- Email: [email protected]
Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis virus (IHHNV)
Journal of Virological Methods 224 (2015) 16–19
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis virus (IHHNV) Yong-Wei Wei a , Dong-Dong Fan a , Jiong Chen a,b,∗ a b
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China The Donghai Sea Collaborative Innovation Center for Industrial Upgrading Mariculture, Ningbo University, Ningbo 315211, China
a b s t r a c t Article history: Received 10 March 2015 Received in revised form 5 August 2015 Accepted 11 August 2015 Available online 14 August 2015 Keywords: IHHNV Cloning Genome China Molecular variation
Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes shrimp diseases worldwide. However, a rapid method for cloning full-length IHHNV genome sequences is still lacking, which makes it difficult to study the genomics and molecular epidemiology of IHHNV. Here, a novel and rapid PCR technique was developed to determine the complete genomic sequences of IHHNV. The IHHNV genome was amplified in two overlapping fragments which each yielded a 2 kb PCR product covering the first half or the second half of IHHNV genome, respectively. Using this method, six complete genomic sequences of IHHNV, which were collected from different regions of Zhejiang province in China, were cloned and sequenced successfully. The new cloning method will greatly facilitate the study on the genomics and molecular epidemiology of IHHNV. © 2015 Elsevier B.V. All rights reserved.
Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes disease in shrimps. Since the first isolation of IHHNV in Hawaii in the early 1980s (Lightner et al., 1983a,b), the virus has become one of the most prevalent pathogens in the shrimp industry globally (Dhar et al., 2014; Rai et al., 2012; Vega-Heredia et al., 2012). IHHNV infection causes high mortalities up to 90% in cultured Litopenaeus stylirostris postlarvae and juveniles (Lightner et al., 1983a,b). It has been reported that IHHNV does not cause mortality in Litopenaeus vannamei or Penaeus monodon, but causes runt deformity syndrome (RDS) in L. vannamei (Browdy et al., 1993; Kalagayan et al., 1991; Primavera and Quinitio, 2000; Withyachumnarnkul et al., 2006). IHHNV belongs to the family Parvoviridae and is a singlestranded DNA virus (3.9–4 kb) with three open reading frames, encoding the nonstructural protein 1 (NS-1), non structural protein 2 (NS-2) and capsid protein (CP), respectively (Rai et al., 2012). Until now, a number of studies have been carried out to detect IHHNV infection using genome cloning approaches
∗ Corresponding author at: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China. Tel.: +86 574 87609571; fax: +86 574 87600167. E-mail address: [email protected] (J. Chen). http://dx.doi.org/10.1016/j.jviromet.2015.08.004 0166-0934/© 2015 Elsevier B.V. All rights reserved.
(Encinas-Garcia et al., 2015; Krabsetsve et al., 2004; Mrotzek et al., 2010; Nunan et al., 2000; Silva et al., 2014; Saksmerprome et al., 2010; Xia et al., 2015; Yu et al., 2011). However, these strategies are primarily used to clone short genomic fragments mostly ranging only from 300 to 1000 bp (Kim et al., 2012; Rai et al., 2011; Shike et al., 2000). Furthermore, in recent years, it has been reported that partial non-infectious IHHNV sequences are present in the genome of L.vannamei and P. monodon from Thailand (Saksmerprome et al., 2011), India (Rai et al., 2009), East Africa and Australia (Tang and Lightner, 2006). Therefore, the small fragment cloning methods may yield false positive results since positive fragments may be amplified from these IHHNV sequences inserted in the shrimp genome instead of the IHHNV itself. To distinguish infectious IHHNV and inserted IHHNV-related sequences, specific PCR primers (named IHHNV309F/R) and nested PCR (named IHHNV648F/R and IHHNV309F/R) have been developed (Rai et al., 2009; Tang et al., 2007). However, a study of Saksmerprome’s group showed that the position and length of the IHHNV-related sequence insertions in shrimp stocks are random, and the primers mentioned above may lead to false positive results (Saksmerprome et al., 2011). As an alternative, a method which covers most of the regions in IHHNV genome (positions nt 158–3625) was developed to distinguish between viral infection and inserted sequences. Unfortunately, this method is relatively complicated since it requires 7 pairs of primers for 7 PCR reactions, and does not cover the
Y.-W. Wei et al. / Journal of Virological Methods 224 (2015) 16–19
1
2000
17
3909
IHHNV-1942F
Step1
5'
Genome of IHHNV
3' IHHNV-3909R
Step 2
IHHNV-1F
5'
Genome of IHHNV
3'
IHHNV-2020R
IHHNV-Ex
Step 2'
IHHNV-Linker
5'
3'
Genome of IHHNV IHHNV-2020R
Fig. 1. Cloning strategy for the full-length genome of IHHNV. Step 1: PCR amplicons using primers IHHNV-1942F/IHHNV-3909R. Step 2: PCR amplicons using primers IHHNV-1F/IHHNV-2020R. Step 2’: PCR amplicons using primers IHHNV -Ex, IHHNV-Linker and IHHNV-2020R. During the PCR amplification in step 2’, the first cycle of PCR using linker primer created a new template with extension sequence at 5 end of IHHNV genome sequence. These extensions subsequently allow the binding of the highly specific extension primer to the new template. After that, the target sequence could be amplified using the extension primers.
full-length genome of IHHNV (Saksmerprome et al., 2011). Therefore, a simple, efficient and convenient method is required for cloning the full-length sequence of IHHNV. In this study, a novel and simple strategy for cloning the complete genomic sequences of IHHNV was developed. Samples of L. vannamei, P. monodon, Fenneropenaeus chinensis, Macrobrachium rosenbergii and Marsupenaeus japonicus were collected from the hatcheries and culture systems from Ningbo, Wenzhou, Jianxing Zhoushan regions in Zhejiang Province of China in 2014 and kept in −80 ◦ C freezers. For each sample, 30 mg of gills and pleopods were taken, mixed, and homogenized. Then, these homogenized tissue samples were incubated at 65 ◦ C for 4 h in 20 mM Tris–HCl (pH 8.0), 10 mM EDTA, 0.1% sodium dodecyl sulfate (SDS) and 0.8 mg/ml proteinase K. The DNA was then extracted using a DNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. The cloning procedure was carried out in 2 steps separately (Fig. 1). Primers were designed based on the strain of Hawaii (GenBank no. AF218266) and listed in Table 1. Two pairs of primers, IHHNV-1942F/IHHNV-3909R and IHHNV-1F/IHHNV-2020R were used to clone the genome of IHHNV (step 1 and step 2). PCR was performed in 0.2 mL tubes with a final volume of 25 L containing 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 mM dNTP, 0.01% gelatine, 2.5 U of Taq DNA polymerase (Boehringer Mannheim), 10 ng of IHHNV positive genomic DNA template and 1 mM primers of IHHNV-1F/IHHNV-2020R or IHHNV1942F/IHHNV-3909R. The amplification was performed for 35 cycles with denaturation at 94 ◦ C for 30 s, across an annealing temperature gradient at 50 ◦ C, 52 ◦ C, 54 ◦ C, 56 ◦ C, 58 ◦ C or 60 ◦ C for 30 s, and elongation at 72 ◦ C for 1 min with an increment of 2 min after 35 cycles in a gradient thermocycler (Eppendorf Mastercycler pro). Agarose gel analysis of PCR products indicated that single bands were clearly visible when the annealing temperatures were below 58 ◦ C for the IHHNV-1942F/3909R primer set, but there were not any visible bands in the assay using the IHHNV-1F/2020R primer set. The efficiency of PCR can be affected by many other Table 1 Primers used for amplifying the full-length sequences of IHHNV. Primer name
Nucleotide sequence (5 -3 )
IHHNV-1F IHHNV-2020R IHHNV-1942F IHHNV-3909R IHHNV-Linker IHHNV-Ex
5 -TAGAGCGCGAAGCGCGAGTATC-3 5 -GCATATTGTCGTAGTCTGGT-3 5 -GTCACTAATTACAAACCTGCAG-3 5 -CTTCGCAGAAACCGTTAAC-3 5 -GGTACACTGACATGCTGACTTAGAGCGCGAAGCGCGAG-3 5 -GGTACACTGACATGCTGACT-3
Note: The extended sequence in primer IHHNV-Linker was underlined.
factors such as the working concentrations of primers, template and magnesium, buffer ingredients, or the time and temperature of denaturation. All those have been taken into account by optimization of the reaction conditions. However, the fragment which covers the genome of IHHNV from 5 end to nt 2020 was failed to get in multiple attempts by optimizing various parameters. To circumvent this problem and obtain the complete nucleotide sequence of IHHNV, a novel strategy for cloning the upstream fragment of IHHNV genome was employed. Firstly, one linker primer at the 5 end of IHHNV genome was designed, and one highly specific extension primer with the same sequence that was added in the linker primer was synthesized (Table 1). Three primers IHHNV-Ex, IHHNV-Linker and IHHNV-2020R were used to clone the fragment which covers the first half of IHHNV genome (step 2’). Ten 0.2 mL PCR reaction tubes were prepared in a volume of 24 L containing all components as described as above, and 1 mM primers of IHHNV-Linker and IHHNV-2020R. Finally, 1 L of primer different concentrations of IHHNV-Ex were added to each tube (the final concentration ratios between IHHNV-Ex and IHHNV-Linker are 1:1, 1:10, 1:25, 1:50, 1:100, 1:500 and 1:1000). The amplification was performed for 35 cycles with denaturation at 94 ◦ C for 30 s, annealing temperature at 54 ◦ C for 30s, and elongation at 72 ◦ C for 1 min with an increment of 2 min after 35 cycles in a thermocycler. Agarose gel analysis indicated that relatively higher yields of PCR amplicons were detected when the IHHNV-Ex/IHHNV-Linker primer ratios were between 1:25, 1:50 and 1:100 at annealing temperature of 54 ◦ C. No bands were observed when this primer ratio was under 1:20 or above 1:200. PCR fragments with the expected size were extracted from 1% agarose gels and were purified using a DNA purification kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. These purified PCR products were cloned into a pMD18-T vector (TaKaRa, Dalian, China) according to the manufacturer’s instructions to yield DNA template suitable for IHHNV genome sequencing. Sequence analysis (Beijing Genomics Institution, Beijing, China) using three independent positive clones confirmed that the IHHNV genome had been successfully cloned. To determine the analytical sensitivity of the cloning method, PCRs were performed as described above using plasmid DNA containing IHHNV genome, which was serially diluted tenfold in ddH2 O ranging from 100 ng (100 ng/l) to 0.1 fg (0.1 fg/l). These results showed that the detection limit was 1 fg/l (20 copies/l) for the IHHNV-1942F/3909R primer set, and 0.01 ng/l (2 × 105 copies/l) for IHHNV-Linker/Ex/2020R set. These PCRs were routinely used to amplify the target sequences of IHHNV spiked in DNA extracted from L. vannamei, P. monodon, F. chinensis, and M. japonicus, to evaluate the broad-spectrum use
18
Y.-W. Wei et al. / Journal of Virological Methods 224 (2015) 16–19
Ningbo
55 51 45
Wenzhou Jiaxing
52
KF214742
91
Zhoushan
57
JX258653 JN377975
61 57
Lineage III
AY362548 JX840067 AF218266
10045
EF633688
47
AY355308
78
100
AF273215 GQ411199 100
KF031144
100 100 89
Ningbo-22
Lineage II
Ningbo-36
Lineage I
GQ475529 AY124937
Non-infectious type B EU675312 100
DQ228358
Non-infectious type A
0.005 Fig. 2. Phylogenetic analysis of full-length sequences of IHHNV. Sixteen full-length (and nearly full-length) sequences from GenBank of IHHNV were included in the analysis. The sequences were aligned with Clustal W 2.0 and the phylogenetic tree was then generated using the neighbor-joining method with 1000 bootstrap replicates using the MEGA 5.0 software program.
of this method. One microliter of these positive IHHNV DNA samples were mixed with DNA extracted from each kind of shrimp. PCRs were performed as described above using the DNAs mixture as template, and agarose gel electrophoresis showed strong PCR amplifications in all types of shrimp. These results indicated that the IHHNV detection method developed is not only suitable for L. vannamei, but also applicable in other kinds of shrimp. Furthermore, samples of L. vannamei and P. monodon were collected from hatcheries and culture systems from different regions in Zhejiang Province of China in 2014. DNA isolation and full-genome cloning of IHHNV were performed as described as above. Five more full-length genomes of IHHNV (three strains from L. vannamei and two strains from P. monodon) were cloned. All cloned sequences were deposited in the GenBank database (accession numbers for each isolate are KP733862 (Wenzhou), KP733861 (Jiaxing), KP733860 (Ningbo) KP733859 (Zhoushan), KP733858 (Ningbo-22) and KP733857 (Ningbo-36)). These sequences were aligned with 15 IHHNV sequences (five complete genomic sequences and ten partial sequences) from GenBank (Clustal W 2.0) and the phylogenetic tree (Fig. 2) was then generated using the neighbor-joining method with 1000 bootstrap replicates (MEGA 5.0 software program). The overall branching patterns of the phylogenetic tree constructed for the nucleotide sequence of IHHNV was split into three major distinct branches: one encompassing non-infectious IHHNV type A (EU675312 and
DQ228358), the other containing type B (AY124937) and the third containing infectious IHHNV strains. The infectious IHHNVs were split into three major lineages as follows: lineage I, lineage II, lineage III. The strains of Ningbo, Wenzhou, Jiaxing and Zhoushan strains formed a cluster with lineage III strains. However, Ningbo22 and Ningbo-36 strains formed a cluster with lineage II strains. Analysis of full-length genome sequence identity and evolutionary distance supported the observed tree topology. The deduced amino acid (aa) sequences of CP (329aa), NS1 (666aa) and NS2 (363aa) from the cloned IHHNV strains were aligned with other available CP, NS1 and NS2 sequences in GenBank. Sequence distances data derived from the multiple alignment of the CP revealed that CP of Wenzhou, Ningbo, Zhoushan and Jiaxing strains shared the highest aa identity to IHHNV strains of lineage III (98.2–99.7%), lower aa identity to strains in lineage I and lineage II (96.7–97.3%), and lowest aa identity to non-infectious IHHNV type A and type B (93.0–93.6%). For CP of Ningbo22 and Ningbo 36 strains, sequence distance revealed that Ningbo22 and Ningbo 36 strains shared more aa identity to lineage II KF031144 and GQ411199 strains (97.3–99.7%) than to other lineage strains (91.5–97.3%). Within the genome of IHHNV, two separate and partially overlapping ORFs encode non structural proteins NS1 and NS2. Multiple alignments revealed that NS1 and NS2 aa sequences of Wenzhou, Ningbo, Zhoushan and Jiaxing were closest to lineage III strains (97.3–99.8% identity). For NS1 and NS2 of Ningbo22 and Ningbo36, sequence
Y.-W. Wei et al. / Journal of Virological Methods 224 (2015) 16–19
distance revealed that Ningbo22 and Ningbo36 strains shared more aa identity to lineage II strains (98.9–99.2%) than to other strains. As shown in Supplementary Fig. 1 the 5 -NCR sequences of Wenzhou, Ningbo, Zhoushan and Jiaxing strains shared close identity, with three nucleotides substitutions at positions 142 (T/C for Zhoushan strain), 423 (G/A for Wenzhou strain) and 428 (C/T for Ningbo and Jiaxing strains). 3 -NCRs of Wenzhou, Zhoushan and Jiaxing strains were completely identical with each other. But Ningbo has two substitutions at position 3756 (C/T) and 3769 (A/C). 3 -NCRs of Ningbo22 and Ningbo 36 strains shared completely identical sequences with each other. The striking difference between these sequences for available strains was that both 5 and 3 -NCRs sequences were highly conserved within but not between genetic lineages. Wenzhou, Ningbo, Zhoushan and Jiaxing strains had a six nucleotide (GAAGCG) deletion at position 75, and JN377975 strain has a five nucleotide (TGCGA) insertion at position 77, KF031144 has one nucleotide (C) insertion at position 71 in 5 -NCR and one nucleotide (C) insertion at position 3901 in 3 -NCR. The reason of this difference is unclear yet. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2015.08. 004 Although IHHNV was discovered thirty years ago, cloning of full-length genomes of this virus has been limited to a multi-fragment cloning strategy (6–7 fragments) (Kim et al., 2012; Rai et al., 2011). In addition, it has been a challenge to work with this virus because of IHHNV-related sequence insertions in the genome of shrimp (Rai et al., 2009; Saksmerprome et al., 2011; Tang and Lightner, 2006). Because of the lack of a rapid cloning method of IHHNV, there were only five full-length genomes of IHHNV available in GenBank. Therefore, a new and reliable method that allows for rapid cloning of infectious IHHNV has been developed. Criteria for the design of this new method included sensitivity and simplicity and,also utility for a broad-spectrum of clinical specimens from a variety of shrimp species. Using this over lapping PCR and cloning method, six complete sequences of IHHNV from different regions of Zhejiang Province in China were successfully generated. This new method will facilitate the study of the genomics and molecular epidemiology of IHHNV. Acknowledgements This project was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA092001), the Educational Commission of Zhejiang Province of China (Y201430861), the Natural Science Foundation of Ningbo City of China (2014A610182), and the KC Wong Magna Fund in Ningbo University. We thank Yuanmei Ma for critical reading of the manuscript. References Browdy, C.L., Holloway, J.D., King, C.O., Stokes, A.D., Hopkins, J.S., Sandifer, P.A., 1993. IHHN virus and intensive culture of Penaeus vannamei: effects of stocking density and water exchange rates. Journal of Crustacean Biology 13, 87–94. Dhar, A.K., Robles-Sikisaka, R., Saksmerprome, V., Lakshman, D.K., 2014. Biology, genome organization, and evolution of parvoviruses in marine shrimp. Advances in Virus Research 89, 85–139. Encinas-Garcia, T., Mendoza-Cano, F., Enriquez-Espinoza, T., Luken-Vega, L., Vichido-Chavez, R., Sanchez-Paz, A., 2015. An improved validated SYBR
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green-based real-time quantitative PCR assay for the detection of the penaeus stylirostris densovirus in penaeid shrimp. Journal of Virological Methods 212, 53–58. Kalagayan, G., Godin, D., Kanna, R., Hagino, G., Sweeny, J., Wyban, J., Brock, J., 1991. IHHN virus as an etiological factor in runt deformity syndrome of juvenile Penaeus vannamei cultured in Hawaii. Journal of The World Aquaculture Society 22, 235–243. Kim, J.H., Kim, H.K., Nguyen, V.G., Park, B.K., Choresca, C.H., Shin, S.P., Han, J.E., Jun, J.W., Park, S.C., 2012. Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei. Archives of Virology 157, 369–373. Krabsetsve, K., Cullen, B.R., Owens, L., 2004. Rediscovery of the Australian strain of infectious hypodermal and haematopoietic necrosis virus. Diseases of Aquatic Organisms 61, 153–158. Lightner, D.V., Redman, M., Bell, A., 1983a. Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeid shrimp. Journal of Invertebrate Pathology 42, 62–70. Lightner, D.V., Redman, R.M., Bell, T.A., Brock, J.A., 1983b. Detection of IHHN virus in Penaeus stylirostris and Penaeus vannamei imported into Hawaii. Journal of The World Aquaculture Society 14, 212–225. Mrotzek, G., Haryanti, Koesharyani, I., Tretyakov, A.N., Sugama, K., Saluz, H.P., 2010. Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses. Journal of Virological Methods 168, 262–266. Nunan, L.M., Poulos, B.T., Lightner, D.V., 2000. Use of polymerase chain reaction for the detection of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp. Marine Biotechnology 2, 319–328. Primavera, H., Quinitio, E.T., 2000. Runt deformity syndrome in cultured giant tiger prawn Penaeus monodon. Journal of Crustacean Biology 20, 796–802. Rai, P., Balakrishnan, P., Muhammed, P.S., Karunasagar, I., Karunasagar, I., 2009. Simultaneous presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Type A virus-related sequence in Penaeus monodon from India. Aquaculture 295, 168–174. Rai, P., Safeena, M.P., Karunasagar, I., Karunasagar, I., 2011. Complete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV) from India. Virus Research 158, 37–45. Rai, P., Safeena, M.P., Krabsetsve, K., La Fauce, K., Owens, L., Karunasagar, I., 2012. Genomics, molecular epidemiology and diagnostics of infectious hypodermal and hematopoietic necrosis virus. Indian Journal of Virology: An Official Organ of Indian Virological Society 23, 203–214. Saksmerprome, V., Jitrakorn, S., Chayaburakul, K., Laiphrom, S., Boonsua, K., Flegel, T.W., 2011. Additional random, single to multiple genome fragments of Penaeus stylirostris densovirus in the giant tiger shrimp genome have implications for viral disease diagnosis. Virus Research 160, 180–190. Saksmerprome, V., Orapim, P., Noonin, C., Flegela, T.W., 2010. Detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) in farmed Australian Penaeus monodon by PCR analysis and DNA sequencing. Aquaculture 198, 190–193. Shike, H., Dhar, A.K., Burns, J.C., Shimizu, C., Jousset, F.X., Klimpel, K.R., Bergoin, M., 2000. Infectious hypodermal and hematopoietic necrosis virus of shrimp is related to mosquito brevidensoviruses. Virology 277, 167–177. Silva, D.C., Nunes, A.R., Teixeira, D.I., Lima, J.P., Lanza, D.C., 2014. Infectious hypodermal and hematopoietic necrosis virus from Brazil: sequencing, comparative analysis and PCR detection. Virus Research 189, 136–146. Tang, K.F., Lightner, D.V., 2006. Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus Research 118, 185–191. Tang, K.F., Navarro, S.A., Lightner, D.V., 2007. PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and virus-related sequences in the genome of Penaeus monodon. Diseases of Aquatic Organisms 74, 165–170. Vega-Heredia, S., Mendoza-Cano, F., Sanchez-Paz, A., 2012. The infectious hypodermal and haematopoietic necrosis virus: a brief review of what we do and do not know. Transboundary and Emerging Diseases 59, 95–105. Withyachumnarnkul, B., Chayaburakul, K., Lao-Aroon, S., Plodpai, P., Sritunyalucksan, K., Nash, G., 2006. Low impact of infectious hypodermal and hematopoietic necrosis virus (IHHNV) on growth and reproductive performance of Penaeus monodon. Diseases of Aquatic Organisms 69, 129–136. Xia, X., Yu, Y., Hu, L., Weidmann, M., Pan, Y., Yan, S., Wang, Y., 2015. Rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time, isothermal recombinase polymerase amplification assay. Archives of Virology 160, 987–994. Yu, X.W., Wang, J.P., Zhang, W., Shi, Z.L., 2011. Prevalence of three shrimp viruses in Zhejiang Province in 2008. Virologica Sinica 26, 67–71.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis virus (IHHNV) Yong-Wei Wei a , Dong-Dong Fan a , Jiong Chen a,b,∗ a b
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China The Donghai Sea Collaborative Innovation Center for Industrial Upgrading Mariculture, Ningbo University, Ningbo 315211, China
a b s t r a c t Article history: Received 10 March 2015 Received in revised form 5 August 2015 Accepted 11 August 2015 Available online 14 August 2015 Keywords: IHHNV Cloning Genome China Molecular variation
Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes shrimp diseases worldwide. However, a rapid method for cloning full-length IHHNV genome sequences is still lacking, which makes it difficult to study the genomics and molecular epidemiology of IHHNV. Here, a novel and rapid PCR technique was developed to determine the complete genomic sequences of IHHNV. The IHHNV genome was amplified in two overlapping fragments which each yielded a 2 kb PCR product covering the first half or the second half of IHHNV genome, respectively. Using this method, six complete genomic sequences of IHHNV, which were collected from different regions of Zhejiang province in China, were cloned and sequenced successfully. The new cloning method will greatly facilitate the study on the genomics and molecular epidemiology of IHHNV. © 2015 Elsevier B.V. All rights reserved.
Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes disease in shrimps. Since the first isolation of IHHNV in Hawaii in the early 1980s (Lightner et al., 1983a,b), the virus has become one of the most prevalent pathogens in the shrimp industry globally (Dhar et al., 2014; Rai et al., 2012; Vega-Heredia et al., 2012). IHHNV infection causes high mortalities up to 90% in cultured Litopenaeus stylirostris postlarvae and juveniles (Lightner et al., 1983a,b). It has been reported that IHHNV does not cause mortality in Litopenaeus vannamei or Penaeus monodon, but causes runt deformity syndrome (RDS) in L. vannamei (Browdy et al., 1993; Kalagayan et al., 1991; Primavera and Quinitio, 2000; Withyachumnarnkul et al., 2006). IHHNV belongs to the family Parvoviridae and is a singlestranded DNA virus (3.9–4 kb) with three open reading frames, encoding the nonstructural protein 1 (NS-1), non structural protein 2 (NS-2) and capsid protein (CP), respectively (Rai et al., 2012). Until now, a number of studies have been carried out to detect IHHNV infection using genome cloning approaches
∗ Corresponding author at: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China. Tel.: +86 574 87609571; fax: +86 574 87600167. E-mail address: [email protected] (J. Chen). http://dx.doi.org/10.1016/j.jviromet.2015.08.004 0166-0934/© 2015 Elsevier B.V. All rights reserved.
(Encinas-Garcia et al., 2015; Krabsetsve et al., 2004; Mrotzek et al., 2010; Nunan et al., 2000; Silva et al., 2014; Saksmerprome et al., 2010; Xia et al., 2015; Yu et al., 2011). However, these strategies are primarily used to clone short genomic fragments mostly ranging only from 300 to 1000 bp (Kim et al., 2012; Rai et al., 2011; Shike et al., 2000). Furthermore, in recent years, it has been reported that partial non-infectious IHHNV sequences are present in the genome of L.vannamei and P. monodon from Thailand (Saksmerprome et al., 2011), India (Rai et al., 2009), East Africa and Australia (Tang and Lightner, 2006). Therefore, the small fragment cloning methods may yield false positive results since positive fragments may be amplified from these IHHNV sequences inserted in the shrimp genome instead of the IHHNV itself. To distinguish infectious IHHNV and inserted IHHNV-related sequences, specific PCR primers (named IHHNV309F/R) and nested PCR (named IHHNV648F/R and IHHNV309F/R) have been developed (Rai et al., 2009; Tang et al., 2007). However, a study of Saksmerprome’s group showed that the position and length of the IHHNV-related sequence insertions in shrimp stocks are random, and the primers mentioned above may lead to false positive results (Saksmerprome et al., 2011). As an alternative, a method which covers most of the regions in IHHNV genome (positions nt 158–3625) was developed to distinguish between viral infection and inserted sequences. Unfortunately, this method is relatively complicated since it requires 7 pairs of primers for 7 PCR reactions, and does not cover the
Y.-W. Wei et al. / Journal of Virological Methods 224 (2015) 16–19
1
2000
17
3909
IHHNV-1942F
Step1
5'
Genome of IHHNV
3' IHHNV-3909R
Step 2
IHHNV-1F
5'
Genome of IHHNV
3'
IHHNV-2020R
IHHNV-Ex
Step 2'
IHHNV-Linker
5'
3'
Genome of IHHNV IHHNV-2020R
Fig. 1. Cloning strategy for the full-length genome of IHHNV. Step 1: PCR amplicons using primers IHHNV-1942F/IHHNV-3909R. Step 2: PCR amplicons using primers IHHNV-1F/IHHNV-2020R. Step 2’: PCR amplicons using primers IHHNV -Ex, IHHNV-Linker and IHHNV-2020R. During the PCR amplification in step 2’, the first cycle of PCR using linker primer created a new template with extension sequence at 5 end of IHHNV genome sequence. These extensions subsequently allow the binding of the highly specific extension primer to the new template. After that, the target sequence could be amplified using the extension primers.
full-length genome of IHHNV (Saksmerprome et al., 2011). Therefore, a simple, efficient and convenient method is required for cloning the full-length sequence of IHHNV. In this study, a novel and simple strategy for cloning the complete genomic sequences of IHHNV was developed. Samples of L. vannamei, P. monodon, Fenneropenaeus chinensis, Macrobrachium rosenbergii and Marsupenaeus japonicus were collected from the hatcheries and culture systems from Ningbo, Wenzhou, Jianxing Zhoushan regions in Zhejiang Province of China in 2014 and kept in −80 ◦ C freezers. For each sample, 30 mg of gills and pleopods were taken, mixed, and homogenized. Then, these homogenized tissue samples were incubated at 65 ◦ C for 4 h in 20 mM Tris–HCl (pH 8.0), 10 mM EDTA, 0.1% sodium dodecyl sulfate (SDS) and 0.8 mg/ml proteinase K. The DNA was then extracted using a DNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. The cloning procedure was carried out in 2 steps separately (Fig. 1). Primers were designed based on the strain of Hawaii (GenBank no. AF218266) and listed in Table 1. Two pairs of primers, IHHNV-1942F/IHHNV-3909R and IHHNV-1F/IHHNV-2020R were used to clone the genome of IHHNV (step 1 and step 2). PCR was performed in 0.2 mL tubes with a final volume of 25 L containing 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 mM dNTP, 0.01% gelatine, 2.5 U of Taq DNA polymerase (Boehringer Mannheim), 10 ng of IHHNV positive genomic DNA template and 1 mM primers of IHHNV-1F/IHHNV-2020R or IHHNV1942F/IHHNV-3909R. The amplification was performed for 35 cycles with denaturation at 94 ◦ C for 30 s, across an annealing temperature gradient at 50 ◦ C, 52 ◦ C, 54 ◦ C, 56 ◦ C, 58 ◦ C or 60 ◦ C for 30 s, and elongation at 72 ◦ C for 1 min with an increment of 2 min after 35 cycles in a gradient thermocycler (Eppendorf Mastercycler pro). Agarose gel analysis of PCR products indicated that single bands were clearly visible when the annealing temperatures were below 58 ◦ C for the IHHNV-1942F/3909R primer set, but there were not any visible bands in the assay using the IHHNV-1F/2020R primer set. The efficiency of PCR can be affected by many other Table 1 Primers used for amplifying the full-length sequences of IHHNV. Primer name
Nucleotide sequence (5 -3 )
IHHNV-1F IHHNV-2020R IHHNV-1942F IHHNV-3909R IHHNV-Linker IHHNV-Ex
5 -TAGAGCGCGAAGCGCGAGTATC-3 5 -GCATATTGTCGTAGTCTGGT-3 5 -GTCACTAATTACAAACCTGCAG-3 5 -CTTCGCAGAAACCGTTAAC-3 5 -GGTACACTGACATGCTGACTTAGAGCGCGAAGCGCGAG-3 5 -GGTACACTGACATGCTGACT-3
Note: The extended sequence in primer IHHNV-Linker was underlined.
factors such as the working concentrations of primers, template and magnesium, buffer ingredients, or the time and temperature of denaturation. All those have been taken into account by optimization of the reaction conditions. However, the fragment which covers the genome of IHHNV from 5 end to nt 2020 was failed to get in multiple attempts by optimizing various parameters. To circumvent this problem and obtain the complete nucleotide sequence of IHHNV, a novel strategy for cloning the upstream fragment of IHHNV genome was employed. Firstly, one linker primer at the 5 end of IHHNV genome was designed, and one highly specific extension primer with the same sequence that was added in the linker primer was synthesized (Table 1). Three primers IHHNV-Ex, IHHNV-Linker and IHHNV-2020R were used to clone the fragment which covers the first half of IHHNV genome (step 2’). Ten 0.2 mL PCR reaction tubes were prepared in a volume of 24 L containing all components as described as above, and 1 mM primers of IHHNV-Linker and IHHNV-2020R. Finally, 1 L of primer different concentrations of IHHNV-Ex were added to each tube (the final concentration ratios between IHHNV-Ex and IHHNV-Linker are 1:1, 1:10, 1:25, 1:50, 1:100, 1:500 and 1:1000). The amplification was performed for 35 cycles with denaturation at 94 ◦ C for 30 s, annealing temperature at 54 ◦ C for 30s, and elongation at 72 ◦ C for 1 min with an increment of 2 min after 35 cycles in a thermocycler. Agarose gel analysis indicated that relatively higher yields of PCR amplicons were detected when the IHHNV-Ex/IHHNV-Linker primer ratios were between 1:25, 1:50 and 1:100 at annealing temperature of 54 ◦ C. No bands were observed when this primer ratio was under 1:20 or above 1:200. PCR fragments with the expected size were extracted from 1% agarose gels and were purified using a DNA purification kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. These purified PCR products were cloned into a pMD18-T vector (TaKaRa, Dalian, China) according to the manufacturer’s instructions to yield DNA template suitable for IHHNV genome sequencing. Sequence analysis (Beijing Genomics Institution, Beijing, China) using three independent positive clones confirmed that the IHHNV genome had been successfully cloned. To determine the analytical sensitivity of the cloning method, PCRs were performed as described above using plasmid DNA containing IHHNV genome, which was serially diluted tenfold in ddH2 O ranging from 100 ng (100 ng/l) to 0.1 fg (0.1 fg/l). These results showed that the detection limit was 1 fg/l (20 copies/l) for the IHHNV-1942F/3909R primer set, and 0.01 ng/l (2 × 105 copies/l) for IHHNV-Linker/Ex/2020R set. These PCRs were routinely used to amplify the target sequences of IHHNV spiked in DNA extracted from L. vannamei, P. monodon, F. chinensis, and M. japonicus, to evaluate the broad-spectrum use
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Ningbo
55 51 45
Wenzhou Jiaxing
52
KF214742
91
Zhoushan
57
JX258653 JN377975
61 57
Lineage III
AY362548 JX840067 AF218266
10045
EF633688
47
AY355308
78
100
AF273215 GQ411199 100
KF031144
100 100 89
Ningbo-22
Lineage II
Ningbo-36
Lineage I
GQ475529 AY124937
Non-infectious type B EU675312 100
DQ228358
Non-infectious type A
0.005 Fig. 2. Phylogenetic analysis of full-length sequences of IHHNV. Sixteen full-length (and nearly full-length) sequences from GenBank of IHHNV were included in the analysis. The sequences were aligned with Clustal W 2.0 and the phylogenetic tree was then generated using the neighbor-joining method with 1000 bootstrap replicates using the MEGA 5.0 software program.
of this method. One microliter of these positive IHHNV DNA samples were mixed with DNA extracted from each kind of shrimp. PCRs were performed as described above using the DNAs mixture as template, and agarose gel electrophoresis showed strong PCR amplifications in all types of shrimp. These results indicated that the IHHNV detection method developed is not only suitable for L. vannamei, but also applicable in other kinds of shrimp. Furthermore, samples of L. vannamei and P. monodon were collected from hatcheries and culture systems from different regions in Zhejiang Province of China in 2014. DNA isolation and full-genome cloning of IHHNV were performed as described as above. Five more full-length genomes of IHHNV (three strains from L. vannamei and two strains from P. monodon) were cloned. All cloned sequences were deposited in the GenBank database (accession numbers for each isolate are KP733862 (Wenzhou), KP733861 (Jiaxing), KP733860 (Ningbo) KP733859 (Zhoushan), KP733858 (Ningbo-22) and KP733857 (Ningbo-36)). These sequences were aligned with 15 IHHNV sequences (five complete genomic sequences and ten partial sequences) from GenBank (Clustal W 2.0) and the phylogenetic tree (Fig. 2) was then generated using the neighbor-joining method with 1000 bootstrap replicates (MEGA 5.0 software program). The overall branching patterns of the phylogenetic tree constructed for the nucleotide sequence of IHHNV was split into three major distinct branches: one encompassing non-infectious IHHNV type A (EU675312 and
DQ228358), the other containing type B (AY124937) and the third containing infectious IHHNV strains. The infectious IHHNVs were split into three major lineages as follows: lineage I, lineage II, lineage III. The strains of Ningbo, Wenzhou, Jiaxing and Zhoushan strains formed a cluster with lineage III strains. However, Ningbo22 and Ningbo-36 strains formed a cluster with lineage II strains. Analysis of full-length genome sequence identity and evolutionary distance supported the observed tree topology. The deduced amino acid (aa) sequences of CP (329aa), NS1 (666aa) and NS2 (363aa) from the cloned IHHNV strains were aligned with other available CP, NS1 and NS2 sequences in GenBank. Sequence distances data derived from the multiple alignment of the CP revealed that CP of Wenzhou, Ningbo, Zhoushan and Jiaxing strains shared the highest aa identity to IHHNV strains of lineage III (98.2–99.7%), lower aa identity to strains in lineage I and lineage II (96.7–97.3%), and lowest aa identity to non-infectious IHHNV type A and type B (93.0–93.6%). For CP of Ningbo22 and Ningbo 36 strains, sequence distance revealed that Ningbo22 and Ningbo 36 strains shared more aa identity to lineage II KF031144 and GQ411199 strains (97.3–99.7%) than to other lineage strains (91.5–97.3%). Within the genome of IHHNV, two separate and partially overlapping ORFs encode non structural proteins NS1 and NS2. Multiple alignments revealed that NS1 and NS2 aa sequences of Wenzhou, Ningbo, Zhoushan and Jiaxing were closest to lineage III strains (97.3–99.8% identity). For NS1 and NS2 of Ningbo22 and Ningbo36, sequence
Y.-W. Wei et al. / Journal of Virological Methods 224 (2015) 16–19
distance revealed that Ningbo22 and Ningbo36 strains shared more aa identity to lineage II strains (98.9–99.2%) than to other strains. As shown in Supplementary Fig. 1 the 5 -NCR sequences of Wenzhou, Ningbo, Zhoushan and Jiaxing strains shared close identity, with three nucleotides substitutions at positions 142 (T/C for Zhoushan strain), 423 (G/A for Wenzhou strain) and 428 (C/T for Ningbo and Jiaxing strains). 3 -NCRs of Wenzhou, Zhoushan and Jiaxing strains were completely identical with each other. But Ningbo has two substitutions at position 3756 (C/T) and 3769 (A/C). 3 -NCRs of Ningbo22 and Ningbo 36 strains shared completely identical sequences with each other. The striking difference between these sequences for available strains was that both 5 and 3 -NCRs sequences were highly conserved within but not between genetic lineages. Wenzhou, Ningbo, Zhoushan and Jiaxing strains had a six nucleotide (GAAGCG) deletion at position 75, and JN377975 strain has a five nucleotide (TGCGA) insertion at position 77, KF031144 has one nucleotide (C) insertion at position 71 in 5 -NCR and one nucleotide (C) insertion at position 3901 in 3 -NCR. The reason of this difference is unclear yet. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2015.08. 004 Although IHHNV was discovered thirty years ago, cloning of full-length genomes of this virus has been limited to a multi-fragment cloning strategy (6–7 fragments) (Kim et al., 2012; Rai et al., 2011). In addition, it has been a challenge to work with this virus because of IHHNV-related sequence insertions in the genome of shrimp (Rai et al., 2009; Saksmerprome et al., 2011; Tang and Lightner, 2006). Because of the lack of a rapid cloning method of IHHNV, there were only five full-length genomes of IHHNV available in GenBank. Therefore, a new and reliable method that allows for rapid cloning of infectious IHHNV has been developed. Criteria for the design of this new method included sensitivity and simplicity and,also utility for a broad-spectrum of clinical specimens from a variety of shrimp species. Using this over lapping PCR and cloning method, six complete sequences of IHHNV from different regions of Zhejiang Province in China were successfully generated. This new method will facilitate the study of the genomics and molecular epidemiology of IHHNV. Acknowledgements This project was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA092001), the Educational Commission of Zhejiang Province of China (Y201430861), the Natural Science Foundation of Ningbo City of China (2014A610182), and the KC Wong Magna Fund in Ningbo University. We thank Yuanmei Ma for critical reading of the manuscript. References Browdy, C.L., Holloway, J.D., King, C.O., Stokes, A.D., Hopkins, J.S., Sandifer, P.A., 1993. IHHN virus and intensive culture of Penaeus vannamei: effects of stocking density and water exchange rates. Journal of Crustacean Biology 13, 87–94. Dhar, A.K., Robles-Sikisaka, R., Saksmerprome, V., Lakshman, D.K., 2014. Biology, genome organization, and evolution of parvoviruses in marine shrimp. Advances in Virus Research 89, 85–139. Encinas-Garcia, T., Mendoza-Cano, F., Enriquez-Espinoza, T., Luken-Vega, L., Vichido-Chavez, R., Sanchez-Paz, A., 2015. An improved validated SYBR
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