This study aims to establish a method for counting the viral particles in adenovirus vector-based vaccines. Nano-flow cytometry was employed to analyze the viral particles in adenovirus-based vector vaccines at the single-particle level. Monodisperse silica nanoparticles with a refractive index close to that of the virus were selected as the particle size standard to calculate the viral particle size, which was then compared with the results obtained from transmission electron microscopy (TEM) to determine the gating strategy. Subsequently, a particle count standard was employed to calculate the viral particle concentration. The established method demonstrated good linearity, accuracy, precision, and specificity. The results of determined viral particle concentration showed a good correlation with the infectious titer. Compared with the conventional OD₂₆₀ method, nano-flow cytometry can directly measure the viral particle concentration and indicate whether the sample has been disassembled according to changes in viral particle concentration and size, thus more accurately reflecting the actual infectious potency of the sample. The novel quantification method established in this study is capable of indicating the efficacy of adenovirus vector-based vaccines and provides effective technical support for the quality control of such products.
Adenoviridae/genetics* ; Genetic Vectors ; Flow Cytometry/methods* ; Virion/isolation & purification* ; Particle Size ; Nanoparticles ; Viral Vaccines

BACKGROUND: We evaluated the analytical and clinical performances of the SD BIOLINE Rota/Adeno Rapid kit (SD Rota/Adeno Rapid; Standard Diagnostics, Inc., Korea), an immunochromatographic assay (ICA), for the simultaneous detection of rotaviruses and adenoviruses in human stool samples. METHODS: We tested 400 clinical stool samples from patients with acute gastroenteritis and compared the ICA results with the results obtained by using ELISA, enzyme-linked fluorescent assays (ELFA), PCR, and multiplex reverse transcription-PCR (mRT-PCR). To assess the analytical performance of the SD BIOLINE Rota/Adeno Rapid kit, we determined its detection limit, reproducibility, cross-reactivity, and analytical reactivity for adenovirus subtypes, and performed interference studies. RESULTS: The overall agreement rates among the tested methods were 91.5% for rotavirus and 85.5% for adenovirus. On the basis of mRT-PCR, the overall agreement, positive agreement, and negative agreement rates of the ICA were 95.6%, 100%, and 94.9% for rotavirus, and 94.0%, 71.4%, and 94.8% for adenovirus, respectively. Using the ICA, we detected all the subtypes of adenovirus tested, but the analytical reactivities for adenovirus subtypes were different between the 4 adenovirus detection methods. The high reproducibility was confirmed, and no cross-reactivity or interference was detected. CONCLUSIONS: The SD BIOLINE Rota/Adeno Rapid kit showed acceptable analytical and clinical performances. However, interpretation of adenovirus positive/negative result should be cautious because of different detectability for adenovirus subtypes among adenovirus detection methods.
Acute Disease ; Adenoviridae/genetics/*immunology/isolation & purification ; Cross Reactions ; DNA, Viral/analysis ; Enzyme-Linked Immunosorbent Assay ; Feces/*virology ; Gastroenteritis/diagnosis/virology ; Humans ; *Immunochromatography ; Multiplex Polymerase Chain Reaction ; RNA, Viral/analysis ; Reagent Kits, Diagnostic ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus/genetics/*immunology/isolation & purification

Bats are important reservoir animals and more than 60 viruses have been identified in bats with many of them highly pathogenic to human. In order to understand the natural background, genetic diversity of bat viruses in China and discover potential viral pathogens, Solexa sequencing based viral metagenomics focusing on bats tissues was established and to analyze the virome of bats collected from Jilin, Yunnan and Hunan province. By Solexa sequencing, 116 442 324 useful reads were obtained and assembled into 4 872 contigs, of which 8.2% (4 002/4 4872) were annotated to 36 viral families, including 19 vertebrate virus families, 6 plant virus families, 4 insect virus families and 4 phages. Further contigs analyses showed that some adenovirus, bocavirus, picobirnavirus, parvovirus contigs sequences were similar with known viruses. However, part of them shared limited identities to these viruses implying the discovery of new viruses. Moreover, PCR validation of adenovirus and bocavirus confirmed the results obtained by viral metagenomics. This study aimed to understand bat virome in China by viral metagenomics and could be helpful to establish effective surveillance on wildlife-associate zoonoses.
Adenoviridae ; genetics ; isolation & purification ; Animals ; Bunyaviridae ; genetics ; isolation & purification ; China ; Chiroptera ; virology ; Genome, Viral ; genetics ; Metagenome ; genetics ; Metagenomics ; methods ; Picornaviridae ; genetics ; isolation & purification

This study was performed to characterize respiratory viral infections in pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). Study samples included 402 respiratory specimens obtained from 358 clinical episodes that occurred in the 116 children of the 175 consecutive HSCT cohort at Seoul National University Children's Hospital, Korea from 2007 to 2010. Multiplex reverse-transcription polymerase chain reactions were performed for rhinovirus, respiratory syncytial virus (RSV), parainfluenza viruses (PIVs), adenovirus, human coronavirus (hCoV), influenza viruses and human metapneumovirus. Viruses were identified in 89 clinical episodes that occurred in 58 patients. Among the 89 clinical episodes, frequently detected viruses were rhinovirus in 25 (28.1%), RSV in 23 (25.8%), PIV-3 in 16 (18.0%), adenovirus in 12 (13.5%), and hCoV in 10 (11.2%). Lower respiratory tract infections were diagnosed in 34 (38.2%). Neutropenia was present in 24 (27.0%) episodes and lymphopenia was in 31 (34.8%) episodes. Sixty-three percent of the clinical episodes were hospital-acquired. Three patients died of respiratory failure caused by respiratory viral infections. Respiratory viral infections in pediatric patients who have undergone HSCT are common and are frequently acquired during hospitalization. Continuous monitoring is required to determine the role of respiratory viruses in immunocompromised children and the importance of preventive strategies.
Adenoviridae/genetics/isolation & purification ; Adolescent ; Adult ; Child ; Child, Preschool ; Cohort Studies ; Coronavirus/genetics/isolation & purification ; Female ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology ; Hospitalization ; Humans ; Infant ; Lymphopenia/epidemiology ; Male ; Neutropenia/epidemiology ; Parainfluenza Virus 3, Human/genetics/isolation & purification ; Prevalence ; Respiratory Syncytial Viruses/genetics/isolation & purification ; Respiratory Tract Infections/epidemiology/therapy/*virology ; Reverse Transcriptase Polymerase Chain Reaction ; Rhinovirus/genetics/isolation & purification ; Seasons ; Young Adult

OBJECTIVEThe study was designed to evaluate adenovirus infection in hospitalized children with diarrhea.

METHODStool specimens were collected from 519 hospitalized children with diarrhea during 2010, including those defined as community-acquired diarrhea (CAD) who developed diarrhea symptoms within 48 hours after admission, and those defined as hospital-acquired diarrhea (HAD) whose symptoms of diarrhea occurred beyond 48 hours after admission. PCR was employed to identify adenovirus in fecal samples by using universal primers for adenoviruses of all types, and specific primers for adenovirus group F. PCR products with expected size were sequenced for adenovirus typing. Clinical data for children with adenovirus positive specimens were analyzed.

RESULTA total of 519 hospitalized children, including 289 with CAD and 230 with HAD, were enrolled in the study. Out of 519 stool specimens, 76 showed PCR products with expected 301 bp and identified as adenovirus by sequencing, and the overall positive rate was 14.6%. Out of 289 CAD samples, 43 were positive (positive rate was 14.9%). Of them, 20 were identified as enteric adenovirus infection (adenovirus type 41, Ad41). Thirty-three out of 230 HAD samples were positive (positive rate was 14.3%). Of them, 13 were characterized as enteric adenovirus infection (one was Ad40 and others were Ad41). Ad41 in this study could be divided into two genotypes by phylogenetic tree analysis. Non-enteric adenoviruses were identified in 43 specimens (43/76, 56.6%) including 5 of serotype 1, 8 of serotype 2, 15 of serotype 3, 10 of serotype 7, 1 of serotype 12, and 4 of serotype 31. In this study, the positive rate of adenovirus between CAD children and HAD children did not differ (χ(2) = 0.03, P > 0.05), while the positive rate of enteric adenovirus was high in CAD children.

CONCLUSIONAdenovirus infection was the main cause of diarrhea in hospitalized children. In this study, the positive rate of adenovirus was similar between children with CAD and with HAD. Enteric adenovirus (adenovirus group F) was the most common adenovirus serotype detected in 2010 in Beijing, and Ad41 was the dominant type.

Adenoviridae ; classification ; genetics ; isolation & purification ; Adenoviridae Infections ; epidemiology ; Age Distribution ; Child, Hospitalized ; Child, Preschool ; China ; epidemiology ; DNA, Viral ; analysis ; Diarrhea ; epidemiology ; virology ; Diarrhea, Infantile ; epidemiology ; virology ; Feces ; virology ; Female ; Humans ; Infant ; Male ; Polymerase Chain Reaction ; Sex Distribution

Adenovirus remains a significant threat to public health. Recent studies showed that bats can harbor diverse adenoviruses. To further investigate the distribution and genetic diversity of bat adenoviruses in China, we collected throat and anal swab samples of 11 bat species from 6 provinces of China, including Beijing, Hunan, Jiangxi, Yunnan, Guizhou and Hainan. Nested PCR was used to identify potential bat adenoviruses from the samples, and positive results were cloned and sequenced for genetic diversity study. In addition, nucleotide sequence alignments based on corresponding amino acid sequence similarities were used for phylogenetic analyses. Our results showed that about 20% of bat species in China are positive to adenoviruses, and Myotis ricketti is likely to be the most important host of bat adenoviruses in all locations. Moreover, we identified two diverse sequences of bat adenoviruses from the same sample of Ia io in Guizhou province of China. In general, the average nucleotide and amino acid sequence similarities of the conserved region of DNA polymerases of bat adenoviruses are 66.6% and 74.7%, respectively. The differences between bat species and their residences environments may have driven the adaptive evolution of the viruses, leading to the genetic diversity of the bat adenoviruses.
Adenoviridae ; classification ; genetics ; isolation & purification ; physiology ; Animals ; China ; Chiroptera ; virology ; Genetic Variation ; Host Specificity ; Phylogeny

UNLABELLEDTo investigate the epidemiological features and types of human adenoviruses (ADV) in children with acute respiratory tract infection in Nanjing area, China. Nasopharyngeal aspirates and nasopharyngeal swabs were collected from 644 outpatients or hospitalized pediatric patients with ARTI at the Children Hospital of Nanjing, Jiangsu Province, China, between August 2010 and July 2011. Adenoviruses were identified and typed from the collected clinical specimens by nested-PCR based on the partial region of the hexon gene. Other 12 respiratory viruses including human bocavirus (HBoV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza viruses 1-4 (PIV1-4), influenza virus A/B (IFVA/B), human metapneumovirus (HMPV), human coronavirus NL63 and HKU1 (HCoV-HKU1 and HCoV-NL63) were also identified by PCR method. All PCR positive products were sequenced and phylogenetic analysis was conducted. It was showed that adenoviruses were detected in 171 patients out of 644 (26. 55%) children, 120 (70.18%, 120/171) for ADV3, 16 (9.36%,16/171) for ADV7, 12 (7.02%, 12/171) for ADV1, 10 (5.85%, 10/171) for ADV2, 6 (3.51%, 6/171) for ADV5, 3 (1.75%, 3/171) for ADV6, 3 (1.75%, 3/171) for ADV57, and 1 (0.58%,1/171) for ADV41. ADV infection could occur in any season. There was a higher possibility of ADV infection from April to July in 2011. Most cases (96.49%) were younger than 7 years old. A total of 99 of the 171 ADV-positive children (57.89%) were co-infected with other respiratory viruses. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were the most common additional respiratory viruses, Lower respiratory tract infections were the most frequent diagnoses made in the hospital, in which there were 52 pneumonia (30.4%) cases.

CONCLUSIONADV is one of the most important pathogens of acute respiratory tract infection in children in Nanjing area, and adenovirus type 3 was the most prevalent serotype. It is important to develop long-term surveillance.

Adenoviridae ; classification ; genetics ; isolation & purification ; Adenoviridae Infections ; epidemiology ; virology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Respiratory Tract Infections ; epidemiology ; virology

Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0 x 1(9) PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P < 0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.
Adenoviridae ; genetics ; metabolism ; Animals ; Epithelial Cells ; metabolism ; Female ; Genetic Vectors ; genetics ; Goats ; HEK293 Cells ; Humans ; Mammary Glands, Animal ; cytology ; Parathyroid Hormone-Related Protein ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

PURPOSE: Early identification of causative agents in lower respiratory infection of pediatric patients can reduce morbidity and prevent an overuse of antimicrobials. Two kinds of multiplex polymerase chain reaction (PCR) and a commercial shell vial viral culture were performed to identify causative agents in pediatric patients. MATERIALS AND METHODS: Nasopharyngeal aspirates of 220 children diagnosed with viral pneumonia were obtained. Two kinds of multiplex PCR (Seeplextrade mark RV detection kit, and Labopasstrade mark RV detection kit), and a shell vial culture by R-Mix were performed. RESULTS: Positive samples from 220 total samples by two multiplex PCRs were 52.7% and 46.4%, respectively. We also cultured 103 samples that showed positive results of the adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus (RSV) by two multiplex PCR. The RSV was most frequently detected in 53.0% (Seeplex) and 51.7% (Labopass) of patients. The detection rate of adenovirus (AdV) was 10.3% and 12.1%, influenza virus (IFV) A and B was 12.5% and 3.4%, and parainfluenza virus (PIFV) 1, 2, and 3 were 2.9% and 2.6%. Shell vial cultures showed concordant results with each multiplex PCR by 96.1% and 77.7%, respectively. Sequencing results were 90% consistent with multiplex PCR. CONCLUSION: Multiplex PCR showed more positivity than the shell vial culture and it can be an effective primary test. Other complementary efforts such as viral cultures and sequencing analysis could be considered, according to clinical and laboratory conditions.
Adenoviridae/genetics/*isolation & purification ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Orthomyxoviridae/genetics/*isolation & purification ; Pneumonia, Viral/*virology ; Polymerase Chain Reaction/*methods ; Respiratory Syncytial Viruses/genetics/*isolation & purification ; Respirovirus/genetics/*isolation & purification

OBJECTIVETo investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.

METHODSFour non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.

RESULTSThe adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.

CONCLUSIONThe non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.

Adenoviridae ; genetics ; isolation & purification ; physiology ; Cell Line ; DNA, Recombinant ; genetics ; Humans ; Serial Passage ; Transgenes ; Virus Replication