Dengue

Dengue Bulletin Volume 28 (2004)

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Identification and Phylogenetic Analysis of DEN-1 Virus Isolated in Guangzhou, China, in 2002

Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An^#

Department of Microbiology, College of Medicine, Third Military Medical University,
Chongqing, 400038, People’s Republic of China

Introduction

Dengue (DEN) viruses belong to the genus Flavivirus. There are four closely related, but antigenically distinct, virus serotypes (DEN-1, DEN-2, DEN-3 and DEN-4). The viral genome is a positive-sense single-stranded
RNA, approximately 11,000 nucleotides long, encoding 10 distinct proteins. The gene order is 5’-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3’, expressed as a single polyprotein that is cleaved by both viral and cellular proteases to form the viral polypeptides. The three 5’ proteins are
three structural proteins: capsid (C), membrane (M) and envelope (E), and the remaining seven are nonstructural (NS) proteins^[1]. The DEN viruses cause classical dengue fever (DF) and dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). DF is a non-specific viral febrile illness while DHF/DSS is a severe and fatal haemorrhagic disease. Up to 100 million cases of DF occur annually in the world, with some 2 billion people at risk of the infection in tropical and subtropical regions of Africa, Asia and the Americas^[2,3].

The pathogenesis of the DEN virus infection is not clear. Although many factors are proposed to be involved in the DEN virus epidemiology and occurrence of DHF/DSS, the evolution of the DEN viral virulence and the spread of DEN viruses in different geographical areas are two important features in the epidemiology and pathogenesis of the disease. Studies have suggested that specific viral structures might contribute to the replication capability in the target cells and to their transmission^[4]. Co-circulation of novel DEN genotype, another genotype of the same serotype or different serotypes of DEN viruses in the same area, might lead tothedisplacement of the native genotype by a new genotype and thus to the occurrence of DHF/DSS^[5].

Several studies have shown that the phylogenetic trees obtained from small gene fragment sequences are congruent with the trees obtained from an entire gene sequence, although there might be minor rearrangements in the terminal branches^[6-13]. Using a partial sequence from Envelope-Nonstructural protein 1 (E/NS1) junction region, Rico-Hesse defined five genotypes for DEN-1 viruses isolated worldwide: I) America, Africa and South-East Asia; II) Sri Lanka; III) Japan; IV) South-East Asia, the South Pacific, Australia and Mexico; and V) Taiwan and Thailand^[14]. Recently, Goncalvez set up the phylogeny of 36 DEN-1 viruses according full E gene sequences. Statistical analyses of the validity of branching patterns (bootstrap) also suggested five classifications:1) from Asia; 2) from Thailand; 3) from sylvatic/Malaysia; 4) from South Pacific; and 5) from the Americas/Africa, in which three genotypes indicated as 1), 4) and 5) were consistent to I), IV) and V) mentioned by Rico-Hesse^[15]. Aviles-G and collaborators sequenced Capsid-pre Membrane (C/prM) and E/NS1 regions of 24 recent isolates of DEN-1 from South America, suggesting that the recent epidemics in Argentina and Paraguay were due to the re-emergence of a previously circulated strain^[16]. This indicated that small gene fragment sequences are available for analysis of viral genetic characterization.

Current methods for obtaining DEN virus sequences no longer require a viable virus isolate. In fact, the entire genome sequences can be obtained by enzymatic amplification of viral RNA template in the patient’s blood sample. Unfortunately, there are numerous sequences that contain errors, which have led to serious mistakes in their interpretation. Therefore, it is wise to obtain virus isolates for further genetic characterization^[17]. In this study, a viral strain that circulated in Guangzhou, China, in 2002 was isolated first, then identified by amplifying and sequencing 539 nucleotide (nt) in NS1 region of DEN virus genome using universal primers. Also, E/NS1 regions of the isolated virus were sequenced to determine their origin.

Materialsand methods

Collection of blood samples

Twenty patients with suspected DEN virus infection admitted to the Eighth People’s Hospital of Guangzhou were enrolled – 12 males and 8 females aged between 23-57 years. The blood samples were collected on day 1 to 11 after the onset of fever. The sera were separated by centrifugation and stored at –70 °C until use.

Viral isolation

Aedes albopictus mosquito cell line, C6/36, was cultured in RPMI 1640（pH6.8～7.0）containing 10% fetal bovine serum (FBS) and 2 mM glutamine with 5% CO₂ at 28 °C. 30μl of the serum sample was diluted with 1 ml RPMI 1640 containing 2% FBS and was incubated with overnight C6/36 cells for 1 hour at 28 °C. After removing the serum, the cells were cultured in RPMI 1640（pH6.8～7.0）containing 2% FBS at 28 °C. The cells were observed daily for cytopathic effect (CPE)^[18]. If CPE was not seen initially, the cells were passaged several times to confirm whether there was virus in the serum samples or not.

RNA extraction

Viral RNA was extracted from the culture supernatants of infected C6/36 with ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions.

RT-PCR amplification

Nucleotides from positions 2503~3041 coding a fragment of the NS1 gene were amplified using RT-PCR. The universal primer sequences are: 5′GTG CAC ACA TGG ACA GAA CA 3′(forward) and 5′CTT TCT ATC CAA TAA CCC AT 3′(reverse). Their combination generated a 539nt fragment. Briefly, 5 µl of the extracted RNA was mixed with 50 pmol (2µl) of reverse primer at 70 °C for 5 minutes and cooled down on ice. Then 13 µl of RT mix containing 4µl of 5X RT XL buffer, 0.5 µl of 10mM dNTP, 6.0 µl of sterile UHQ water, 0.5µl RNase inhibitor and 20 U of AMV reverse transcriptase XL (TaKaRa, Japan) were added. The mixture was incubated at 42 °C for 1 hour for RT reaction, then heated to 95 °C to inactivate AMV and cooled down on ice. PCR was subsequently carried out by adding 24 µl of PCR mix containing 2.5 µl of 10X Taq Polymerase buffer (PROMEGA, USA), 2.5 µl of 25mM MgCl2, 12.5 pmol each of sense and reverse primers, 0.5 µl of 10mM dNTP, 2U of Taq DNA Polymerase (PROMEGA, USA) and 17.5 µl of sterile UHQ water to the 1st strand synthesis tube containing 1 µl of cDNA. PCR with denaturation at 94 °C for 30 seconds; annealing at 53 °C for 30 seconds; and extension at 72 °C for 1 minute 30 seconds for 30 cycles. The RT-PCR conditions for the amplification of nucleotides from positions 2107~2701 coding for the E/NS1 fragment were similar to those used for NS1 fragment, but with an annealing temperature of 58 °C instead of 53 °C.

DNA purification and sequencing

PCR-amplified DNA products were electrophoresed in 1% agarose gels and stained with 1μg/ml ethidium bromide. The bands of predicted size (539nt for the NS1 fragment and 593nt for E/NS1 fragment) were excised from the gel and purified using a Golden Beads Product Purification Kit (Songon, CN) according to the manufacturer’s instructions. Purified PCR products were cloned on to pMD18-T Vector (TaKaRa, Japan) and sent to Co. Bioasia, Shanghai, CN, in 15% Glycerol to perform sequencing using an ABI Prism 377 Genetic Analyzer. Amplifying primers were M13-48.

Computer analysis 

The multiple sequence alignment programme Clustalx, version 1.8^[19], was used to obtain an optimal nucleotide sequence alignment file. Phylogenetic analysis of nucleotide sequences from the NS1 region and the E/NS1 junction of our isolates were carried out with the neighbor-joining method (NJ)^[20], calculating bootstrap confidence intervals of 1,000 replicates. Character state tree-building algorithms (PHYLIP package) were also tested. A strict consensus bootstrap tree was obtained by using the following programmes: (i) SEQBOOT to generate 100 replicas, (ii) DNADIST and NEIGHBOR to acquire the tree of each reiterated data, and (iii) CONSENSE to build a strict consensus bootstrap tree. Phylogenetic trees were drawn using TreeView^[21]. Published sequences used in the analysis are listed in Table 1.

Table 1. Dengue viral sequences from GenBank used in the phylogenetic analysis

* The full-length gene segment sequence of this viral strain is unavailable in GenBank and the NS1 region in the phylogenetic analysis was sequenced by us

The resulting unrooted trees were out-grouped to the sequence of DEN-2 (TR1751), which was kindly provided by Dr Oya A (National Institute of Infectious Diseases, Japan) and isolated from a DF patient^[22,23]. The RT-PCR amplification of the NS1 region of this virus strain using universal primers and sequencing of amplified products was done as described above.

Biological character detection 

The virus isolated from the serum samples was propagated in C6/36 cells (MOI = 1) and the titer was determined by the plaque assay using Vero cells monolayer culture under methylcellulose overlay medium. TCID₅₀ of the virus was also measured by the plaque assay and calculated using Reed-Muench method^[24]. Eighteen suckling mice were intracerebrally (ic) inoculated with 10⁴ pfu/mouse of the virus for observing clinical signs and their survival time.

Results

General observation 

After two passages, 5 of the 20 serum specimens caused CPE on C6/36 cells. Infected C6/36 cells became syncytia and cell-cell fusion. Some cells showed necrosis and got detached from the plate at a late stage of the infection (Figure 1). The virus was propagated in C6/36 cells and the highest titer was 5×10⁵pfu/ml atday 6 post-infection (pi). Using the Reed-Muench method, TCID₅₀ of the virus was 4.37 log pfu/0.2ml. Plaque formation on Vero cells was detected on day 8 or 9 after infection and they were small and unclear (Figure 2). After ic inoculation with isolated virus, all of the suckling mice showed kyphoscoliosisand paralysis of hind legs and died on days 10 to 11 after infection.

Figure 1. Cultured C6/36 cells:  (a) C6/36 cells without DEN-1 infection; (b) Cell-cell fusion and syncytia on C6/36 cells was seen on day 6 after infection with DEN-1/GZ2002

Figure 1 (a)      

Figure 1 (b)      

Figure 2. Plaque formation on Vero cells was detected on day 8 after infection A. Blank control; B. Vero cells were infected with DEN-2/Tr1751; C and D. Vero cells were infected with DEN-1/GZ2002

Comparison of nucleotide sequences

As shown in Figure 3, a 539nt fragment from the NS1 region (positions 2503 to 3041) was amplified using the universal primer by RT-PCR and the amplified products were sequenced to determine the relationships among the 5 isolates and their origin. Comparisons of the NS1 region of the 5 isolates virus strains showed little divergence, so we chose one as the representative strain to compare with the sequences published in GenBank. The nucleotide sequence of this strain was the closest torDEN-1 dalte30 (AY145123), which was an attenuation strain of DEN-1 virus. According to the full-length gene sequences of rDEN-1 dalte30 (AY145123), we designed the primers to amplify the nucleotides of a fragment from the E/NS1 region (positions 2107 to 2701). The 240nt from the E/NS1 region (position 2309 to 2548) of this DEN-1 virus genome is the closest to DEN-1/T14 strain from Australia isolated in 1981 (M32931), which was genotype IV, their divergence was 2%. Based on sequence information and definition by Rico-Hesse, our isolates belonged to DEN-1 and genotype IV, named DEN-1/GZ2002.

Figure 3.Agarose gel analysis of the cDNA products from RT-PCR of RNA samples isolated from the supernatant of the infected C6/36 cells

(a) After amplification with the universal primer, a band of 539nt for the NS1 fragment was seen. Lanes 2-6: PCR amplified DNA products from different samples respectively; lane 7: blank control; lanes 1 and 8: 1Kb DNA Ladder marker (GeneRuler^TM)

(b) After amplification with a primer for 593nt fragment of E/NS1 region, an expected band about 593nt was seen (lane 1), Lane 2: 1Kb DNA Ladder marker (GeneRuler^TM)

Figure 3 (a)

Figure 3 (b)

Phylogenetic analysis 

We compared 480nt from the NS1 region (position 2516 to 2995) and 240nt from the E/NS1 region (position 2309 to 2548) of this DEN-1 virus genome with sequences of other published DEN-1 viruses. The phylogenetic analysis of the NS1 (480nt) and E/NS1 (240nt) regions of the gene nucleotide sequences was performed using NJ methods respectively. Two NJ trees were set up and they showed a similar result, except for a little difference in terminal branches (Figure 4). From the NJ trees in our study, 11 strains of the DEN-1 virus were divided into three genotypes coinciding with I, IV and V as defined by Rico-Hesse or Asia, South Pacific and Americas/Africa, as defined by Goncalvez. Our isolated DEN-1/GZ2002 strain belonged to genotype IV or South Pacific genotype. In addition, it was found that an arrangement of the S275/90 strain showed a major difference between the two trees, which supports the previous hypothesis that the S275/90 strain is a recombined strain^[25].

Figure 4. Phylogenetic relationship of DEN-1/GZ2002 to previously characterized DEN-1 viruses. Two phylogenetic trees were set up using 240nt from the E/NS1 region, position 2309 to 2548 (a) and using 480nt from the NS1 region, position 2516 to 2995 (b) of these DEN-1 virus genomes. The resulting unrooted trees were out-grouped to the sequence of DEN-2 (TR1751)

Discussion

It is evident that the DEN virus epidemiology is determined by many factors, including those in the host, the virus, the vector and the environment. DEN virus evolution is also determined by many complex interactions. Some genetic changes that occur during the natural transmission cycles of the DEN virus might affect its virulence and cause the disease. Therefore, understanding DEN virus variation is especially important to clarify the pathogenesis of DEN virus infection. Although current methods of obtaining DEN virus sequences no longer require viable virus isolates, virus isolates are necessary for further studies of the biological and genetic characteristics^[17]. In this study, we isolated DEN-1 virus that circulated in Guangzhou in 2002 and analysed its possible origin and partial biological features.

In an In vitro experiment, the virus caused typical CPE on C6/36 cells such as syncytia and cell-cell fusion, which was continuously shown when the viruspassaged several times. Their titer maintained a level about 5×10⁵pfu/ml during passages. On Vero cells, a small and unclear plaque, a biological marker of attenuation, was seen. The suckling mice survived for 11 days after ic inoculation.Interestingly, the sequences of 539nt from the NS1 were very close to rDEN-1 dalte30 (AY145123), which was an attenuation strain of DEN-1 virus. Combined with clinical data where thousands of patients only showed DF clinical signs, our results indicated that the isolated virus might be a low-virulence strain.

As is known, a comparison of full-length gene segment sequences is impractical, especially when looking at a large number of samples. Recently, several studies have shown that the phylogenetic trees obtained from small gene fragment sequences are highly congruent with those obtained from an entire gene sequence, except for a minor rearrangement in the terminal branches, suggesting thereby that small gene fragment sequences were effective for the study of viral genetic characteristics^[6-13]. The E/NS1 junction sequences were a useful indicator in DEN virus evolution and the NS1 sequences were a highly conserved region. In our study, after sequencing E/NS1 (240nt) and the NS1 (480nt) gene region, we analysed the phylogenetic relationship of 11 DEN-1 virusrepresentatives of three genotypes coincidence with I, IV, and V as referred to by Rico-Hesse (1990), or Asia, South Pacific and Americas/Africa, as defined by Goncalvez. This indicated that the isolated virus belonged to genotype IV/South Pacific.

Tolou completed the genome sequence analysis to demonstrate the likelihood of recombination between different strains of DEN-1 virus. The region of 1295~2592nt was considered to be a hot spot for the recombination^[25]. We sequenced one region outside (2516~2995nt) and one region inside (2309~2548nt) the possible recombination site. No recombination event occurred in our strains, at least in the regions analysed. However, no virus isolates meeting the stringent criteria for recombination have yet been described and it remains to be determined whether and at what frequency DEN viruses undergo recombination in nature^[17].

The evolution of DEN viruses has had a major impact on their virulence for humans and on the epidemiology of the DEN virus around the world. It is necessary that a more complete and systematic survey of DEN-1 samples be undertaken before a link between specific genotypes and the virulence of these viruses can be established.

Acknowledgment

This work was partially supported by grants nos. 30170848 and 30300303 from the National Science Foundation of China (NSFC).

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