Evaluation of type-specific real-time PCR assays using the LightCycler and J.B.A.I.D.S. for detection of adenoviruses in species HAdV-C

Sporadically, HAdVs from species HAdV-C are detected in acute respiratory disease outbreaks. To rapidly type these viruses, we designed real-time PCR assays that detect and discriminate between adenovirus types HAdV-C1, -C2, -C5, and -C6. Sixteen clinical isolates from the California Department of Public Health were used to validate the new assays. Type-specific TaqMan real-time PCR assays were designed and used independently to successfully identify 16 representative specimens. The lower limit of detection for our LightCycler singleplex real-time PCR assays were calculated to be 100, 100, 100, and 50 genomic copies per reaction for HAdV-C1, HAdV-C2, HAdV-C5 and HAdV-C6, respectively. The results for the singleplex J.B.A.I.D.S. assays were similar. Our assays did not cross-react with other adenoviruses outside of species HAdV-C, respiratory syncytial virus, influenza, or respiratory disease causing bacteria. These assays have the potential to be useful as diagnostic tools for species HAdV-C infection.     

Adenoviruses Associated with Acute Respiratory Diseases Reported in Beijing from 2011 to 2013

Background

Adenovirus is one of the most common causes of viral acute respiratory infections. To identify the types of human adenoviruses (HAdVs) causing respiratory illness in Beijing, a sentinel surveillance project on the viral aetiology of acute respiratory infection was initiated in 2011.

Principal findings

Through the surveillance project, 4617 cases of respiratory infections were identified during 2011-2013. Throat swabs (pharynx and tonsil secretions) were collected from all the patients, and 15 different respiratory viruses were screened by multiplex one-step PCR method. 45 were identified as adenovirus-positive from sporadic and outbreak cases of respiratory infection by a multiplex one-step RT-PCR method, and a total of 21 adenovirus isolates were obtained. Five HAdV types among three species, including HAdV-3 (species HAdV-B), HAdV-4 (species HAdV-E), HAdV-7 (species HAdV-B), HAdV-55 (species HAdV-B), and an undefined HAdV type (species HAdV-C) were identified. The comparison results of the penton base, hexon, and fiber gene sequences of the Beijing HAdV-3, HAdV-4, HAdV-7, and HAdV-55 strains in this study and those from the GenBank database indicated significant spatial and temporal conservation and stability of sequences within the genome; however, the phylogenetic relationship indicated that both strain BJ04 and strain BJ09 isolated in 2012 and 2013, respectively, may have recombined between HAdV-1 genome and HAdV-2 genome within species HAdV-C, indicating intraspecies recombination.

Conclusions

This study confirmed that at least 5 HAdV types including HAdV-3, HAdV-4, HAdV-7, HAdV-55 and an undefined HAdV type were co-circulating and were the causative agents of respiratory tract infections in recent years in Beijing. HAdV-3, HAdV-4, HAdV-7, and HAdV-55 showed the apparent stability of the genomes, while intraspecies recombination was identified in strain BJ04 and BJ09. The recombinants carrying penton base gene of HAdV-1 as well as hexon and fiber genes of HAdV-2 might be a novel type of HAdV worthy of further study.     

Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

From January to December 1998, nasopharyngeal aspirates were obtained from 482 children with acute respiratory infections attended in emergence department and wards of a teaching hospital in the city of Salvador, Brazil. The samples were tested for the presence of adenovirus by isolation in tissue culture and indirect immunofluorescence assay. Eleven adenoviruses were detected by both methods in the same clinical samples. Infections by adenovirus were observed during seven months of the year without association with rainy season. Genome analysis was performed on these 11 isolates. Species C was represented by serotypes 1, 2 and 5. Within species B, only serotype 7 (Ad7) was detected. Two genomic variants of Ad1, two variants of Ad2, one of Ad5, and one of Ad7 (7h) were identified. This is the first study of molecular epidemiology of adenovirus associated to acute respiratory infections in children living in Northeast Brazil, and contributes to a better understanding of adenovirus infections in the country.     

Adenovirus infections in Manitoba

This review of adenovirus infections in Manitoba over a five-year period is introduced with a brief presentation on the structure-function relationships of the adenoviruses. Extensive research has resulted in the accumulation of a great deal of basic information, far surpassing our knowledge about the infections that these viruses produce in man. More research is needed on the epidemiological and clinical aspects of these diseases, including methods for prevention and treatment, and earlier methods of diagnosis.Features of 216 adenovirus respiratory infections diagnosed from 1963 to 1967 are reviewed and presented. All infections were diagnosed by showing a four-fold or greater increase. In addition, an adenovirus was isolated from many of the infections. These infections occurred every year in substantial numbers and they were seen during each month of the year, although they were more frequent and severe in the winter. Infants and younger persons were often affected. The most severe disease was a pneumonitis which occurred mainly in infants. Associated symptoms usually arose from the gastrointestinal tract, and were sufficiently common to indicate that one should suspect an adenovirus infection when respiratory and gastrointestinal symptoms occur together in a child.The possible role of adenoviruses in the etiology of other than respiratory diseases is discussed and the features of 96 possible non-respiratory infections are presented. Further work will be required to establish a definite etiologic role of the adenoviruses in most of these infections.     

Identification and typing of respiratory adenoviruses in Guangzhou, Southern China using a rapid and simple method

Human adenoviruses (HAdVs), especially HAdV-B3, -E4 and -B7, are associated with Acute Respiratory Disease in Chinese children, and occasionally in adults. In order to establish and document the profiles of the respiratory adenovirus pathogen among children in Guangzhou, Southern China, a rapid, simple and practical method for identification and typing of respiratory adenoviruses was developed and evaluated. One pair of universal PCR primers was designed according to the conserved region of the hexon gene, which can detect not only HAdV-B3, -E4 and -B7, but also HAdV-B14, -F40 and -F41, with a specific 300bp PCR product. Three pairs of type-specific PCR primers were also designed according to the hypervariable regions of the hexon gene to type HAdV-B3, -E4 and -B7 by three independent PCR reactions, making it easy to optimize the PCR conditions. By using this method, one hundred throat swab specimens collected during Oct 2010 to Dec 2011 and suspected of being positive for adenoviral infection were identified and typed for adenoviruses. Of these samples, fifty-five were adenovirus-positive. The most common HAdV type was HAdV-B3, identified in 92.7% of samples, which is not only consistent with the data reported in 2004–2006, but also consistent with the recent report in Hangzhou, eastern China, indicating that HAdV-B3 has been circulating in Guangzhou, and maybe in eastern China, for many years. The method for the respiratory adenovirus identification and typing we developed is rapid, simple and practical, which has a potential in the real-time surveillance of circulating adenovirus strains and also to provide etiological evidence for the adenovirus-relative disease control and prevention in China.     

Aetiology of Acute Respiratory Tract Infections in Hospitalised Children in Cyprus

In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV (30.4%) and Rhinovirus (27.4%). RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections.     

CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7

The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.     

南京地区2010～2011年度呼吸道感染患儿腺病毒的流行病学研究

本研究为了解南京地区儿童腺病毒(ADV)感染的流行特点及型别,收集2010年8月至2011年7月南京医科大学附属南京儿童医院住院及门诊呼吸道感染患儿的鼻咽抽吸物(NPA)及咽拭子(NPS)共644例,采用巢氏聚合酶链反应法(Nested-PCR)检测ADV hexon基因,将阳性PCR扩增产物进行测序、同源性和进化分析。同时对12种其他呼吸道相关病毒进行PCR检测,包括人博卡病毒(HBoV),呼吸道合胞病毒(RSV),人鼻病毒(HRV),副流感病毒1～4型(PIV1-4),流感病毒A和B(IFVA/B),人偏肺病毒(HMPV),冠状病毒NL63和HKU1(HCoV-NL63和HCoV-HKU1)。结果显示:644例标本中共检出ADV阳性扩增产物171份,检出率为26.55%,3型120例(70.18%,120/171),7型16例(9.36%,16/171),1型12例(7.02%,12/171),2型10例(5.85%,10/171),5型6例(3.51%,6/171),6型3例(1.75%,3/171),57型3例(1.75%,3/171),41型1例(0.58%,1/171)。ADV感染呈全年散发,其发病高峰主要在4～7月。以7岁以下儿童多见(96.49%)。171例ADV感染患儿中有99例(57.89%)存在混合感染,其中以呼吸道合胞病毒(RSV)、人鼻病毒(HRV)多见。ADV阳性患儿诊断以下呼吸道感染为主(63.16%),肺炎占30.41%。结论:ADV是2010年8月到2011年7月南京地区儿童呼吸道感染的重要病原之一,其优势流行株为3型,长期监测其流行型别具有重要意义。     

Aetiology of upper respiratory tract infections in children in Arak city: a community based study

Viruses are frequent causes of upper respiratory tract infections in children. We investigated the viral aetiology of community-acquired upper respiratory tract infections (URIs) in young children treated as outpatients in community settings. During November 2008, nasal swab specimens were taken from children with recent onset of upper respiratory tract infections. The patients attended day care or primary schools; the specimens were randomly obtained by pediatricians from schools and childcare institutions and sent for identification by PCR method. A total of 300 specimens were collected. From all samples, 40.67% were positive for at least 1 virus, viz. adenovirus 11.76%, rhinovirus 9.8%, respiratory syncytial virus 6.08%, influenza virus 5.56%, parainfluenza virus 4.9%, enterovirus 2.94% and a combination of 2 viruses 2%. Clinical manifestations of the respiratory infections were as follows: 70.7% of the patients had coryza, 69.3% cough, 26% sneezing, 19.7% sore throat, 2.7% headache, 7.7% fever, 2.3% conjunctivitis, 1.3% abdominal pain and 1% hoarseness. The results of this study demonstrate that adenoviruses and rhinoviruses are the two most common viral agents isolated from pediatric outpatients with acute URIs in autumn in Arak City. Coryza and cough were the most common symptoms in children. Sore throat and hoarseness were more prevalent in infections caused by influenza virus, conjunctivitis in parainfluenza, and coryza in rhinovirus infections.     

Genetic Analysis of Human Adenovirus Type 7 Strains Circulating in Different Parts of China

To investigate the molecular epidemiology and genetic variation of human adenovirus type 7(HAdV-7) in children with acute respiratory infections(ARI) in China. HAdV-7-positive respiratory samples collected from children with ARI in Beijing, Shijiazhuang, Wenzhou and Guangzhou from 2014–2018 were selected for gene amplification and sequence analysis. Fifty-seven HAdV-7 clinical strains with hexon, penton base and fiber gene sequences were obtained. Meanwhile17 strains were selected randomly from different cities for whole genome sequencing. Phylogenetic and variation analyses were performed based on the obtained sequences, HAdV-7 prototype strain Gomen(AY594255), vaccine strains(AY495969 and AY594256) and representative sequences of strains. The phylogenetic trees constructed based on whole genome sequences, major capsid protein genes(hexon, penton base and fiber) and the early genes(E1, E2, E3 and E4) were not completely consistent. The HAdV-7 strains obtained in this study always clustered with most of the circulating strains worldwide from the 1980 s to the present. Compared with the HAdV-7 prototype strain Gomen(AY594255), some amino acid mutations in loop1 and loop2 of hexon and the RGD loop region of the penton base gene were observed. Recombination analysis showed that partial regions of 55 k Da protein and 100 kDa hexon-assembly associated protein genes among all HAdV-7 strains in this study were from HAdV-16 and HAdV-3, respectively. Our study demonstrated the molecular evolution characteristics of HAdV-7 strains circulating in China and provided basic reference data for the prevention, control and vaccine development of HAdV-7.     

Microbiological and epidemiological aspects of rotavirus and enteric adenovirus infections in hospitalized children in Italy

Rotaviruses and enteric adenoviruses are the most important causative agents of acute infantile gastroenteritis worldwide. From July 2005 to June 2007, 445 stool specimens from pediatric patients hospitalized with acute diarrhea were collected and tested for the presence of rotaviruses and enteric adenoviruses using an immunochromatographic assay. Rotavirus infection was detected in 123 cases (27.6%, ranging from 31.7% in 2005-2006 to 24.2% in 2006-2007); adenovirus infection occurred in 17 cases (3.8%, 13 cases in 2005-2006 (6.3%) and 4 cases in 2006-2007 (1.7%). The highest prevalence was seen in children from 13 to 24 months for rotaviruses, and in children from 25 to 36 months for adenoviruses. Rotavirus infection was detected with significantly higher frequency in children up to 36 months old (32.0%) compared to the older children (19.9%) (P < 0.01). Mixed infections were observed in 10 cases (6 rotavirus-adenovirus, and 4 rotavirus-Salmonella spp.). Rotavirus infection was found predominantly in winter and spring with respect to autumn (P < 0.001) or summer (P < 0.05), with a peak in February. Adenovirus infection had a major epidemic period in spring 2006, peaking in March. Finally, this study indicates that many patients acquired rotavirus infection (37.4%), and enteric adenovirus infection (41.2%) during hospitalization for other underlying diseases.     

Host Cell Autophagy Modulates Early Stages of Adenovirus Infections in Airway Epithelial Cells

Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lysosomal degradation pathway for recycling intracellular components that is upregulated during periods of cell stress. Autophagic cargo is sequestered in double-membrane structures called autophagosomes that fuse with endosomes to form amphisomes which then deliver their content to lysosomes. Autophagy is an important adaptive response in airway epithelial cells targeted by many common adenovirus serotypes. Using two established tissue culture models, we demonstrate here that adaptive autophagy enhances expression of the early region 1 adenovirus protein, induction of mitogen-activated protein kinase signaling, and production of new viral progeny in airway epithelial cells infected with adenovirus type 2. We have also discovered that adenovirus infections are tightly regulated by endosome maturation, a process characterized by abrupt exchange of Rab5 and Rab7 GTPases, associated with early and late endosomes, respectively. Moreover, endosome maturation appears to control a pool of early endosomes capable of fusing with autophagosomes which enhance adenovirus infection. Many viruses have evolved mechanisms to induce autophagy in order to aid their own replication. Our studies reveal a novel role for host cell autophagy that could have a significant impact on the outcome of respiratory infections.     

The role of respiratory tract viruses in the etiology of obstructive bronchitis in infants

Out of 524 children with acute respiratory infections in 141 obstructive bronchitis was diagnosed (OZO). Seventy cases could be linked to viral infection. Viral infections tested (influenza virus A, B, parainfluenza typ 1-3, RSV, adenoviruses) were more frequently associated with OZO than other acute respiratory infections of unknown etiology. Majority infections induced by influenza virus A and parainfluenza virus typ 2 were accompanied by OZO symptoms. Of the highest risk of acquiring OZO despite of viral infection participation, were children of 4-12 months of age. OZO associated viral infections prevailed during autumn-winter season, while in spring-summer period undetermined factors were the major cause of OZO. In serum samples of children with OZO, despite of etiology of the disease, higher level of IgE was found than in a group of children without the symptoms. In the case of OZO of unestablished etiology the level of serum IgE was significantly higher than in the cases when viral etiology of the disease was found.     

人腺病毒工程化应用研究进展

人腺病毒流行广泛,感染常诱发呼吸道疾病、流行性结膜炎等疾病。腺病毒是研究较为深入的一类病原体,并且作为病毒载体已广泛应用,也是基因治疗最常用的病毒载体,尤其在肿瘤基因治疗和病毒溶瘤方面具有较大的研究价值和临床应用潜力。文中对腺病毒的生物学特性以及应用范围等方面进行综述,以期为腺病毒的工程化与临床应用提供参考。     

Epidemiology and Clinical Characteristics of Respiratory Infections Due to Adenovirus in Children Living in Milan,Italy, during 2013 and 2014

To evaluate the predominant human adenovirus (HAdV) species and types associated with pediatric respiratory infections, nasopharyngeal swabs were collected from otherwise healthy children attending an emergency room in Milan, Italy, due to a respiratory tract infection from January 1 to February 28 of two subsequent years, 2013 and 2014. The HAdVs were detected using a respiratory virus panel fast assay (xTAG RVP FAST v2) and with a HAdV-specific real-time polymerase chain reaction; their nucleotides were sequenced, and they were tested for positive selection. Among 307 nasopharyngeal samples, 61 (19.9%) tested positive for HAdV. HAdV was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. Human Enterovirus/human rhinovirus and respiratory syncytial virus were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. Overall, the HAdV strain sequences analyzed were highly conserved. In comparison to HAdV-negative children, those infected with HAdV had a reduced frequency of lower respiratory tract involvement (36.1% vs 55.2%; p = 0.007), wheezing (0.0% vs 12.5%; p = 0.004), and hospitalization (27.9% vs 56.1%; p<0.001). Antibiotic therapy and white blood cell counts were more frequently prescribed (91.9% vs 57.1%; p = 0.04) and higher (17,244 ± 7,737 vs 9,565 ± 3,211 cells/μL; p = 0.04), respectively, in children infected by HAdV-C than among those infected by HAdV-B. On the contrary, those infected by HAdV-B had more frequently lower respiratory tract involvement (57.1% vs 29.7%) but difference did not reach statistical significant (p = 0.21). Children with high viral load were absent from child care attendance for a longer period of time (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and had higher C reactive protein levels (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dL; p = 0.03). This study has shown that HAdV infections are diagnosed more commonly than usually thought and that HAdVs are stable infectious agents that do not frequently cause severe diseases. A trend toward more complex disease in cases due to HAdV species C and in those with higher viral load was demonstrated. However, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures.     

Prevalence and structure of acute respiratory virus infections

Information on epidemiology of acute respiratory virus infections (ARVI) is reviewed and analyzed. In addition to influenza viruses, the role of respiratory syncytial viruses (RSV), rhino- and adenoviruses, as well as other viruses, in the development of respiratory diseases, especially in newborns, young children and elderly persons, is emphasized. A high proportion of RSV in the etiology the severe forms of ARVI and in the development of intrauterine infection is pointed out. The conclusion has been made that the identification of the causative agents of ARVI with the use of modern methods makes it possible to determine the real role of each of the pathogens in the formation of the severe forms of diseases, as well as the expediency of vaccinal prophylaxis.     

Etiological structure of acute respiratory viral infections in children hospitalized in 1981-1999

The role of influenza and parainfluenza viruses, respiratory syncytial viruses and adenoviruses in the etiological structure of morbidity in acute respiratory virus infections (ARVI) in children hospitalized during the 19 year period is analyzed. As the result of examination of 56,287 patients by direct immunofluorescent test, respiratory viruses were detected in 21% of cases. The seasonal character, periodicity and level of ARVI morbidity were established. According to medical records, in the 1990s ARVI took a more severe course in children than that observed in the 1980s. In addition, the data on morbidity among children regarding rotavirus infection and ARVI were found to be similar.     

Molecular Typing and Epidemiology Profiles of Human Adenovirus Infection among Paediatric Patients with Severe Acute Respiratory Infection in China

Background

Human adenoviruses (HAdVs) have been recognised as pathogens that cause a broad spectrum of diseases. The studies on HAdV infection among children with severe acute respiratory infection (SARI) are limited.

Objective

To investigate the prevalence, epidemiology, and genotype of HAdV among children with SARI in China.

Study Design

Nasopharyngeal aspirates (NPAs) or induced sputum (IS) was collected from hospitalised children with SARIs in Beijing (representing Northern China; n = 259) and Zhejiang Province (representing Eastern China; n = 293) from 2007 to 2010. The prevalence of HAdV was screened by polymerase chain reaction (PCR), followed by sequence typing of PCR fragments that targeted the second half of the hexon gene. In addition, co-infection with other human respiratory viruses, related epidemiological profiles and clinical presentations were investigated.

Results and Conclusions

In total, 76 (13.8%) of 552 SARI patients were positive for HAdV, and the infection rates of HAdV in Northern and Eastern China were 20.1% (n = 52) and 8.2% (n = 24), respectively. HAdV co-infection with other respiratory viruses was frequent (infection rates: Northern China, 90.4%; Eastern China, 70.8%). The peak seasons for HAdV-B infection was winter and spring. Additionally, members of multiple species (Human mastadenovirus B, C, D and E) were circulating among paediatric patients with SARI, of which HAdV-B (34/52; 65.4%) and HAdV-C (20/24, 83.3%) were the most predominant in Northern and Eastern China, respectively. These findings provide a benchmark for future epidemiology and prevention strategies for HAdV.