Evaluation of a silver-amplified immunochromatography kit for adenoviral conjunctivitis.

OBJECTIVE: To compare and evaluate the sensitivity of a newly developed silver-amplified immunochromatography (SAI) kit with various immunochromatography (IC) kits for adenoviruses based on the detection limit (copies/test). METHODS: An SAI kit and four ophthalmic IC kits were evaluated. The detection limits of the five kits were determined using the limiting dilution method for 15 conjunctivitis-associated adenoviruses (adenoviruses 1, 2, 3, 4, 5, 7, 8, 11, 37, 53, 54, 56, 64, 81, and 85). The detection limits were presented as numerical values as determined by real-time polymerase chain reaction (PCR). RESULTS: The detection limit of the SAI kit for the adenovirus types ranged from 1.0 × 103 -5.0 × 10 4 copies/test (geometric mean, 4.7 × 10 3 ). SAI had a 10-250-fold lower detection limit than the four IC kits for all adenoviruses studied. There were also differences in detection limits among the adenovirus types for each kit. DISCUSSION: The detection limit of the SAI kit was drastically reduced because the silver-amplification reaction increased the color development sensitivity. The results revealed the high sensitivity of SAI for detecting adenoviruses and suggested its usefulness for conjunctivitis examination.

Herpes simplex virus type 1 virion-derived US11 inhibits type 1 interferon-induced protein kinase R phosphorylation.

The herpes simplex virus type 1 (HSV-1) VRTK(-) strain that was previously isolated in our laboratory as an acyclovir-resistant thymidine kinase (TK)-deficient mutant, is more sensitive to type 1 interferon than is the parent strain VR3. The properties of this mutant were investigated to clarify the mechanism for its hyper-sensitivity to interferon (IFN). It was found that: (i) IFN-pretreated cells, but not those treated with IFN after adsorption, are hyper-sensitive to IFN; (ii) the mutant cannot inhibit protein kinase R phosphorylation efficiently during the early stage of replication (2 hrs post-infection); (iii) expression of US11 in infected cells and its incorporation into the virion is reduced in the mutant compared to the wild type, despite the fact that a similar degree of DNA synthesis occurs during replication of both strains and; (iv) over-expression of wild-type viral TK has no effect on the phenotype of the VRTK(-) strain, indicating that the phenotype is induced by a mutation(s) that does not involve the TK gene. These results suggested that the presence of US11 in the virion, but not that expressed after infection, plays an important role in the escape function of HSV-1 from the antiviral activity of type 1 IFN.

[Bioinformatics on new human adenoviruses causing nosocomial infection].

Human adenovirus (HAdV) causing epidemic keratoconjunctivitis is limited to D and E species. Recent progress in bioinformatics revealed that these viruses attach to the host with fibers, infiltrate the host cells via RGD (Arg-Gly-Asp) motif of penton base, and reveal their serological reaction by hexons. Loops 1 and 2 are the variable regions of each hexon. The possibility that a novel adenovirus later named HAdV-52 was transmitted over the wall of species' from monkeys to humans was reported. The recombination of the above three hot spots introduces novel types such as HAdV-53, -54, and -56. Boinformatics may provide rapid genotyping in nosocomial infection, predicting future epidemics, and an estimate of the therapeutic target molecules in the near future.

Anti-viral and anti-bacterial activities of an extract of blackcurrants (Ribes nigrum L.).

The inhibitory effects of an extract of the blackcurrant (Ribes nigrum L.) against pathogens associated with oral, nasopharyngeal and upper respiratory infectious diseases; namely respiratory syncytial virus (RSV), influenza virus A and B (IFV-A and IFV-B), adenovirus (AdV), herpes simplex virus type 1, Haemophilus influenzae type B, Streptococcus pneumoniae and Streptococcus mutans, were investigated. Less than 1% concentration of extract of blackcurrant inhibited replication of RSV, IFV-A and -B and HSV-1 by over 50% and a 10% extract inhibited adsorption of these viruses onto the cell surface by over 95%. The effects on AdV were much less pronounced; the half minimal inhibitory concentration of AdV replication was 2.54 ± 0.26, and a 10% concentration of the extract inhibited AdV adsorption on the cell surface by 72.9 ± 3.4%. The antibacterial activities of the blackcurrant were evaluated based on its efficacy as a disinfectant. A 10% extract disinfected 99.8% of H. Influenzae type B and 78.9% of S. pneumoniae in 10 min, but had no demonstrable effect against S. mutans. The blackcurrant extract still showed antiviral and antibacterial activities after the pH had been made neutral with sodium hydroxide, suggesting that these activities are not the result of acidic reactions or of components precipitated at a neutral pH. These findings demonstrate the potential of blackcurrant extract as a functional food for oral care.

Recombination analysis of intermediate human adenovirus type 53 in Japan by complete genome sequence.

Human adenovirus type 53 (HAdV-53) has commonly been detected in samples from epidemic keratoconjunctivitis (EKC) patients in Japan since 1996. HAdV-53 is an intermediate virus, containing hexon-chimeric, penton base and fiber structures similar to HAdV-22 and -37, HAdV-37 and HAdV-8, respectively. HAdV-53-like intermediate strains were first isolated from EKC samples in Japan in the 1980s. Here, the complete genome sequences of three such HAdV-53-like intermediate strains (870006C, 880249C and 890357C) and four HAdV-53 strains were determined, and their relationships were analysed. The seven HAdV strains were classified into three groups, 870006C/880249C, 890357C and the four HAdV-53 strains, on the basis of phylogenetic analyses of the partial and complete genome sequences. HAdV strains within the same group showed the highest nucleotide identities (99.87-100.00 %). Like HAdV-53, the hexon loop 1 and 2 regions of 870006C, 880249C and 890357C showed the highest identity with HAdV-22. However, these strains did not show a hexon-chimeric structure similar to HAdV-22 and -37, or a penton base similar to HAdV-37. The fiber genes of 870006C and 880249C were identical to that of HAdV-37, but not HAdV-8. Thus, the three intermediate HAdVs isolated in the 1980s were similar to each other but not to HAdV-53. The recombination breakpoints were inferred by the Recombination Detection Program (rdp) using whole-genome sequences of these seven HAdV and of 12 HAdV-D strains from GenBank. HAdV-53 may have evolved from intermediate HAdVs circulating in the 1980s, and from HAdV-8, -22 and -37, by recombination of sections cut at the putative breakpoints.

Complete genome analysis of a novel intertypic recombinant human adenovirus causing epidemic keratoconjunctivitis in Japan.

For 4 months from September 2008, 102 conjunctival swab specimens were collected for surveillance purposes from patients across Japan suspected of having epidemic keratoconjunctivitis (EKC). Human adenovirus (HAdV) DNA was detected in 61 samples by PCR, though the HAdV type for 6 of the PCR-positive samples could not be determined by phylogenetic analysis using a partial hexon gene sequence. Moreover, for 2 months from January 2009, HAdV strains with identical sequences were isolated from five conjunctival swab samples obtained from EKC patients in five different regions of Japan. For the analyses of the 11 samples mentioned above, we determined the nucleotide sequences of the entire penton base, hexon, and fiber genes and early 3 (E3) region, which are variable regions among HAdV types, and compared them to those of other HAdV species D strains. The nucleotide sequences of loops 1 and 2 in the hexons of all 11 samples showed high degrees of identity with those of the HAdV type 15 (HAdV-15) and HAdV-29 prototype strains. However, the fiber gene and E3 region sequences showed high degrees of identity with those of HAdV-9, and the penton base gene sequence showed a high degree of identity with the penton base gene sequences of HAdV-9 and -26. Moreover, the complete genome sequence of the 2307-S strain, which was isolated by viral culture from 1 of the 11 samples, was determined. The 2307-S strain was a recombinant HAdV between HAdV-9, -15, -26, -29, and/or another HAdV type; however, the recombination sites in the genome were not obvious. We propose that this virus is a novel intertypic recombinant, HAdV-15/29/H9, and may be an etiological agent of EKC.

[Three cases of acute conjunctivitis caused by human adenovirus in medical workers].

We report three cases of acute conjunctivitis due to different types of human adenovirus (HAdV) in medical workers. Case 1: A 37-year-old man had epidemic keratoconjunctivitis and urethritis caused by human adenovirus type 37 (HAdV-37). Case 2: A 27-year-old woman had acute conjunctivitis due to human adenovirus type 15 (HAdV-15), Case 3:A 32-year-old woman had acute follicular conjunctivitis leading to a serotype of human adenovirus-53 serotype (HAdV-53). Note that HAdV-37 infection may cause keratoconjunctrivitis and urethritis. HAdV-15, -37, and -53 are important causative agents of nosocomial outbreak, and the application of a rapid diagnostic kit or PCR to a rapid diagnosis and proper infection-control measures can significantly reduce infection spread.

DSCG reduces RSV-induced illness in RSV-infected mice.

Respiratory syncytial virus (RSV) is one of the pathogens generally associated with the common cold, lower respiratory infection, and exacerbation of asthma. Disodium cromoglycate (DSCG) is a safe and widely used drug for the prevention of bronchial asthma and allergic rhinitis attacks. The effect of DSCG on acute upper respiratory tract viral infections remains controversial. The purpose of the study was to investigate the effects of DSCG on parameters of RSV induced-illness. Using a well-characterized murine model of RSV infection, the effect of DSCG on RSV-induced illness was evaluated by body weight, respiratory function, viral replication, level of IFN-gamma in lungs, serology, and histopathology. Mice treated with DSCG were protected against RSV-induced weight loss. The baseline Penh in RSV-infected mice treated with DSCG was less than that in mice treated with saline. In methacholine challenge, the increase in Penh in RSV-infected mice treated with DSCG was suppressed to the same level as that in the mock-infected group. Further, there were no differences in viral replication between the mice treated with DSCG and those treated with saline, and the level of inflammation observed in the lungs in RSV-infected mice treated with DSCG was not as severe as that in mice treated with saline. These findings indicate that DSCG may be an effective agent for the prevention of RSV induced disease and the relief of symptoms of RSV infection.

An improved rapid quantitative detection and identification method for a wide range of fungi.

To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 10(1) and 10(7) copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 5' end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 3'-terminal fragments, derived using restriction enzyme ScrFI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.

Discrimination of herpes simplex virus type 2 strains by nucleotide sequence variations.

We determined the polymorphous 400-bp regions in UL53, US1, and US4 for the discrimination of herpes simplex virus type 2 (HSV-2) strains. Thirty-six HSV-2 clinical strains could be differentiated into 35 groups using these three regions and into 36 groups by additional analysis of three noncoding regions previously reported as polymorphous.

Epidemic keratoconjunctivitis due to the novel hexon-chimeric-intermediate 22,37/H8 human adenovirus.

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 (epsilon 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.

Identification of a highly conserved region in the human cytomegalovirus glycoprotein H gene and design of molecular diagnostic methods targeting the region.

Genomic polymorphism of human cytomegalovirus (HCMV) leads to difficulties in the design of molecular diagnostic systems; therefore, a suitable target region was determined in the glycoprotein H (gH) gene, which has been reported to be the most conserved gene. A highly conserved region was identified from codon 1,282 to 1,988 of the gH gene by alignment of 23 nucleotide sequences (14 registered in the DNA Data Bank of Japan and 9 sequenced in this laboratory). Diagnostic methods based on nested PCR, real-time PCR and loop-mediated isothermal temperature amplification (LAMP) were designed for this region. Primers and a probe for nested and real-time PCR were designed for the completely conserved sequences in all HCMV strains. However, a few mismatched nucleotides could not be excluded from the LAMP primers due to the need for eight primer-binding sites in a 200bp-region. The sensitivities of the nested PCR, real-time PCR and LAMP reactions were 5, 10 and 100 copies/tube, respectively. An analysis of clinical specimens showed that both nested and real-time PCR detected HCMV with greater sensitivity than did a pp65 antigenemia assay and were expected to minimize the incidence of false-negative results, whereas the sensitivity of the LAMP reaction was comparable with that of the antigenemia assay.

Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances.

To simplify the molecular detection of micro-organisms, we evaluated the tolerance of loop-mediated isothermal amplification (LAMP) to a culture medium and some biological substances. The sensitivity of LAMP was less affected by the various components of the clinical samples than was polymerase chain reaction (PCR); therefore, DNA purification from samples could be omitted.

[The study of prevalence of neutralization antibody against human adenovirus in ophthalmological medical workers].

PURPOSE: We studied the prevalence of neutralization antibodies against human adenovirus (hADV) in ophthalmological medical workers as one measure for the prevention of nosocomial infection. METHODS: The prevalence of neutralization antibodies against hADV-3, -4, -7, -8, -11, -19, and -37, which can cause conjunctivitis, was measured in 288 workers at ten ophthamological facilities in Japan. We studied the prevalence in different facilities, different generations, different types of workers (doctors, nurses, and others), and their medical history of hADV conjunctivitis. RESULTS: Among the workers, the prevalence of neutralization antibodies against hADV-3, -4, -7, -8, -11, -19, and -37 was 70.1%, 43.8%, 18.8%, 16.3%, 16.3%, 8.7%, and 6.3%, respectively. The prevalence of neutralization antibodies against hADV-8 was two times higher in doctors than in other workers. People who have a history of hADV conjunctivitis have a high prevalence of neutralization antibodies against hADV-8, -19, and-37. CONCLUSIONS: The prevalence of neutralization antibodies against hADV-8, -19, and-37 was low in the ophthalmological medical workers. These seroepidemiological data indicate the high possibility of an epidemic of conjunctivitis and occurrence of nosocomial infection caused by these serotypes.

[Clinical and virological studies of nosocomial conjunctivitis infection caused by adenovirus type 37 variant].

PURPOSE: To study the clinical features and virological analysis of the nosocomial adenoviral conjunctivitis cases occurring in the ophthalmology ward of Fukushima Medical University Hospital. MATERIALS AND METHODS: We studied the symptoms and clinical course of 61 patients who had adenoviral conjunctivitis caused by nosocomial infections in our hospital. We attempted to detect the adenovirus antigen, analyze the viral DNA, and isolate the virus from conjunctival swabs. RESULTS: The clinical symptoms of adenoviral conjunctivitis were mainly conjunctival hyperemia, discharge and conjunctival follicles. Adenoviral conjunctivitis patients who had undergone ophthalmic surgery had conjunctivitis in the operated eye. The sensitivity of Adeno-check was 78.9% in the in-patients. Adenovirus type 37 variant was detected by molecular analysis and viral isolation. CONCLUSIONS: Adenoviral conjunctivitis can often lead to outbreaks of nosocomial infection in the ophthalmic ward and sometimes requires makes necessary restriction of hospitalization and closing of the ward. Therefore, patients need to be observed carefully. The virological analysis of specimens from conjunctival swabs detected pathogens and provided useful information concerning adenoviral conjunctivitis.

The cotton rat model for adenovirus ocular infection: antiviral activity of cidofovir.

To determine the antiviral effects of compounds against ocular adenovirus (AdV) infection, we established an animal model of AdV infection in cotton rat eyes. Cotton rat eyes were inoculated intrastromally and topically with four AdV serotypes 4, 5, 8, and 37, and treated topically with 1% HPMPC (cidofovir) eye drops twice a day. The infected corneas were extracted and homogenized, and virus titers in the cornea specimens were determined by a plaque assay. The virus titer in AdV type 5-inoculated eyes peaked on days 0 through 3 after inoculation and virus shedding was detected for 18.0+/-2.8 days. AdV 5 antigen in the infected corneas was demonstrated in the corneal epithelial cells by immunofluorescence stain. However, for AdV serotypes 4, 8, and 37, no evidence of continued virus replication in cotton rat eyes was noted. Specimens from cidofovir-treated eyes infected with AdV 5 demonstrated a statistically significant reduction in the mean virus titer (days 3-15) (P=0.028) and virus shedding duration (P=0.0014), as compared with those of the control group.

[Antiviral effect of sulfated sialyl lipid against a clinical strain of adenovirus].

PURPOSE: Currently, there is no antiviral drug for adenovirus(AdV). We have reported that sulfated sialyl lipid(NMSO) 3, a NMSO, has an antiviral effect against AdV prototype strains. We evaluated the antiviral inhibitory effect and the mechanism of NMSO 3 against AdV strains from patients with conjunctivitis in vitro. METHODS: Viruses used for the experiment were clinically isolated AdV type 3(AdV 3), AdV type 4(AdV 4), type 8(ADV 8), AdV type 19(AdV 19), and type 37(AdV 37). We examined three antiviral agents, i.e., NMSO 3, cidofovir(HPMPC), and zalcitabine(ddC). 50% effective concentration(EC50), 50% cytotoxic concentration(CC50), and selectivity index(SI) of compounds were determined for AdV infection in HEp-2 cells using 3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods. We also evaluated the anti-AdV activity of NMSO 3 when it was added during the stage of virus adsorption. RESULTS: NMSO 3, HPMPC, and ddC showed an inhibitory effect against all five AdV clinical strains. The EC50 values of NMSO3 were lower than those of HPMPC and ddC. NMSO 3 exhibited minimal cytotoxicity. NMSO 3 inhibited AdV infection only when it was added during the stage of virus adsorption. CONCLUSIONS: NMSO 3 inhibited the replication of all clinical AdV serotypes tested. NMSO 3 was the most potent and selective anti-AdV compound. The mechanism of anti-AdV activity by NMSO 3 was inhibition of viral adsorption to cells.