto, an indoor system is also used for imported bulls around 300-350 kg. Mycoplasmas, in particular and , contribute to BRD in young calves, but their role in the disease in older cattle has not been investigated. In this study, ten heads of cattle were selected from each of the 24 groups kept in 13 different farms. Bulls were sampled by nasal swabbing at 0, 15, and 60 days after arrival for isolation. Identification was carried out by 16S-rDNA PCR followed by denaturing gradient gel electrophoresis. , , and were identified, and prevalence was analysed by mixed-effects logistic regression models. This showed that most bulls arrived free of , but within two weeks, approximately 40% became infected, decreasing to 13% by the last sampling. In contrast, the prevalence of was not dependent on time or seasonality, while only showed a seasonality-dependent trend. The Italian fattening system creates an ideal environment for infection with , probably originating from previously stabled animals.
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http://dx.doi.org/10.3390/pathogens9070537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399803PMC
July 2020

Genomic Sequence of a New Detected in Comber (Serranus cabrilla).

Microbiol Resour Announc 2020 Jan 9;9(2). Epub 2020 Jan 9.

Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Department of Comparative Biomedical Sciences, Legnaro, Padua, Italy

The comber alphavirus was isolated from a fish cell line from the brain of an apparently healthy specimen collected during wild fish surveillance in southern Italy. The comber alphavirus is a new member of the genus , family .
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http://dx.doi.org/10.1128/MRA.01294-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952657PMC
January 2020

Evaluation of Minimum Inhibitory Concentrations for 154 Mycoplasma synoviae isolates from Italy collected during 2012-2017.

PLoS One 2019 7;14(11):e0224903. Epub 2019 Nov 7.

Istituto Zooprofilattico Sperimentale delle Venezie, viale Dell'Università 10, Legnaro (PD), Italy.

Mycoplasma synoviae (MS) is a highly prevalent bacterial species in poultry causing disease and severe economic losses. Antibiotic treatment is one of the control strategies that can be applied to contain clinical outbreaks in MS-free flocks, especially because this bacterium can be transmitted in ovo. It becomes, then, very important for veterinarians to know the antibiotic susceptibility of the circulating strains in order to choose the most appropriate first-line antibiotic molecule as a proactive role in fighting antibiotic resistance. We evaluated the Minimum Inhibitory Concentrations (MICs) of enrofloxacin, oxytetracycline, doxycycline, erythromycin, tylosin, tilmicosin, spiramycin, tiamulin, florfenicol and lincomycin for MS isolates collected between 2012 and 2017 in Italy. A total of 154 MS isolates from different poultry commercial categories (broiler, layer, and turkey sectors) was tested using commercial MIC plates. All MS isolates showed very high MIC values of erythromycin (MIC90 ≥8 μg/mL) and enrofloxacin (MIC90 ≥16 μg/mL). MIC values of doxycycline and oxytetracycline obtained were superimposable to each other with only a one-fold dilution difference. Discrepancies between MIC values of tylosin and tilmicosin were observed. Interestingly, seven isolates showed very high MIC values of lincomycin and tilmicosin, but not all of them showed very high MIC values of tylosin. Most of the MS isolates showed low MIC values of spiramycin, but seven strains showed a MIC ≥16 μg/mL. In the observation period, the frequency of the different MIC classes varied dependently on the tested antibiotic. Interestingly, tilmicosin MICs clearly showed a time-dependent progressive shift towards high-concentration classes, indicative of an on-going selection process among MS isolates. Until standardized breakpoints become available to facilitate data interpretation, it will be fundamental to continue studying MIC value fluctuations in the meantime in order to create a significant database that would facilitate veterinarians in selecting the proper drug for treating this impactful Mycoplasma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224903PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837496PMC
March 2020

Development and evaluation of two multi-antigen serological assays for the diagnosis of bovine tuberculosis in cattle.

J Microbiol Methods 2018 10 21;153:118-126. Epub 2018 Sep 21.

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy. Electronic address:

There is currently an increased interest in the use of serological approaches in combination with traditional cell-mediated immunity-based techniques to improve the detection of tuberculosis (TB)-infected animals. In the present study, we developed and validated two different serological TB-detection assays using four antigens, MPB70, MPB83, ESAT6 and CFP10, and the tuberculin PPDb. A conventional multi-antigen TB-ELISA method and a novel TB multiplex test, based on Luminex technology, were developed to detect antibodies to multiple antigen targets. The performance levels of the two tests were evaluated and compared using selected panels of samples having known TB states. The TB-ELISA test (containing five antigens, including PPDb) had a sensitivity (Se) of 74.2% and a specificity (Sp) of 94.9%, while the TB-Luminex test had higher Se (79.0%) and Sp (99.1%) rates even when only one reactive antigen was used to classify the test as positive. If a more restrictive criterion, requiring two positive antigens to classify the test as positive, was used, then the TB-ELISA's Sp rate increased to 99.8% but the Se decreased to 61.3%, while the TB-Luminex test's Sp rate increased to 100% but the Se decreased to 51.2%. TB-ELISA and TB-Luminex were applied to a panel of 257 sera collected from bTB-positive herds, as determined by a post-mortem inspection. They showed good performance levels, identifying 49 (80.3%) and 48 (78.7%), respectively, of 61 samples that had tested positive by the intradermal tuberculin (IDT) test and/or interferon-gamma assay. In addition, TB-ELISA and TB-Luminex were able to identify 60 and 42 samples as positive, respectively, out of the 196 samples that tested negative to IDT and interferon-gamma at the time of serum collection. Subsequent IDT tests performed after 1-2 months, confirmed the positivity of 18 samples, indicating the strategic value of having two serological assays to detect TB-infected herds that were not reactive to initial IDT testing, thereby allowing for the rapid control of outbreaks and eradication of the disease.
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http://dx.doi.org/10.1016/j.mimet.2018.09.013DOI Listing
October 2018

Identification of a newly described OsHV-1 µvar from the North Adriatic Sea (Italy).

J Gen Virol 2018 05 26;99(5):693-703. Epub 2018 Mar 26.

Department of Biology, University of Padova, Padova (PD), Italy.

The surveillance activities for abnormal bivalve mortality events in Italy include the diagnosis of ostreid herpesvirus type 1 (OsHV-1) in symptomatic oysters. OsHV-1-positive oysters (Crassostrea gigas) were used as a source for in vivo virus propagation and a virus-rich sample was selected to perform shotgun sequencing based on Illumina technology. Starting from this unpurified supernatant sample from gills and mantle, we generated 3.5 million reads (2×300 bp) and de novo assembled the whole genome of an Italian OsHV-1 microvariant (OsHV-1-PT). The OsHV-1-PT genome encodes 125 putative ORFs, 7 of which had not previously been predicted in other sequenced Malacoherpesviridae. Overall, OsHV-1-PT displays typical microvariant OsHV-1 genome features, while few polymorphisms (0.08 %) determine its uniqueness. As little is known about the genetic determinants of OsHV-1 virulence, comparing complete OsHV-1 genomes supports a better understanding of the virus pathogenicity and provides new insights into virus-host interactions.
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http://dx.doi.org/10.1099/jgv.0.001042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994699PMC
May 2018

Interplay between co-divergence and cross-species transmission in the evolutionary history of bat coronaviruses.

Infect Genet Evol 2018 03 30;58:279-289. Epub 2018 Jan 30.

National Reference Centre, OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Universita' 10, Legnaro, Padova 35020, Italy.

Coronaviruses (CoVs) have been documented in almost every species of bat sampled. Bat CoVs exhibit both extensive genetic diversity and a broad geographic range, indicative of a long-standing host association. Despite this, the respective roles of long-term virus-host co-divergence and cross-species transmission (host-jumping) in the evolution of bat coronaviruses are unclear. Using a phylogenetic approach we provide evidence that CoV diversity in bats is shaped by both species richness and their geographical distribution, and that CoVs exhibit clustering at the level of bat genera, with these genus-specific clusters largely associated with distinct CoV species. Co-phylogenetic analyses revealed that cross-species transmission has been more common than co-divergence across coronavirus evolution as a whole, and that cross-species transmission events were more likely between sympatric bat hosts. Notably, however, an analysis of the CoV RNA polymerase phylogeny suggested that many such host-jumps likely resulted in short-term spill-over infections, with little evidence for sustained onward transmission in new co-roosting host species.
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http://dx.doi.org/10.1016/j.meegid.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106311PMC
March 2018

Ultrastructural and molecular characterization of Vairimorpha austropotamobii sp. nov. (Microsporidia: Burenellidae) and Thelohania contejeani (Microsporidia: Thelohaniidae), two parasites of the white-clawed crayfish, Austropotamobius pallipes complex (Decapoda: Astacidae).

J Invertebr Pathol 2018 01 6;151:59-75. Epub 2017 Nov 6.

Department of Earth and Environmental Sciences, University of Pavia, Via Adolfo Ferrata 7, I-27100 Pavia, Italy.

The microsporidiosis of the endangered white-clawed crayfish Austropotamobius pallipes complex has generally been attributed to only one species, Thelohania contejeani, the agent of porcelain disease. Species identification was mostly assessed by macroscopic examination or microscopic evaluation of muscle samples rather than by molecular or ultrastructural analyses. A survey conducted on A. pallipes complex populations in Northern Italy highlighted the presence of two different microsporidia causing similar muscular lesions, T. contejeani and an undescribed octosporoblastic species Vairimorpha austropotamobii sp. nov. Mature spores and earlier developmental stages of V. austropotamobii sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, which gave rise to a rosette-shaped plasmodium, and eight uninucleate spores were produced within the persistent SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and an average of 3.9 × 2.2 µm in size. The polar filament was coiled 11-14 times, lateral to the posterior vacuole. The small subunit ribosomal RNA gene (SSU rRNA) and the large subunit RNA polymerase II gene (RPB1) of V. austropotamobii sp. nov. were sequenced and compared with other microsporidia. The highest sequence identity of SSU rRNA (99%) and RPB1 (74%) genes was with the amphipod parasite Nosema granulosis and subsequently with V. cheracis, which infects the Australian yabby Cherax destructor. In our work we discuss about the reasons for placing this new species in the genus Vairimorpha. In addition, we provide for T. contejeani a RPB1 gene sequence, supplemental sequences of SSU rRNA gene and ultrastructural details of its sporogony in the host A. pallipes complex.
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http://dx.doi.org/10.1016/j.jip.2017.11.002DOI Listing
January 2018

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces.

J Microbiol Methods 2011 Mar 21;84(3):413-7. Epub 2011 Jan 21.

Istituto Zooprofilattico Sperimentale delle Venezie, Sezione di Verona, Via San Giacomo 5, 37135 Verona, Italy.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10(4)-10(-1)) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10(2) and 10(3)MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6weeks by real-time PCR.
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http://dx.doi.org/10.1016/j.mimet.2011.01.019DOI Listing
March 2011

Multiplexed typing of Mycobacterium avium subsp. paratuberculosis types I, II, and III by Luminex xMAP suspension array.

J Clin Microbiol 2011 Jan 17;49(1):389-91. Epub 2010 Nov 17.

Istituto Zooprofilattico Sperimentale delle Venezie-Sezione di Verona, Via San Giacomo 5, 37135 Verona, Italy.

Differentiation among types I, II, and III is the primary step in typing Mycobacterium avium subsp. paratuberculosis. We propose an innovative approach based on detection of gyrase B (gyrB) gene polymorphisms by suspension array technology, with high discriminatory power and high-throughput potential.
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http://dx.doi.org/10.1128/JCM.01761-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020487PMC
January 2011

Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35.

J Virol 2010 May 17;84(10):5336-50. Epub 2010 Mar 17.

Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here we show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, alphanu integrins, and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.
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http://dx.doi.org/10.1128/JVI.02494-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863792PMC
May 2010

Genetic reconstitution of the human adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape.

Virol J 2009 Oct 27;6:174. Epub 2009 Oct 27.

Institute of Zoology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Human adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C adenoviruses Ad2 and Ad5 (Ad2/5) cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L) in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC) gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein), which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for genetic analyses of distinct host requirements for Ad endocytosis and escape from endosomes.
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http://dx.doi.org/10.1186/1743-422X-6-174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771014PMC
October 2009

Infectious adenovirus type 2 transport through early but not late endosomes.

Traffic 2008 Dec 22;9(12):2265-78. Epub 2008 Oct 22.

Institute of Zoology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Receptor-mediated endocytosis is a major gate for pathogens into cells. In this study, we analyzed the trafficking of human adenovirus type 2 and 5 (Ad2/5) and the escape-defective temperature-sensitive Ad2-ts1 mutant in epithelial cancer cells. Ad2/5 and Ad2-ts1 uptake into endosomes containing transferrin, major histocompatibility antigen 1 and the Rab5 effector early endosome antigen 1 (EEA1) involved dynamin, amphiphysin, clathrin and Eps15. Cointernalization experiments showed that most of the Ad2/5 and Ad2-ts1 visited the same EEA1-positive endosomes. In contrast to Ad2/5, Ad2-ts1 required functional Rab5 for endocytosis and lysosomal transport and was sensitive to the phosphatidyl-inositol-3 (PI3)-kinase inhibitor wortmannin or the ubiquitin-binding protein Hrs for sorting from early to late endosomes. Endosomal escape of Ad2 was not affected by incubation at 19 degrees C, which blocked membrane sorting in early endosomes and inhibited Ad2-ts1 transport to lysosomes. Unlike Semliki Forest Virus (SFV), sorting of Ad2-ts1 to late endosomes was independent of Rab7 and Ad2/5 infection independent of EEA1. The data indicate that Ad2/5 and Ad2-ts1 use an invariant machinery for clathrin-mediated uptake to early endosomes. We suggest that the infectious Ad2 particles are either directly released from early endosomes to the cytosol or sorted by a temperature-insensitive and PI3-kinase-independent mechanism to an escape compartment different from late endosomes or lysosomes.
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http://dx.doi.org/10.1111/j.1600-0854.2008.00835.xDOI Listing
December 2008

Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3.

EMBO J 2008 Apr 6;27(7):956-69. Epub 2008 Mar 6.

Institute of Zoology, University of Zürich, Zürich, Switzerland.

Endocytosis supports cell communication, growth, and pathogen infection. The species B human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, and fatal respiratory and systemic disease. Here we show that Ad3 uses dynamin-independent endocytosis for rapid infectious entry into epithelial and haematopoietic cells. Unlike Ad5, which uses dynamin-dependent endocytosis, Ad3 endocytosis spatially and temporally coincided with enhanced fluid-phase uptake. It was sensitive to macropinocytosis inhibitors targeting F-actin, protein kinase C, the sodium-proton exchanger, and Rac1 but not Cdc42. Infectious Ad3 macropinocytosis required viral activation of p21-activated kinase 1 (PAK1) and the C-terminal binding protein 1 of E1A (CtBP1), recruited to macropinosomes. These macropinosomes also contained the Ad3 receptors CD46 and alpha v integrins. CtBP1 is a phosphorylation target of PAK1, and is bifunctionally involved in membrane traffic and transcriptional repression of cell cycle, cancer, and innate immunity pathways. Phosphorylation-defective S147A-CtBP1 blocked Ad3 but not Ad5 infection, providing a direct link between PAK1 and CtBP1. The data show that viruses induce macropinocytosis for infectious entry, a pathway used in antigen presentation and cell migration.
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http://dx.doi.org/10.1038/emboj.2008.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323250PMC
April 2008

Junctional gating: the achilles' heel of epithelial cells in pathogen infection.

Cell Host Microbe 2007 Sep;2(3):143-6

Institute of Zoology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Mucosal epithelial cells are a major barrier restricting pathogen entry and, paradoxically, an important entry port for respiratory and enteric viruses. Elegant studies in this issue of Cell Host & Microbe describe how coxsackievirus B3 (related to human poliovirus) infects polarized epithelial cells by engaging two transmembrane proteins of the tight junctions, occludin and CAR. A distinctive endocytic mechanism opens the junctions and gates infectious virus entry.
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http://dx.doi.org/10.1016/j.chom.2007.08.004DOI Listing
September 2007

Early steps of clathrin-mediated endocytosis involved in phagosomal escape of Fcgamma receptor-targeted adenovirus.

J Virol 2005 Feb;79(4):2604-13

Zoologisches Institut, University of Zürich, 8057 Zürich, Switzerland.

Adenovirus type 2 (Ad2) and Ad5 enter epithelial cells via the coxsackievirus B Ad receptor (CAR) and alpha(v) integrin coreceptors. In the absence of CAR, they can be directed to the Fcgamma receptor 1 of hematopoietic cells by an adaptor comprising the extracellular CAR domain and the Fc portion of a human immunoglobulin G (CARex-Fc). This gives rise to Ad aggregates and single particles which together enhance gene delivery up to 250-fold compared to adaptor-less viruses. A small interfering RNA knockdown of the clathrin heavy chain and quantitative electron microscopy of hematopoietic leukemia cells showed that the majority of Ads were phagocytosed as clusters of 1 to 3 microm in diameter and that about 10% of the particles entered cells by clathrin-mediated endocytosis. The clathrin knockdown did not affect phagocytosis but, surprisingly, inhibited viral escape from phagosomes. Similarly, blocking an early stage of clathrin-coated pit assembly inhibited phagosomal escape and infection but not aggregate uptake, unlike blocking of a late stage of clathrin-coated pit formation. We propose a cooperative interaction of clathrin-mediated endocytosis and phagocytosis triggering phagosomal lysis and infection.
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http://dx.doi.org/10.1128/JVI.79.4.2604-2613.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC546601PMC
February 2005