Quantitative Detection and Rapid Identification of Human Adenoviruses

Abstract

We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 ؋ 10 2 to 1.6 ؋ 10 Human adenoviruses (hAdVs) of the genus Mastadenovirus of the family Adenoviridae infect billions of people worldwide and cause various diseases such as conjunctivitis, respiratory infectious disease, diarrhea in infants and young children, hemorrhagic cystitis, etc. (1,8,33,39,40). Most of these diseases heal naturally, but sometimes the infection may also provoke serious illnesses such as pneumonia caused by AdV type 7 (AdV-7) or epidemic keratoconjunctivitis (EKC) due to . These more serious outcomes occur in all age groups and can possibly trigger highly contagious nosocomial infections (5,6,19,27,42). Therefore, it is important to establish a rapid method of virological diagnosis. Furthermore, hAdV infection in immunosuppressed patients, such as graft recipients and immunodeficient patients including those with AIDS, has been a major problem in recent years and has been lethal in many cases (7,9,10,17,23,40,41). Nosocomial infection is also a serious problem which may require restriction of hospitalization and closing of hospital wards (15). Therefore, it is essential to monitor these viruses, and a rapid method of identifying serotypes is urgently needed.hAdVs were initially grouped into six subgenera (A to F) on the basis of several biochemical and biophysical criteria (1, 39). In 1999, reclassification on the basis of nucleotide and deduced amino acid sequences was approved by the International Committee on Taxonomy of Viruses; under this reclassification, the 51 serotypes of hAdVs in the genus Mastadenovirus were grouped into six species, hAdV-A to hAdV-F (38). Virus isolation followed by a neutralization test has been the standard method of serotyping (39); however, these procedures are complicated and time-consuming, and the standardized antisera are in limited supply. Recent advances in amplifying hAdV genes and decoding nucleotide sequences have allowed us to develop a PCR-restriction fragment length polymorphism method with which we have succeeded in distinguishing 14 hAdVs including AdV-3, -4, -8, -19a, and -37, all of which cause eye infections in humans (31). Furthermore, nucleotide sequence analysis of the partial hexon genes (916 bp) of all 33 prototype strains in hAdV-D and hAdV-E allowed us to construct a database for the phylogeny-based identification of hAdVs from patients with conjunctivitis (34). However, this method requires several overlapping sequences to determine 9...

Molecular epidemiology of the attachment glycoprotein (G) gene in respiratory syncytial virus in children with acute respiratory infection in Japan in 2009/2010

“…We primarily isolated various viruses from the samples using cell-culture methods for Vero E6, RD18S and Madin-Derby canine kidney cells. Next, in the samples negative for isolation, we detected various respiratory viruses such as influenza A (H1N1) pdm09 virus, seasonal influenza virus (subtypes A, B and C), human parainfluenza virus (HPIV; types 1-4), adenovirus (AdV), human rhinovirus (HRV), human metapneumovirus (HMPV), enterovirus (EV) and human bocavirus (HBoV) by (RT)-PCR, as described previously (Allander et al, 2005;Bellau-Pujol et al, 2005;Echevarría et al, 1998;Miura-Ochiai et al, 2007;Nakauchi et al, 2011;Olive et al, 1990;Takao et al, 2004;Zhang & Evans, 1991). In addition, we attempted to isolate and/or detect various respiratory bacteria such as Haemophilus influenzae, Legionella pneumophila, Neisseria spp.…”

Epidemic Keratoconjunctivitis Due to the Novel Hexon-Chimeric-Intermediate 22,37/H8 Human Adenovirus

“…Serotyping of HAdVs is generally performed by virus isolation followed by a neutralization test (NT) and hemagglutination inhibition test (HI) using type-specific antiserum (29). To date, amplification of the genome by PCR and determination of the nucleotide sequences followed by phylogenetic analysis have become the general procedure for both the classification and identification of HAdVs (21,22,28). In 2003, we encountered an EKC outbreak in Japan (Infectious Agents Surveillance Report [Weekly reports of adenovirus isolation/detection from epidemic keratoconjunctivitis cases, [2001][2002][2003][2004][2005][2006][2007]; http://idsc.nih.go.jp/iasr/prompt/graph/ad3.gif]) and detected HAdV DNA in 67 swabs from EKC patients who visited five eye clinics in different parts of Japan.…”

“…However, the nucleotide sequence of the fiber gene was identical to that of the HAdV-8 prototype strain. 22 …”

Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus

“…The nucleotide sequence was 100% identical to the HAdV-4 isolated from basic training recruits in United States of America during the end of the 1990s (Blasiole et al 2004). This serotype was also found in Japan, where it was associated with cases of conjunctivitis (Miura-Ochiai et al 2007). To our knowledge, this is the first description of serotype HAdV-4 in Brazil.…”