EKC due to HAdV-54 can result in epidemics; therefore, it should be accurately diagnosed and monitored as an emerging infection worldwide.
We determined the complete genome sequence of epidemic keratoconjunctivitis (EKC)-related human adenoviruses (HAdVs). We analysed a total of 12 HAdV strains; three prototype strains and two HAdV-8, three HAdV-19 and three HAdV-37 clinical isolates from EKC patients in Japan, and one novel serotype of HAdV. Genome organization of these serotypes was identical to those of the recently determined HAdV-19 and HAdV-37. The identities of the whole genome were over 99 % among strains from the same serotype, except for HAdV-19p, which is not associated with conjunctivitis, resulting in the formation of a distinct cluster in the phylogenetic analysis. The penton, loop 1 and loop 2 of hexon, early region 3 (E3) and fiber were hypervariable regions between serotypes. Results suggest that the HAdV-19 clinical strain is a recombinant of HAdV-19p-like and HAdV-37-like strains, and that the acquisition of the penton, E3 or fiber may be related to ocular tropism.Adenoviruses are nonenveloped, double-stranded DNA viruses with icosahedral capsids (Swenson et al., 2003). Human adenoviruses (HAdVs) belong to the genus Mastadenovirus of the family Adenoviridae and are classified into six species, A to F (HAdV-A to HAdV-F) (Benkö et al., 2000;Wold & Horwitz, 2007). Adenoviral conjunctivitis is mainly caused by HAdV-3 (in HAdV-B), HAdV-4 (in HAdV-E), and HAdV-8, , with the three HAdV-D serotypes being known to cause epidemic keratoconjunctivitis (EKC). HAdV-8 is the original causative agent of EKC and remains the predominant HAdV serotype isolated in association with EKC in many countries (Ishii et al., 1987;Chang et al., 2001;Vainio et al., 2001;Aoki & Tagawa, 2002; Jin et al., 2006). In Japan, although HAdV-8 and HAdV-19 have been described, a novel serotype of HAdV recently isolated from EKC patients and HAdV-37 are the predominant causative serotype of EKC in Japan (Higuchi et al., 1987;Aoki & Tagawa, 2002;Ishiko et al., 2008;Kaneko et al., 2008).Nucleotide polymorphisms in HAdV strains isolated from EKC patients can be classified into discrete genotypes within a specific HAdV serotype on the basis of their restriction endonuclease cleavage pattern (Wadell et al., 1980;Adrian et al., 1986). It has been speculated that the appearance of new genotypes might contribute to the incidence of outbreaks of each serotype (Aoki & Tagawa, 2002;Ariga et al., 2005). DNA sequence analysis has allowed us to appreciate the molecular evolution of HAdV in greater detail, and revealed that the penton, hexon and fiber genes were the most variable among the different serotypes (Pring-Akerblom & Adrian, 1995;Arnberg et al., 1997;Ebner et al., 2005;Madisch et al., 2005Madisch et al., , 2007 MiuraOchiai et al., 2007). The complete genome sequences of 24 HAdV serotypes 2, 3, 4, 5, 7, 9, 11, 12, 14, 16, 17, 19, 21, 26, 34, 35, 37, 40, 41, 46, 48, 49 and 50) have now been determined.In this study, we describe the complete genome sequences of the prototype strains HAdV-8p, HAdV-19p and HAdV37p together with a novel HAdV serotype and eight clinical
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65°C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV) are alphaherpesviruses that infect, establish lifelong latency in, and subsequently reactivate from human sensory neuronal ganglia (1,20). The reactivation of a latent HSV or VZV infection can occur spontaneously or in association with physical or emotional stress and immune suppression. Following reactivation from latent ganglion reservoirs, each of these herpesviruses may cause significant clinical symptoms in the individual and may spread to uninfected persons. Symptomatic VZV reactivation is an infrequent, usually once-in-a-lifetime event in 10% of the population that results in zoster (shingles), while HSV-1 and HSV-2 reactivation occurs frequently and results in numerous symptomatic and asymptomatic recurrences.HSV-1 and HSV-2 infections and even some VZV infections cause similar clinical symptoms, for example, cutaneous vesicles, keratitis, and acute retinal necrosis (ARN), and the causative agent cannot be distinguished based on the clinical features. However, different clinical courses and prognoses are caused by each virus. HSV-2 genital herpes tends to recur, but HSV-1 genital herpes does not (7). VZV ARN is severer and has a worse prognosis than HSV ARN (4). The identification of the virus is important for the determination of treatment and for an understanding of the clinical progress and prognosis; therefore, an effective laboratory method is urgently needed for the diagnosis of HSV-1, HSV-2, and V...
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.
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