Investigation of Human and Animal Viruses in Water Matrices from a Rural Area in Southeastern Region of Brazil and Their Potential Use as Microbial Source-Tracking Markers.

This study assessed the sources of contamination of water matrices in a rural area using detection of a host-specific virus (human adenovirus [HAdV], porcine adenovirus [PAdV] and bovine polyomaviruses [BoPyV]) as potential microbial source-tracking tool, and rotavirus A [RVA], given its epidemiological importance in Brazil. From July 2017 to June 2018, 92 samples were collected from eight points (P1-P8) of surface and raw waters in southeastern region of Brazil. Fifty-five (59.8%) were positive for HAdV, 41 (44.5%) for RVA, 10 (10.9%) for PAdV and four (4.3%) for BoPyV. HAdV and RVA were detected at all sites, and over the entire sampling period, PAdV was detected at a porcine breeding area and at Guarda River site, presenting high concentrations up to 2.6 × 109 genome copies per liter [GC/L], and viral concentrations ranging from 9.6 × 101 to 7.1 × 107, while BoPyV (1.5 × 104 GC/L-9.2 × 105 GC/L) was only detected in samples from the bovine breeding areas. The combination of human and animal virus circulation presents a potential impact in the environment due to raw sewage discharge from regional communities, as well as potential hazard to human and animal health.

First Isolation and Identification of Homologous Recombination Events of Porcine Adenovirus from Wild Boar.

Porcine adenoviruses (PAdVs) are distributed in pig populations and classified into five immunologically distinct serotypes (PAdV-1 to 5). In this study, a PAdV was isolated from a fecal sample of wild boar for the first time. Whole-genome analysis revealed that this strain (Ino5) has sequence homology (approximately > 93%) throughout the genome with the PAdV-5 strain HNF-70 that was isolated from a pig in Japan in 1987, except for the hexon, E3 612R, and fiber coding regions. Two possible recombination breakpoints were detected in the hexon and E3 612R regions, which were found to have reduced GC content. Structural prediction analysis showed that a part of the hexon protein corresponding to the tower region of Ino5 had structural differences when compared with HNF-70, suggesting antigenic heterogeneity between these strains. PAdVs were detected in 1.77% (2/113) and 12% (12/100) of the fecal samples from wild boars and pigs collected in Japan by PCR, respectively. Phylogenetic analyses of the hexon and fiber genes revealed that some samples showed different grouping in the hexon and fiber genes, suggesting that these viruses have recombination events. These findings suggest that the PAdV-5 has evolved with homologous recombination events in the same manner as human adenoviruses among not only pig populations, but also wild boars in Japan.

Characterization of the First Genome of Porcine mastadenovirus B (HNU1 Strain) and Implications on Its Lymphoid and Special Origin.

Porcine adenoviruses (PAdVs) are classified into three species, PAdV-A, PAdV-B, and PAdV-C. The genomes of PAdV-A and PAdV-C have been well characterized. However, the genome of PAdV-B has never been completely sequenced, and the epidemiology of PAdV-B remains unclear. In our study, we have identified a novel strain of PAdV-B, named PAdV-B-HNU1, in porcine samples collected in China by viral metagenomic assay and general PCR. The genome of PAdV-B-HNU1 is 31,743 bp in length and highly similar to that of California sea lion adenovirus 1 (C. sea lion AdV-1), which contains typical mastadenoviral structures and some unique regions at the carboxy-terminal end. Especially, PAdV-B-HNU1 harbors a dUTPase coding region not clustering with other mastadenoviruses except for C. sea lion AdV-1 and a fiber coding region homologous with galectin 4 and 9 of animals. However, the variance of GC contents between PAdV-B-HNU1 (55%) and C. sea lion AdV-1 (36%) indicates their differential evolutionary paths. Further epidemiologic study revealed a high positive rate (51.7%) of PAdV-B-HNU1 in porcine lymph samples, but low positive rates of 10.2% and 16.1% in oral swabs and rectal swabs, respectively. In conclusion, this study characterized a novel representative genome of a lymphotropic PAdV-B with unique evolutionary origin, which contributes to the taxonomical and pathogenic studies of PAdVs.

Experimental infection of conventional newly-weaned piglets with porcine astrovirus.

The presence of porcine astroviruses in diarrheic and healthy pigs has been reported, however, the consequences of the astrovirus infection during the weaning process have not been described. In this study, eight healthy conventional newly-weaned piglets were used to evaluate effects of astrovirus infection during the first five days. Four piglets were infected with the porcine astrovirus PoAstV/PUJP5 strain and the other four represented the control group. Body weight, rectal temperature, diarrhea and other clinical signs were monitored every 24 hours. The results showed that all animals gained body weight, the occurrence of mild diarrhea on the 3rd day post-infection, and the astroviral presence in diarrheic samples. On the 5th day post-infection all the piglets were euthanized and then intestinal and extra-intestinal tissues were analyzed for the presence of PoAstV/PUJP5. The cytoplasmic antigen of PoAstV/PUJP5 was observed in the enterocytes of infected piglets from jejunum, ileum, colon and in inflammatory cells from mesenteric lymph nodes. In addition, villi atrophy, fusion, epithelial hyperplasia and incipient virus detection in mesenteric lymph were observed. RNAemia could not be proved. This study shows for the first time the effects of porcine astrovirus infection on conventional newly-weaning piglets. Keywords: porcine astrovirus; newly-weaned piglets.

Molecular epidemiology and characterization of porcine adenoviruses in pigs with diarrhea in Thailand.

Pigs have been assumed as a source of human viral infections. Surveillance of viruses in animals is essential to evaluate the risk to human and animal health and to determine economic impact. A number of studies focused mainly on well- known enteritis viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRVA), however, little data is available for porcine adenovirus (PAdV). In this study, the presence of PAdV was investigated in fecal samples collected from piglets with and without diarrhea from 31 commercial pig farms in northern Thailand. A total of 781 fecal specimens (516 from diarrheic piglets and 265 from non-diarrheic piglets) were screened for adenovirus using nested-PCR. Initial screening both in diarrheic and non-diarrheic piglets showed the overall prevalence of PAdV infection in piglets at 16.9% (132/781). Co-infection with PRVA was found in 24 out of 132 (18.2%) PAdV positive cases whereas PAdV mono-infection was observed at 81.8% (108/132). The prevalence of PAdV infection in diarrheic piglets (24.2%, 102/516) was significantly higher than those detected in non-diarrheic piglets (2.6%, 7/265). Most of PAdV detected in this study (97%, 128/132) were genotype 3 while the other 4 PAdV positive samples were non identifiable genotype. Phylogenetic analysis revealed that the viruses detected in diarrheic and non-diarrheic piglets displayed a closely related (95.4 to 100%) nucleotide sequence identity. To our knowledge, this is the first report on the epidemiology and molecular characterization of PAdV in piglets in Thailand.

Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions.

The adenovirus E3 region encodes proteins that are not essential for viral replication in vitro The porcine adenovirus type 3 (PAdV-3) E3 region encodes three proteins, including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K protein is a structural protein, as it is incorporated in CsCl-purified virions. The 13.7K protein appears to be essential for PAdV-3 replication, as mutant PAV13.73A expressing a mutated 13.7K could be isolated only in VIDO AS2 cells expressing the 13.7K protein. Analysis of PAV13.73A suggested that even in the presence of reduced levels of some late viral proteins, there appeared to be no effect on virus assembly and production of mature virions. Further analysis of CsCl-purified PAV13.73A by transmission electron microscopy revealed the presence of disrupted/broken capsids, suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that the PAdV-3 E3 region-encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions.IMPORTANCE Although E3 region-encoded proteins are involved in the modulation of leukocyte functions (N. Arnberg, Proc Natl Acad Sci U S A 110:19976-19977, 2013) and inducing a lytic infection of lymphocytes (V. K. Murali, D. A. Ornelles, L. R. Gooding, H. T. Wilms, W. Huang, A. E. Tollefson, W. S. Wold, and C. Garnett-Benson, J Virol 88:903-912, 2014), none of the E3 proteins appear to be a component of virion capsid or required for replication of adenovirus. Here, we demonstrate that the 13.7K protein encoded by the E3 region of porcine adenovirus type 3 is a component of progeny virion capsids and appears to be essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that an adenovirus E3-encoded protein is an essential structural protein.

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.

F-coliphages, porcine adenovirus and porcine teschovirus as potential indicator viruses of fecal contamination for pork carcass processing.

There are concerns about the zoonotic transmission of viruses through undercooked pork products. There is a lack of information on suitable indicator viruses for fecal contamination with pathogenic enteric viruses in the meat processing chain. The study compared the incidence and levels of contamination of hog carcasses with F-coliphages, porcine teschovirus (PTV), and porcine adenovirus (PAdV) at different stages of the dressing process to assess their potential as indicator viruses of fecal contamination. One hundred swab samples (200cm2) were collected from random sites on hog carcasses at 4 different stages of the dressing process and from retail pork over the span of a year from 2 pork processing plants (500/plant). Viable F-coliphages, PAdV DNA and PTV RNA were each detected on ≥99% of the incoming carcasses at both plants and were traceable through the pork processing chain. Significant correlations were observed between viable F-coliphages and PAdV DNA and between F-coliphages and PTV RNA but not between PAdV DNA and PTV RNA at the various stages of pork processing. Detection of viable F-coliphages was more sensitive than genomic copies of PAdV and PTV at low levels of contamination, making F-coliphages a preferred indicator in the pork slaughter process as it also provides an indication of infectivity. For plant A, F-RNA coliphages were detected in 25%, 63%, and 21% of carcass swabs after pasteurization, evisceration, and retail pork products, respectively. For plant B, F-coliphages were detected in 33%, 25%, and 13% of carcass swabs after skinning, evisceration, and retail pork samples, respectively. Viable F-RNA coliphages were genotyped. Viable F-RNA GII and GIII were generally not detected at the earlier stages of the slaughter process but they were detected on 13% of carcasses after evisceration and 2% of retail pork samples at plant A, which raises concerns of potential food handler contamination during pork processing. Consumers could be at risk when consuming undercooked meat contaminated with pathogenic enteric viruses.

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication.

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/ß transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.

A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620 ± 49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

Incorporation of porcine adenovirus 4 fiber protein enhances infectivity of adenovirus vector on dendritic cells: implications for immune-mediated cancer therapy.

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.

A novel strain of porcine adenovirus detected in urinary bladder urothelial cell culture.

Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize and as such require special attention. The present study describes the detection of porcine adenovirus (PAdV), strain PAdV-SVN1 in cultures of normal porcine urothelial (NPU) cells isolated from urinary bladders of domestic pigs. NPU cell cultures were evaluated by light microscopy (LM), polymerase chain reaction (PCR), and additionally assessed by transmission electron microscopy (TEM). Characteristic ultrastructure of virions revealed the infection with adenovirus. The adenoviral contamination was further identified by the sequence analysis, which showed the highest similarity to recently described PAdV strain PAdV-WI. Additionally, the cell ultrastructural analysis confirmed the life-cycle characteristic for adenoviruses. To closely mimic the in vivo situation, the majority of research on in vitro models uses cell cultures isolated from human or animal tissue and their subsequent passages. Since the donor tissue could be a potential source of contamination, the microbiological screening of the excised tissue and harvested cell cultures is highly recommended.

Comparison of ZetaPlus 60S and nitrocellulose membrane filters for the simultaneous concentration of F-RNA coliphages, porcine teschovirus and porcine adenovirus from river water.

Increasing attention is being paid to the impact of agricultural activities on water quality to understand the impact on public health. F-RNA coliphages have been proposed as viral indicators of fecal contamination while porcine teschovirus (PTV) and porcine adenovirus (PAdV) are proposed indicators of fecal contamination of swine origin. Viruses and coliphages are present in water in very low concentrations and must be concentrated to permit their detection. There is little information comparing the effectiveness of the methods for concentrating F-RNA coliphages with concentration methods for other viruses and vice versa. The objective of this study was to compare 5 current published methods for recovering F-RNA coliphages, PTV and PAdV from river water samples concentrated by electronegative nitrocellulose membrane filters (methods A and B) or electropositive Zeta Plus 60S filters (methods C-E). Method A is used routinely for the detection of coliphages (Méndez et al., 2004) and method C (Brassard et al., 2005) is the official method in Health Canada's compendium for the detection of viruses in bottled mineral or spring water. When river water was inoculated with stocks of F-RNA MS2, PAdV, and PTV to final concentrations of 1×10(6) PFU/100 mL, 1×10(5) gc/100 mL and 3×10(5) gc/100 mL, respectively, a significantly higher recovery for each virus was consistently obtained for method A with recoveries of 52% for MS2, 95% for PAdV, and 1.5% for PTV. When method A was compared with method C for the detection of F-coliphages, PAdV and PTV in river water samples, viruses were detected with higher frequencies and at higher mean numbers with method A than with method C. With method A, F-coliphages were detected in 11/12 samples (5-154 PFU/100 mL), PTV in 12/12 samples (397-10,951 gc/100 mL), PAdV in 1/12 samples (15 gc/100 mL), and F-RNA GIII in 1/12 samples (750 gc/100 mL) while F-RNA genotypes I, II, and IV were not detected by qRT-PCR.

Tracing enteric viruses in the European berry fruit supply chain.

In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses. A total of 785 samples were collected throughout the food production chain of four European countries (Czech Republic, Finland, Poland and Serbia) during two growing seasons. Samples were taken during the production phase, the processing phase, and at point-of-sale. Samples included irrigation water, animal faeces, food handlers' hand swabs, swabs from toilets on farms, from conveyor belts at processing plants, and of raspberries or strawberries at points-of-sale; all were subjected to virus analysis. The samples were analysed by real-time (reverse transcription, RT)-PCR, primarily for human adenoviruses (hAdV) to demonstrate that a route of contamination existed from infected persons to the food supply chain. The analyses also included testing for the presence of selected human (norovirus, NoV GI, NoV GII and hepatitis A virus, HAV), animal (porcine adenovirus, pAdV and bovine polyomavirus, bPyV) and zoonotic (hepatitis E virus, HEV) viruses. At berry production, hAdV was found in 9.5%, 5.8% and 9.1% of samples of irrigation water, food handlers' hands and toilets, respectively. At the processing plants, hAdV was detected in one (2.0%) swab from a food handler's hand. At point-of-sale, the prevalence of hAdV in fresh raspberries, frozen raspberries and fresh strawberries, was 0.7%, 3.2% and 2.0%, respectively. Of the human pathogenic viruses, NoV GII was detected in two (3.6%) water samples at berry production, but no HAV was detected in any of the samples. HEV-contaminated frozen raspberries were found once (2.6%). Animal faecal contamination was evidenced by positive pAdV and bPyV assay results. At berry production, one water sample contained both viruses, and at point-of-sale 5.7% and 1.3% of fresh and frozen berries tested positive for pAdV. At berry production hAdV was found both in irrigation water and on food handler's hands, which indicated that these may be important vehicles by which human pathogenic viruses enter the berry fruit chain. Moreover, both zoonotic and animal enteric viruses could be detected on the end products. This study gives insight into viral sources and transmission routes and emphasizes the necessity for thorough compliance with good agricultural and hygienic practice at the farms to help protect the public from viral infections.

High occurrence of hepatitis E virus in samples from wastewater treatment plants in Switzerland and comparison with other enteric viruses.

Hepatitis E virus (HEV) is responsible for many enterically transmitted viral hepatitides around the world. It is currently one of the waterborne diseases of global concern. In industrialized countries, HEV appears to be more common than previously thought, even if it is rarely virulent. In Switzerland, seroprevalence studies revealed that HEV is endemic, but no information was available on its environmental spread. The aim of this study was to investigate -using qPCR- the occurrence and concentration of HEV and three other viruses (norovirus genogroup II, human adenovirus-40 and porcine adenovirus) in influents and effluents of 31 wastewater treatment plants (WWTPs) in Switzerland. Low concentrations of HEV were detected in 40 out of 124 WWTP influent samples, showing that HEV is commonly present in this region. The frequency of HEV occurrence was higher in summer than in winter. No HEV was detected in WWTP effluent samples, which indicates a low risk of environmental contamination. HEV occurrence and concentrations were lower than those of norovirus and adenovirus. The autochthonous HEV genotype 3 was found in all positive samples, but a strain of the non-endemic and highly pathogenic HEV genotype I was isolated in one sample, highlighting the possibility of environmental circulation of this genotype. A porcine fecal marker (porcine adenovirus) was not detected in HEV positive samples, indicating that swine are not the direct source of HEV present in wastewater. Further investigations will be necessary to determine the reservoirs and the routes of dissemination of HEV.

Identification and promoter analysis of PERV LTR subtypes in NIH-miniature pig.

Porcine endogenous retroviruses (PERVs) are integrated into the genomes of all pigs. Since some PERVs can also infect human cells, they represent a potential risk for xenotransplantation involving pig cells or organs. The long terminal repeat (LTR) elements of PERVs show promoter activity that can affect human functional genes; therefore, we examined these elements in this study. We detected several expressed LTRs in the NIH-miniature pig liver, among which we identified 9 different subtypes. When these LTRs were compared, distinct structures that contained several insertion and deletion (INDEL) events and tandem repeats were identified in the U3 region. The transcriptional activity of the 9 LTR subtypes was analyzed in the PK15 porcine cell line and in the HepG2 and Hep3B human liver cell lines, and transcriptional regulation was found to be different in the 3 cell lines. The D LTR subtype was found to have stronger promoter activity than all other types in 4 different human cell lines (HepG2, Hep3B, U251, and 293). Using computational approaches, the D type was shown to contain 4 transcription factor-binding sites distinct from those in the U3 regions of the other subtypes. Therefore, deletion mutants were constructed and examined by a transient transfection luciferase assay. The results of this analysis indicated that the binding site for the Hand1:E47 transcription factor might play a positive role in the transcriptional regulation of PERV LTR subtype D in human liver cell lines.

An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

Detection of circoviruses and porcine adenoviruses in water samples collected from swine manure treatment systems.

Samples collected from two swine manure treatment systems including: swine manure treatment system and demonstrative unit (SMTS and DU), were analyzed by qPCR to quantify the amount of porcine adenovirus (PAdV) and porcine circovirus (PCV2) present. Positive samples were tested for virus integrity using DNase assay. Fifty-six water samples were collected monthly from March 2009 to May 2010. PAdV genome was found 66% of the samples in the SMTS and in 78% of the samples in the DU system. PCV2 was detected in 96% of samples collected from the SMTS system and in 86% of samples from DU. DNase assay revealed that there were undamaged virus particles of both PAdV and PCV2 in all sampling sites in the SMTS. However, undamaged particles of both viruses were detected in samples from the DU system in the affluent and middle sites, though undamaged PCV2 was absent in the effluent samples.

Persistence and the state of bovine and porcine adenoviral vector genomes in human and nonhuman cell lines.

The state of vector genome in transduced cells influences the duration of transgene expression and can be a safety concern if it gets integrated randomly into the host genome. Although human adenovirus (Ad) serotype 5 (HAd5) mainly persists in a linear episomal form, information regarding the state of bovine Ad serotype 3 (BAd3) and porcine Ad serotype 3 (PAd3) vector genomes in human and nonhuman cells is currently unknown. To address this issue, MDA-MB-231 (human), MDBK (bovine), PK-15 (porcine), MT1A2 (mouse) and NIH-3T3 (mouse) cell lines were infected with replication-defective BAd3, PAd3 or HAd5 vectors carrying the green fluorescent protein (GFP) gene. The persistence and the state of vector genome were assessed by quantitative real-time PCR and Southern blot hybridization, respectively. Levels of transgene and Ad gene expressions were quantified using real-time RT-PCR. Persistence of BAd3 or PAd3 vectors was comparable to that of HAd5 vector. Only the linear episomal form of the vector genome was observed with each vector. In addition, expression levels of transgene as well as viral genes by all three vectors were comparable and correlated with their transduction levels in each cell type. These results indicate comparable biologic behavior of BAd3, PAd3 and HAd5 vectors in cell culture.

A porcine adenovirus with low human seroprevalence is a promising alternative vaccine vector to human adenovirus 5 in an H5N1 virus disease model.

Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5.