Serotyping of adenoviruses on conjunctival scrapings by PCR and sequence analysis.
(1/69)
To detect and identify adenovirus (Ad), we investigated hypervariable regions (HVRs) of Ad by using a combination of PCR and direct sequencing (PCR-sequence) method. Primers for nested PCR to amplify the conserved region in the hexon protein containing HVRs were designed based on hexon gene sequences derived from GenBank. These two primer sets amplified a DNA fragment of 7 HVRs from 16 prototypes of Ad, which were divided into five subgenera, including seven serotypes that are the predominant causative agents of acute conjunctivitis in Japan, and from 31 recent conjunctival scraping specimens from patients with adenoviral conjunctivitis. HVR DNA sequences were determined by means of universal sequence primers. Analysis of the predicted amino acid homology of HVRs among Ad prototypes suggested three regions, HVR4, -5, and -7, to be candidates for the neutralization epitopes. The clinical serotype of specimens was determined by the PCR-sequence method with reference to these three HVRs. The serotype determined according to this method was identical to that obtained by culture isolation and the neutralization test (NT) in all scraping samples, whereas the results of this method did not match PCR and restriction fragment length polymorphism (PCR-RFLP) analysis in five samples. It took only three days to detect Ad and to identify the serotype, in contrast to culture isolation-NT, which took at least 2 weeks. These findings indicate that our newly developed PCR-sequence method is applicable for the detection and serotyping of human Ads. (+info)
Laboratory investigations on viral and Chlamydia trachomatis infections of the eye: Sankara Nethralaya experiences.
(2/69)
PURPOSE: To review our experiences on the laboratory investigations of viral and chlamydial conjunctivitis, congenital cataract and acute retinal inflammations seen from 1990 to 1998 at Sankara Nethralaya, Chennai, India. METHODS: Conjunctival swabs/scrapings from 1061 patients with conjunctivitis were investigated. Nested polymerase chain reaction (nPCR) and restriction fragment length polymorphism (RFLP) techniques were applied on 74 conjunctival swabs during the 1996 outbreak of acute viral conjunctivitis. The occurrence of Rubella virus in 86 lens aspirates of congenital cataract was investigated. Tests were performed for the association of Herpes simplex virus (HSV), Varicella zoster virus (VZV) and Cytomegalovirus (CMV) with acute retinal inflammation in 32 patients. RESULTS: The causative agents of conjunctivitis were Adenovirus in 13.8%, HSV in 2.2% and C. trachomatis in 20.9% of the patients. Epidemics were due to Adenovirus type 4 in 1991, type 3 in 1992-93 and type 7a in 1996. PCR was 37.9% more sensitive in detecting Adenovirus than virological methods. RFLP identified the conjunctivitis epidemic strain of 1996 as Adenovirus 7a. Rubella virus was isolated from 8.1% of lens aspirates from congenital cataract. Nineteen of the 32 patients with acute retinitis had confirmed virus infections (VZV: 8; HSV: 5; and CMV: 6) and the rapid detection of the virus agent helped institute specific chemotherapy resulting in useful vision in some patients. CONCLUSION: Laboratory investigations for diagnosis of viral and C. trachomatis ocular infections were useful in establishing the aetiology and determining the incidence of causative agents of specific ocular diseases. (+info)
Molecular evidence of ocular Epstein-Barr virus infection.
(3/69)
Ocular manifestations have been attributed to the Epstein-Barr virus (EBV), largely on the basis of seroepidemiologic data. Two patients who developed conjunctival disease as the presenting feature of EBV infection are reported, each confirmed by in situ hybridization of EBV genome in affected tissue biopsy specimens. Recognition of EBV-induced ocular disease as an initial presentation of clinical EBV infection is important to the practitioner because of the ubiquitous nature of this herpesvirus. (+info)
Clinical and epidemiological features of acute follicular conjunctivitis with special reference to that caused by herpes simplex virus type 1.
(4/69)
BACKGROUND/AIMS: It is reported by the national surveillance of ocular infectious diseases in Japan that 4.3% of cases of epidemic keratoconjunctivitis (EKC) diagnosed clinically were caused by herpes simplex virus (HSV). Clinical and virological studies of patients with HSV conjunctivitis were carried out. METHODS: The study population consisted of 478 patients with acute follicular conjunctivitis. Virological analysis was carried out for adenovirus (Ad) and HSV by the cell culture method and fluorescein antibody (FA) method. Polymerase chain reaction for Chlamydia trachomatis was also carried out. RESULTS: From 23 patients, HSV type 1 was isolated but Ad or C trachomatis was not isolated. 87% of cases were unilateral. Most cases showed clinical resolution within 9 days. Early corneal lesions and preauricular lymphadenopathy were less frequent in HSV conjunctivitis than in adenoviral conjunctivitis, especially that due to subgenus D. No case showed a positive result for HSV by the FA method using conjunctival swabs; however, the FA test was positive in all strains isolated by cell culture. CONCLUSIONS: These results indicate that it is difficult clinically to differentiate HSV conjunctivitis from adenoviral conjunctivitis in the acute stage, since the clinical features of adenoviral conjunctivitis are similar to those of HSV conjunctivitis. A biological difference may exist between HSV strains causing keratitis and conjunctivitis. (+info)
No sequence variation in part of the hexon and the fibre genes of adenovirus 8 isolated from patients with conjunctivitis or epidemic keratoconjunctivitis (EKC) in Norway during 1989 to 1996.
(5/69)
BACKGROUND/AIMS: Several local epidemics of keratoconjunctivitis/conjunctivitis caused by adenovirus type 8 (Ad8) occurred in Norway from August 1995 to May 1996. A smaller epidemic occurred in 1992. The Ad8 hexon forms the surface of the virion and contains the hypervariable regions loop I(1) and loop I(2). The fibre mediates the primary contact with cells. Sequence variation in hexon and fibre genes might play an important role in the pathogenicity of adenoviruses. The aim of this study was to investigate the genetic variability at the hexon and fibre genes in 26 strains of Ad8 isolated from 1989 to 1996. METHODS: The genetic variability of 26 strains of Ad8 isolated from 1989 to 1996 was studied by sequencing part of the hexon and fibre genes. The Ad8 sequences were compared with each other and with two Ad8 strains from the EMBL database. In addition, 14 of the 26 isolates were subjected to restriction endonuclease analysis. RESULTS: No significant sequence variation was seen during the six year period. CONCLUSION: The Ad8 strains causing epidemics of keratoconjunctivitis/conjunctivitis in Norway are genetically stable. (+info)
The antiviral resistance and replication of cidofovir-resistant adenovirus variants in the New Zealand White rabbit ocular model.
(6/69)
PURPOSE: To determine the antiviral resistance of three cidofovir (CDV)-resistant variants of adenovirus type 5 (Ad5) and their ability to replicate in the New Zealand White rabbit ocular model. METHODS: Rabbits were inoculated topically in both eyes with the CDV-resistant variants R1, R2, and R3, and the Ad5 parental strain. On day 1, rabbits from each virus inoculation were divided into two topical treatment groups: 0.5% CDV and PBS control. Treatment was administered twice daily in both eyes for 7 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Using viral outcome parameters, CDV resistance was determined for each virus by comparing each CDV-treated virus group to its respective PBS control, and altered pathogenesis was assessed by comparing viral replication in the PBS control groups of the Ad5 parent and the three resistant variants. RESULTS: Topical 0.5% CDV treatment demonstrated significant antiviral inhibitory activity in the Ad5 parental group (e.g., reduced total Ad5-positive cultures, reduced daily Ad5-positive cultures on days 5, 9, 11, and 14, and duration of ocular shedding), but had no effect on the three CDV-resistant variants. There were no significant differences in pathogenicity between the Ad5 parent and the CDV-resistant variants. CONCLUSIONS: The Ad5 variants R1, R2, and R3 were resistant to topical treatment with 0.5% cidofovir in the rabbit ocular model. However, the acquisition of CDV resistance did not alter the replication of the three Ad5 CDV variants on the rabbit eye. (+info)
Rapid detection and typing of oculopathogenic strain of subgenus D adenoviruses by fiber-based PCR and restriction enzyme analysis.
(7/69)
PURPOSE: To develop a new detection and typing method of oculopathogenic strains of subgenus D adenoviruses directly from conjunctival scrapings by a combination of polymerase chain reaction (PCR) and restriction enzyme analysis (REA). METHODS: A new PCR method using primer pairs of AF2/AR2, which are specific for the fiber genes, were developed to amplify 1150-bp products from nine oculopathogenic prototypes of subgenus D adenoviruses. Amplicons were cleaved with three restriction enzymes: DdeI, HinfI, and RsaI. Clinical specimens of 102 conjunctival scrapings were also evaluated by this PCR method. Restriction patterns of prototypes were used for the typing of clinical samples. Detection limit was determined by the PCR amplification of a known amount of purified adenovirus serotype 8 DNA. RESULTS: A novel PCR method based on the fiber genes allowed the amplification of nine oculopathogenic serotypes of subgenus D (Ad8, Ad9, Ad15, Ad17, Ad19, Ad22, Ad28, Ad37, and Ad39). As little as 38.4 fg of adenovirus type 8 could be detected by this method. Positive results were obtained from 48 of 102 samples (47%) by both hexon- and fiber-based PCR, whereas only 29 of 102 (28.4%) yielded positive results by culture isolation/neutralization test (NT). All positive specimens (29 samples) of culture isolation and PCR-RFLP methods showed positive results by our new fiber-based PCR method, and no positive products were detected from other subgenus of adenovirus or nonadenoviral DNA. CONCLUSIONS: A newly developed fiber-based PCR-REA method for the detection and typing of adenoviruses is faster than any former PCR methods. This all-in-1-day detection and typing method will be quite useful to the rapid diagnosis of subgenus D adenovirus infection. (+info)
Detection of antibodies to a disease-associated herpesvirus of the green turtle, Chelonia mydas.
(8/69)
Lung-eye-trachea disease-associated herpesvirus (LETV) is linked with morbidity and mortality in mariculture-reared green turtles, but its prevalence among and impact on wild marine turtle populations is unknown. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of anti-LETV antibodies and could distinguish LETV-exposed green turtles from those with antibodies to fibropapillomatosis-associated herpesvirus (FPHV). Plasma from two captive-reared green turtles immunized with inactivated LETV served as positive controls. Plasma from 42 healthy captive-reared green turtles and plasma from 30 captive-reared green turtles with experimentally induced fibropapillomatosis (FP) and anti-FPHV antibodies had low ELISA values on LETV antigen. A survey of 19 wild green turtles with and 27 without FP (with and without anti-FPHV antibodies, respectively) identified individuals with antibodies to LETV regardless of their FP status. The seroprevalence of LETV infection was 13%. The presence of antibodies to LETV in plasma samples was confirmed by Western blot and immunohistochemical analyses. These results are the first to suggest that wild Florida green turtles are exposed to LETV or to an antigenically closely related herpesvirus(es) other than FPHV and that FPHV and LETV infections are most likely independent events. This is the first ELISA developed to detect antibodies for a specific herpesvirus infection of marine turtles. The specificity of this ELISA for LETV (ability to distinguish LETV from FPHV) makes it valuable for detecting exposure to this specific herpesvirus and enhances our ability to conduct seroepidemiological studies of these disease-associated agents in marine turtles. (+info)