Lyu Jin Jun

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.

Characterization of the DNA nucleotide sequences in the genome of red sea bream iridoviruses isolated in Korea

Abstract The nucleotide sequences of DNA fragments amplified by polymerase chain reaction (PCR) from four different genomic regions of nine red sea bream iridoviruses (RSIVs) isolated from different species of fish, different areas and in different years in Korea were compared with the reported reference sequences. One isolate, RSIV Namhae, showed 100% homology to the reference sequences, while the other eight isolates, which appeared to contain identical nucleotide sequences, showed 96.6–98.9% homology with reference sequences depending upon the target regions of PCR gene amplification. However, differences in nucleotide sequences were not apparent between the RSIVs isolated in different locations, in different years or in different host species. We also cloned and sequenced the 3′ end flanking region (K1) of the DNA polymerase (DPOL) gene using the cassette ligation-mediated PCR method. This sequence was 4436-bp long and possessed two open reading frames (ORF-1 and ORF-2) oriented in opposite directions. The putative proteins encoded by these two ORFs could not be characterized by comparison with the proteins of other species in the data banks. The presence of the ribonucleotide reductase small subunit (RNRS) gene at the 3′ end of the K1 region allowed us to determine that these two genes, RNRS and DPOL, are separated 5508 bp and oriented in the same direction in the genome of RSIV. Moreover, it is of interest that a Pst I-restriction fragment, of which the sequence but not the location within the RSIV genome had previously been reported, is located at nucleotide positions from 1096 to 2054, extending from within the ORF-1 region, spanning the intervening sequence between ORF-1 and ORF-2, and extending into the ORF-2 region. Various repeating sequences up to 86 bp were present at the 3′ ends of ORFs, especially within the nucleotide sequences at the 3′ terminus of ORF-2. No similarities were detected when the DNA sequences of the K1 region were compared to the DNA sequences of a repetitive element in the genome of other iridoviruses.

Influence of temperature shifts on the onset and development of red sea bream iridoviral disease in rock bream Oplegnathus fasciatus.

The effects of various water temperature treatments on the development of red sea bream iridovirus disease (RSIVD) in rock bream Oplegnathus fasciatus challenged with iridovirus Sachun (IVS-1) were determined by measuring the mortality and the viral concentration in the spleen of infected fish. Experimental infections of rock bream with IVS-1 at water temperatures of 18, 21, and 25 degrees C resulted in a cumulative mortality of 100%, but infections at 13 degrees C resulted in 0% mortality, even after 45 d. The disease progressed more rapidly at higher water temperatures; at 25, 21, and 18 degrees C, the mean numbers of days until death were 17, 20, and 30 d, respectively. When the water temperature for fish infected with iridovirus by intramuscular injection was shifted from 13 to 25 degrees C, the cumulative mortality reached 100%, with rapid onset of the disease, independent of the time at which the temperature was shifted, i.e. 7, 14, or 30 d after injection at 13 degrees C. Real-time PCR data revealed that the viral genome copy number in the spleen of rock bream maintained at 13 degrees C increased with time, suggesting the occurrence of viral replication even at 13 degrees C. In the reverse experiment, when the water temperature for fish that were infected at a higher temperature was shifted to 13 degrees C, 3 or 7 d after injection at 25 degrees C, the fish showed 100% cumulative mortality, although the mean number of days until death was higher than that observed for fish maintained at a constant temperature of 25 degrees C. The viral DNA concentration in the spleen of rock bream that had been shifted down to 13 degrees C, 3 or 7 d after injection at 25 degrees C, was not suppressed, but increased and eventually reached levels sufficient to induce mortality at 13 degrees C. However, the level of viral genome copy numbers in the spleen of dead fish at 25 degrees C, regardless of whether those fish were held at a constant temperature of 25 degrees C or shifted up from 13 degrees C, appeared to be greater than the level found in the dead fish shifted down to 13 degrees C after inoculation at 25 degrees C.

Transmission of iridovirus from freshwater ornamental fish (pearl gourami) to marine fish (rock bream)

Freshwater pearl gourami Trichogaster leeri and seawater rock bream Oplegnathus fasciatus infected by the iridoviruses PGIV-SP and IVS-1 were carrying similar numbers of viral particles (2.52 x 10(8) and 2.46 x 10(8) viral genome copies mg(-1) spleen tissue, respectively). The viral genome copy number for both iridoviruses decreased much faster in seawater than in freshwater, reaching a concentration of less than 0.5%, versus 26 to 54% in freshwater, after 4 d of incubation at 25 degrees C. The decrease in copy number altered the infectivity of the viruses, as reflected by the decreased cumulative mortality of rock bream injected intraperitoneally with the incubated iridoviruses. Moreover, uninfected rock bream cohabitated with PGIV-SP-challenged rock bream showed 100% cumulative mortality; a similar experiment using IVS-1 had the same result, implying the potential for iridoviral transmission from freshwater ornamental fish to marine fish even in a marine environment. Of 58 outwardly healthy marine fish groups collected from various markets, 2 rock bream groups and 1 sea perch group Lateolabrax sp. tested positive for PGIV-SP by 2-step polymerase chain reaction (PCR). Thus, PGIV-SP from freshwater ornamental fish may have crossed both environmental and species barriers to infect marine fish such as rock bream.

Monitoring of VHS and RSIVD in Cultured Paralichthys olivaceus of Jeju in 2015

In this study, disease surveillance was performed to monitor the prevalence of viral haemorrhagic septicaemia virus (VHSV) and red seabream iridovirus (RSIV) in olive flounder, Paralichthys olivaceus in 2015. The fish samples were collected in March (60 farms), May (55 farms) and July (52 farms) from different farms in Jeju. Reverse transcription polymerase chain reaction (RT-PCR) (VHSV) or PCR (RSIV) results showed that VHSV detected in 2 farms, but RSIV has not been detected in any farms. The sequences of the nucleocapsid protein (N) and gly-coprotein (G) gene of the 2 VHSV isolates were successfully sequenced. Phylogenetic analysis was included VHSV isolates reported here together with a representative VHSV isolates available in GenBank. Phylogenetic analysis in-dicated that most of Korea VHSV isolates were closely related to the Japan and China genotype Ⅳa which is clearly distinct from the North American genotype Ⅳb.

Monitoring of Emaciation Disease in Cultured Olive Flounder Paralichthys olivaceus in Jeju (2010-2013), Korea

1990년대부터 각종 해수어의 양식이 활발하게 이루어져 각국 에서 종묘 수입이 성해지자 방역체계가 미비한 상태에서 수입 종묘와 같이 질병도 함께 도입되어 우리나라에 없었던 질병들 이 유행하기 시작하였으며(Chun, 2006), 질병의 발병 양상 또 한, 양식 초창기에는 고수온기에 기생충 및 세균에 의한 단독 감 염이 주를 이루었으나 최근에는 수온과 상관없이 연중 다양한 병원체가 혼합감염의 형태로 질병을 일으키고 있어 수산 생물 의 대량 폐사를 유발시키기도 한다(Kim et al., 2006). 최근 들어 외래 질병의 유입 가능성이 증가하고 양식 어류의 질병 연관성 에 대한 관심이 증가하면서 질병을 전반적으로 모니터링하거나 질병과의 상관관계를 구명하고자 하는 연구가 점차 증가하고 있는 추세이다(Cho et al., 2009; Jung et al., 2012; Song et al., 제주의 양식 넙치(Paralichthys olivaceus)를 대상으로 한 여윔증 모니터링(2010-2013)

Increased Resistance to Quinolones in Streptococcus parauberis and Development of a Rapid Assay for Detecting Mutations in Topoisomerase Genes

To investigate the acquisition of quinolone resistance, we examined mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase genes in ciprofloxacin (CIP)-resistant clinical isolates and in vitro mutants of Streptococcus parauberis. The CIP-resistant clinical isolates had one base change responsible for a Ser-79→Thr in the QRDR of parC. However, the CIP-resistant in vitro mutants had an altered QRDR of parC (Ser-79→Ile) that differed from that of the isolates. None of the CIP-resistant S. parauberis clinical isolates or in vitro mutants exhibited amino acid changes in gyrA or gyrB. However, even though involvement in the increased resistance was not clear, an Arg-449→Ser mutation outside of the QRDR of parE was detected in CIP-resistant mutant 2P1. These results suggest that the topoisomerase IV gene, parC (and possibly parE, as well), is the primary ciprofloxacin target in S. parauberis. Additionally we established a high-resolution melting (HRM) assay capable of detecting the dominant mutation in four type II topoisomerase genes conferring ciprofloxacin resistance. These rapid and reliable assays may provide a convenient method of surveillance for genetic mutations conferring antibiotic resistance.