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...
In 2000, we encountered cases of nosocomial infections with epidemic keratoconjunctivitis (EKC) at a university hospital in Kobe, in the western part of Japan. Two human adenovirus (HAdV) strains, Kobe-H and Kobe-S, were isolated from patients with nosocomial EKC infection. They were untypeable by existing neutralizing antisera; however, the isolate was neutralized with homologous antisera. We then encountered several cases of EKC due to nosocomial infections in eye clinics in different parts of Japan. A total of 80 HAdVs were isolated from patients with EKC at eight different hospitals. The partial hexon gene sequences of the isolates were determined and compared to those of the prototype strains of 51 serotypes. All isolates had identical partial hexon nucleotide sequences. Phylogenetic analysis classified these isolates into species of HAdV-D. The isolates showed 93.9 to 96.7% nucleotide identity with HAdV-D prototype strains, while all 32 HAdV-D prototype strains ranged from 93.2 to 99.2% identity. The sequences of the loop 2 and fiber knob regions from the representative strain, Kobe-H, were dissimilar in all prototype strains of 51 serotypes. We believe that this virus is a novel serotype of HAdV that causes EKC.
In a 2-month period in 2003, we encountered an outbreak of epidemic keratoconjunctivitis (EKC) in Japan. We detected 67 human adenoviruses (HAdVs) by PCR from eye swabs of patients with EKC at five eye clinics in different parts of Japan. Forty-one of the 67 HAdV DNAs from the swabs were identified as HAdV-37 by phylogenetic analysis using a partial hexon gene sequence. When the restriction patterns of these viral genomes were compared with that of the HAdV-37 prototype strain, one isolate showed a never-before-seen restriction pattern. Within 1 year, we encountered three more EKC cases caused by a genetically identical virus: two nosocomial infections at two different university hospitals and a sporadic infection at an eye clinic. We determined the nucleotide sequences of the full-length hexon and fiber genes of these isolates and compared them to those of the 51 prototype strains. Surprisingly, the sequence of the hexon ( determinant) loop-1 and -2 regions showed the highest nucleotide identity with HAdV-22, a rare EKC isolate. However, the nucleotide sequence of the fiber gene was identical to that of the HAdV-8 prototype strain. 22 We propose that this virus is a new hexon-chimeric intermediate HAdV-22,37/H8, and may be an etiological agent of EKC.
Dysregulation of cyclin-dependent kinase 5 (Cdk5) by cleavage of its activator p35 to p25 by calpain is involved in the neuronal cell death observed in neurodegenerative disorders, including Alzheimer's disease. However, it is not yet clear how p25/Cdk5 induces cell death, although its cytosolic localization or extended half life are thought to be involved. We show here that endoplasmic reticulum (ER) stress causes the calpaindependent cleavage of p35 to p25 in primary cultured cortical neurons. Generation of p25 occurred at a cell death execution step in ER-stressed neurons. p25 translocated to the nucleus in ER-stressed neurons, whereas p35/Cdk5 was perinuclear in control neurons. Cdk5 inhibitors or dominant-negative Cdk5 suppressed ER stress-induced neuronal cell death. These findings indicate that p25/Cdk5 is a proapoptotic factor that promotes ER stress-induced neuronal cell death in nuclei.
Our study showed types of adenoviruses in patients with ocular infection that occurred in this region of Turkey for the first time. Furthermore, sequence-based typing method is an efficient, accurate, and rapid means of diagnosis and typing of the adenovirus and has significant clinical and epidemiologic implications. HAdV-8 was major type for acute conjunctivitis in Ankara, Turkey. Further studies are required to reveal the major types of HAdVs that cause ocular diseases in this region of the world.
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