The hepatitis E virus (HEV) is an emerging zoonotic agent, for which pigs are the most important reservoir. Complete genome sequences of two swine HEV strains, designated swKOR-1 and swKOR-2, were determined via RT-PCR and RACE-PCR. The strains contained genomes composed of 7,222- and 7,221-bp excluding the poly(A) tails, respectively. The swKOR-1 and swKOR-2 strains were classified into subtype 3a of genotype 3 via phylogenetic analysis. These strains formed a distinctive cluster in the phylogenetic tree with human and swine HEVs isolated in the USA and human HEVs isolated in Japan. Anti-HEV antibodies were identified via ELISA in 8 of 99 (8.1%) cats, whereas, among 115 cattle and 213 dogs, no HEV-specific antibodies were detected. The conserved RNA-dependent RNA polymerase (RdRp) gene of HEV could be detected via RT-PCR in 8.7% of raw oysters collected from coastal regions in Korea. The HEV RNAs detected in oysters were identified as belonging to subtype 3a. The HEV RNAs in oysters most closely resembled that of the swKOR-2 strain. They also showed a close genetic relationship with the swKOR-1 strain and the swine and human HEVs isolated in the USA. This is the first report describing the detection in oysters of HEV that may have originated from genotype 3 swine HEV in Korea. Pigs and cats infected with HEV, as well as oysters contaminated with HEV, are potential risk factors for HEV transmission to humans.

Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk out flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.

Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20-30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 μM of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 μM of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future.

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ABSTRACT. Hepatitis E virus (HEV) infection induces an acute hepatitis or a subclinical disease in humans. It is known that HEV is a zoonotic agent and pigs are major reservoirs of HEV. This study was conducted to determine the fecal shedding rates of HEV in various age groups of pigs and identify the genotypes of swine HEV prevailing in Korea. A total of 565 fecal samples were collected from suckling piglets, post-weaning pigs, growing pigs, and sows at 12 swine farms. RT-PCR was used to detect the presence of swine HEV in the feces. Every swine farm examined in this study had HEV-infected pigs. The fecal shedding rates of the swine HEV at individual farms were in the range of 2.1-35.4%. The overall fecal shedding rate of HEV in individual pigs was 17.5%. The HEV shedding rates of suckling piglets, post-weaning pigs, growing pigs and sows in their feces were 6.3, 16.3, 38.0 and 9.3%, respectively. When the genotypes of swine HEVs identified in this study were determined, they were all grouped into genotype 3. They were further subdivided into subtype 3a together with human and swine HEVs isolated in the U.S.A.