Abstract: Background and Objective: Koi Herpesvirus (KHV) can infect some species of freshwater fish, other than Cyprinidae. As well this possibility, in which tilapia may be infected with KHV, this study aimed to detect KHV from tilapia in Gresik, East Java, Indonesia using clinical and molecular study. Methodology: The study was carried out by examining clinical symptoms of tilapia and PCR examination followed by a sequence analysis and then aligned with BioEdit and the construction of phylogenetic trees was determined using the Neighbors-Joining. Results: The clinical symptoms observed from tilapia were discoloration on the skin and white patches on the gills. A sequence analysis using TK gene and PCR duplex analysis (marker I and marker II) indicated that the KHV genotype of the tilapia was identical to the A1 variants and displayed allele I++ II+ allele of the Asian genotype. Conclusion: The genetic variations of KHV observed from tilapia clearly indicated that the KHV genotype infecting tilapia is an Asian genotype. However, although tilapia was infected with KHV, no specific apparent of clinical symptoms and no mortality was seen in tilapia.
INTRODUCTION
Koi Herpesvirus (KHV), also known as Cyprinid herpesvirus 3 (CyHV-3) is one of the viruses that attack koi and carp1 and infect any sizes of fish2. The KHV was first identified in 1998 in Israel and caused mass mortality of koi in both juvenile and adult stages3. In the Asian regions, KHV infection causes mortality in common carp and koi, in Japan, China, Taiwan, Indonesia, Korea, Malaysia, Singapore and Thailand4-6. The KHV entered Indonesia in 2002 through koi imported from China to Surabaya6. This virus resulted in mass of 80-95% of the population and material losses resulting from the disease reached IDR 15 billions at the end of December6 2003. In addition, KHV also attacked carp cultivated at Lake Kasumigaura with a total loss of 1,200 t or close to 20% of annual production in Japan7. Clinical symptoms of KHV are highly variable and generally non-specific. Symptoms found include fish swimming to the surface to take the air or gasping because of high respiratory frequency. In addition, fish often lose sight and swim with irregular movements8,9, suffer from hepatic dysfunction and osmoregulation systems that are susceptible to secondary pathogen infection2,10.
The KHV can infect freshwater fish other than Cyprinidae, which is believed to be the specific host for KHVD. This allows the tilapia as the KHVD host as well11,12. However, previous studies did not specify that tilapia could be artificially infected by KHV either through injection or oral application2,13,14, several studies have proven that fish other than Cyprinidae is susceptible to KHV infection3,10,15,16. In addition, hybrids are also known to be susceptible to KHV1,17. The KHV can be subdivided into European and Asian genotype or variants and variant of KHV in Indonesia more closely to Asian genotype18. In addition, based on epidemiological investigations, the spread of KHV in Indonesia originated from a single introduction19.
The data was obtained from these results i.e., clinical symptoms and genetic variation can be used as a reference if there are similar infections emerged in other freshwater fish, especially in Indonesia. The prevention and control of KHV distribution in Indonesia has been pursued in both host and non-host. The fact that tilapia may become a KHVD host (or as a carrier) and that there has not been any clarity about the clinical symptoms and genetic variation of KHV-infected tilapia has urged for efforts to KHV disease managements. Thus, this study will report the detection and characterization of KHV DNA in tilapia based on clinical symptoms and molecular assay followed by a sequence analysis.
MATERIALS AND METHODS
Sample collection: The research was conducted between March and August, 2018 in Indonesia. Samples were obtained from Gresik, East Java, where reports on mortalities of carp kept together with tilapia occurred. The carp and tilapia were cultivated for 1 month as reported by the fish owner. Water quality parameters (pH 6, 8; temperature 28°C; DO 4 mg L–1; nitrate 0,2 mg L–1; nitrite 0,1 mg L–1 and ammonia 0,5 mg L–1) showed normal results. Tilapia was grouped into two in a plastic tank and packed using a special sample plastic with oxygen supply and taken to Fish Quarantine Laboratory for dissection on a sterile condition. The dissected organ was taken and kept at -70°C for DNA extraction.
DNA extraction: Total DNA was extracted from the organs according to the procedure of the DNA Easy Blood and Tissue kit (Qiagen). As much as 25 mg of organ was crushed using pellet pestle and homogenized in 180 μL of an AL buffer using a microtube and digested in 20 μL proteinase K at 56°C for 10 min. Then 200 μL of ethanol (100%) and vortex was added to the crushed organ. Then, it moved the supernatant into a new tube, centrifuged it at 8000 rpm for 1 min, took it, then we added 500 μL of buffer AW1 and centrifuged it again at 8000 rpm for 1 min. The step continued as we took supernatant and added 500 μL buffer AW2 and centrifuged it at 14000 rpm for 3 min to dry the DNeasy membrane. The last step was added 200 μL of AE buffer directly onto the DNeasy membrane and incubate it for 1 min at room temperature before centrifuged at 8000 rpm for 1 min. The total DNA produced was dissolved in 50 μL of the AE buffer and stored at -20°C for further process.
DNA amplification and genotype analysis: The KHV DNA amplification was done using the primers specific of TK gene20, Forward (F: 5'-GGG TTA CCT GTA CGA G-3) and Reverse (R: 5'-CAC CCA GTA GAT TAT GC-3'), which would produce amplification products at 409 bp. Amplification was performed using PCR Mix Kappa (Biosystems) with a total volume of 25 μL. Thermal cycler personal (Eppendorf) was used with a temperature setting: 1 cycle at 95°C for 5 min; 35 cycles at 95°C for 30 sec, 52°C for 30 sec and 72°C for 1 min and 1 cycle at 72°C for 10 min.
The genotype analysis was performed on tilapia, which gave positive signals of KHV. Four pairs of primers were used to determine the relationship between KHV in tilapia and other KHV variants21,22. The primer sets used for KHV genotype analysis is presented in Table 1.
| Table 1: | Primer sets and thermal cycling profiles used for KHV genotype analysis |
The amplification product was then electrophoresis in 2% (w/v) agarose gel (Scie-Plus) using 1x TAE buffer (1st Base) for 1 h with 100 V voltage and followed by Sybr Green (Lonza) staining. The DNA signals were visualized using Gel-Documentation (Uvitech). In addition, amplification products were also purified using DNA Clean and Concentrator (Zymo Research) to be used as template sequencing. Sequencing was done at 1st Base Laboratories-Selangor, Malaysia. Sequencing data were analyzed using sequence of BioEdit and NCBI-BLAST program. The construction of phylogenetic trees was determined using the Neighbors-Joining method23.
RESULTS
Clinical symptoms: In the external part of the tilapia, there were no parasites and pathological lesions but it showed the discoloration of the skin (darker skin) when compared with the normal tilapia. While on the internal organs, gill showed the presence of white parts on the ends of the gill (Fig. 1).
KHV detection: The KHV testing was performed on tilapia that showed clinical symptoms such as white parts on the gills and the blackish skin color. The positive signals indicated KHV infection in tilapia by using TK genefor both of 2 tilapia groups (Fig. 2).
As mentioned earlier, 11 published genomic sequences show high sequence analysis24-26, i.e., >99%. In addition, to determine the genotype of tilapia21, primers from Bigarre et al.22 were also used, based on differences in three variable domains within two genetic marker. Marker I (the region between ORF 29 and ORF 30) and Marker II (the regionnear to the start of ORF 133) (Fig. 3).
Figure 3a showed both of 2 tilapia groups amplified using enlarged TK gene at 1.001 bp, while Fig. 3 Bamplifiedin the marker I region displayed allele I++ with the full length 168 bp and marker II region showed allele II+ with fulllength 352 bp. These suggested as allele I++ II+, corresponding to Asian genotype.
| Fig. 1(a-b): | (a) Blackish color of tilapia and (b) White parts on the gill |
| Fig. 2: | TK gene PCR assay of tilapia |
| M: Marker 100 bp, 1: Positive control KHV, 2: Tilapia group I, 3: Tilapia group II | |
Results of sequencing analysis and alignment: Multiple sequence alignment based on enlarged TK gene in Fig. 4 showed that the tilapia had a high similarity with variant A1 of J strain (Asian genotype). Consistent with a previous study, all TK gene sequences clustered into two major genotype: European genotype and Asian genotype21.
| Fig. 3(a-b): | (a) Enlarged TK Gene PCR assay of tilapia. Lane 1: Tilapia group I, 2: Tilapia group II, 3: 100 bp DNA ladder and (b) PCR assay targeting KHV marker I and marker II. Lane 1: 100 bp DNA ladder, 2: Tilapia group I, 3: Tilapia group II |
| Fig. 4: | Based on the alignment of the enlarged TK gene, the tilapia (NBJT_1) is homologous with A1 variant (J strain). Alignment of marker I and marker II of tilapia (NBJT_1) were classified as variant A1 of J strain and displayed the I++ II+allele. The insertion of a nucleotide base A positioned at 161 bp at Marker II |
Compared with three KHV strain, demonstrate that tilapia were identical as variant A1 of J strain. It can be seen that in Marker I, the tilapia has a 168 bp nucleotide length and can be denoted as I++. For Marker II, the tilapia has a length of 353 bp, which can be denoted as II+, identical to the J strain. Although it looks identical to J strain, in marker II, there is an insertion of base A (Adenin) at 161 nt in the middle part of the second motif.
The phylogenetic tree construction was done using a neighbor joining method in MEGA 7 software, after alignment using BioEdit software. The construction of the phylogenetic tree (Fig. 5) showed that the tilapia (code NBJT_1) were in one cluster with the J-strain isolate and the A1-A2 variant, which is a cluster of the Asian genotype. These results similar with previous findings, all TK gene of KHV isolates from Asia have a high homology with each other and there is only 1 nucleotide difference with polymorphisms at 885 nt, although the KHV-infected tilapia in this study showed no such differences21,27.
| Fig. 5: | Phylogenetic tree of KHV-infected tilapia (NBJT_1) with 7 comparative sequences (sequence analysis of TK Gene) |
DISCUSSION
This study described the detection of KHV that emerged in tilapia from Indonesia. The results of the present clinical and molecular examination confirmed that KHV is the causative agent. Although tilapia was infected with KHV, it did not show clinical symptoms and no mortality was seen in tilapia. These finding suggested that KHV can infect tilapia but could not cause death. Bergmann et al.1 also suggested, no clinical symptoms in goldfish (Carassius auratus) infected with KHV. The clinical symptom in the tilapia was only white parts on the gillsindicating the necrosisand skin discoloration. Skin discoloration in tilapia may be due to the proliferation of Melano-Macrophage Centers (MMC) that indicated a defense reaction in fish skin caused by environmental stress conditions. Changes that occur in the environment cause an increase in damaged red blood cells, which ultimately results in changes in pigment28. Changes in tilapia skin color, which becomes darker, is also possible because of an infection of the skin, which results in infiltration of inflammatory cells that extend to the epidermal layer of the body. These finding also suggested that KHV enter through the gills andmultiply. From the gills, the virus is rapidly transferred to the other tissues9.
This present study has found that KHV in the tilapia belongs to the variant A1 of J strain, this resultis similar with previous finding that KHV variants in Indonesia were more closely related to Asian genotype18. This is consistent with previous studies suggesting that all isolates from the Asian region have high homology with only one nucleotide polymorphism that distinguishes A1 and A2 variant based on TK gene analysis21. Based on duplex analysis, it was found that KHV genotype of tilapia identical and displayed the I++ II+ allele. Although at Marker II there is an insertion of base A (Adenine) at 161 nt, this is more related to slippage of the DNA polymerase. These results are similar to the study conducted by Sunarto et al.18, which also found the insertion of base G (Guanine) at 74 nt for KHV originating from Indonesia. In addition, marker I and II can be used to distinguish variants in genetic KHV polymorphisms but it is still unable to inform us about the virulence of KHV or other viruses. This can be proven in such case-although the A1 and A2 variant contains single nucleotide polymorphism, it cannot determine which variants are more virulent. The homogeneity of KHV isolates from Asia showed that KHV invades to this region and adapts to the cooler European region29. This affects the proximity of KHV isolates in the Asian region and gives rise to fewer variations-unlike the KHV isolates derived from European region.
The KHV genotype of tilapia in this study is consistent with the assumption that KHV from Asia are related to each other18,27. However, not all KHV isolates from the Asian regions are included in the Asian genotype. Dong et al.30 isolated KHV from koi, which was designated as KHV-GZ11 and suggested that the KHV outbreak in China in 2011 is caused by KHV belonging to the European lineage; based on the phylogenetic position, KHV GZ-11 is an intermediate variant, which is between the Asian and European cluster27. In addition, European genotype is also found in South Korea27,31. The European genotype found in the Asian regions is believed to be an intermediate variant between Asian and the European genotype. The KHV isolates originating from Indonesia are also believed to be intermediates18. The existence of this intermediate variant is important because it informs us the origin of this KHV. There is no linkage in terms of the spread between Asian and European genotype; this may occur due to koi trading activities from Asia to Europe, which indirectly also causes the spread of the KHV variant in Europe21. To clarify whether the Indonesian KHV from tilapia in this study is an intermediate variant, a complete genome sequence is required as conducted by Li et al.25.
The distribution pattern of KHV variants in some countries is not known clearly; this may be due to international trade involving koi as KHV hosts. Although the KHV variants showed clear geographical boundaries based on molecular markers, the distribution pattern remains unclear. Ongoing detection and monitoring is urgently needed to determine the current condition associated with the presence of KHV in the tilapia and the possibility of KHV to infect other freshwater fish outside of the specific host, as we know that the evolutionary of KHV to infect the other fish is not known yet. Implementation of strict biosecurity and quarantine measures is also indispensable especially to prevent the spread of this disease to all province of Indonesia.
CONCLUSION
This study confirmed the presence of KHV in the tilapia. The fact that KHV can infect the tilapia-as a carrier-without clinical symptoms will make difficult to identify. The KHV genotype of tilapia in this study is consistent with the assumption that KHV isolates from Asia are related to each other. It also recommend avoiding carp cultivation together with other freshwater fish.
SIGNIFICANCE STATEMENTS
This study discovered the clinical symptoms and genetic variation of KHV infected tilapia that can be beneficial for both researchers and policy makers. This study will help the researchers to uncover the critical areas of tilapia infected with KHV that many prior researchers were not able to explore. Thus, a new theory on possibility of other species to KHV may be found.
ACKNOWLEDGMENT
The author would like to thank the Ministry of Fisheries and Marine Affair for financial support in the payment of the Article Processing Charge from Decree of Ministry of Fisheries and Marine Affair No. 101/SJ/KP.530/III/2016.