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Journal of Virology, May 2009, p. 4462-4468, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.00014-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A New Flavivirus and a New Vector: Characterization of a Novel Flavivirus Isolated from Uranotaenia Mosquitoes from a Tropical Rain Forest ^,

Sandra Junglen,^1* Anne Kopp,¹ Andreas Kurth,² Georg Pauli,² Heinz Ellerbrok,² and Fabian H. Leendertz¹

Research Group Emerging Zoonoses, Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany,¹ Center for Biological Safety, Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany²

Received 5 January 2009/ Accepted 9 February 2009

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A novel flavivirus was isolated from Uranotaenia mashonaensis, a mosquito genus not previously known to harbor flaviviruses. Mosquitoes were caught in the primary rain forest of the Taï National Park, Côte d'Ivoire. The novel virus, termed nounané virus (NOUV), seemed to grow only on C6/36 insect cells and not on vertebrate cells. Typical enveloped flavivirus-like particles of 60 to 65 nm in diameter were detected by electron microscopy in the cell culture supernatant of infected cells. The full genome was sequenced, and potential cleavage and glycosylation sites and cysteine residues were identified, suggesting that the processing of the NOUV polyprotein is similar to that of other flaviviruses. Phylogenetic analyses of the whole polyprotein and the NS3 protein showed that the virus forms a distinct cluster within the clade of mosquito-borne flaviviruses. Only a distant relationship to other known flaviviruses was found, indicating that NOUV is a novel lineage within the Flaviviridae.

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The genus Flavivirus (family Flaviviridae) includes 53 viruses (13), some of them responsible for worldwide epidemics and high mortality rates in the human population. The most virulent flaviviruses are yellow fever virus (YFV), dengue virus (types 1 to 4), Japanese encephalitis virus (JEV), and West Nile virus (WNV). Dengue virus is spread over 100 countries, and more than 2.5 billion people live in dengue-endemic areas (17). In almost the same manner, YFV affects more than 200,000 individuals annually, with a case fatality rate of about 20% (25). For all the viruses mentioned above, an increase in incidence and geographic range and spread into new host populations have been reported (23, 27); thus, they are classified as emerging or reemerging infectious pathogens.

Flaviviruses are transmitted mainly by hematophagus arthropods, such as mosquitoes or ticks. Some atypical flaviviruses have been isolated from bats and rodents, such as Entebbe bat virus and Apoi virus (2, 22), but arthropod vectors are unknown. Serologic classification of flaviviruses clearly correlates with the phylogenetic relationship of the virus and with the phylogeny of their natural arthropod vectors. According to this relationship, flaviviruses form a no-known-vector cluster and a vector-borne cluster, with the latter subdivided into tick-borne and mosquito-borne clusters (6, 21). Mosquito-borne viruses could be further subdivided into Aedes- and Culex-transmitted viruses. Interestingly, the Aedes-borne viruses are associated with hemorrhagic fevers and those transmitted by Culex with encephalitic diseases (15).

Among the flaviviruses, some have been isolated from mosquitoes or insect cell lines only, for example, cell fusing agent virus (CFAV), Kamiti River virus (KRV), and Culex flavivirus (8, 19, 33). Clearly, these insect viruses are phylogenetically distinct from other vector-transmitted flaviviruses, and they are considered primordial forms (6). Genomic fragments related to CFAV, KRV, and Culex flavivirus were also found in the genome of Aedes mosquitoes, pointing toward integration of a nonretroviral RNA genome in eukaryotic cells (9).

Common among all flaviviruses is the single-stranded, positive-sense RNA genome consisting of approximately 10.5 kb, with a unique open reading frame (ORF) that is flanked by a type 1 capped 5'-terminal noncoding region (NCR) and a 3'-terminal NCR. The polyprotein is co- and posttranslationally modified into three structural proteins (capsid [C], membrane [M], and envelope [E]) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).

In recent years, many novel flaviviruses have been discovered, such as Ngoye virus and New Mapoon virus, along with the earlier-mentioned KRV and a novel strain of CFAV (7, 8, 16, 26). This indicates a much larger heterogeneity among flaviviruses than previously thought and suggests that a large number of distantly related flaviviruses exist.

In this article, we describe a novel flavivirus isolated from mosquitoes caught in the primary rain forest of the Taï National Park, Côte d'Ivoire. Flavivirus-like particles were detected by electron microscopy (EM) in the cell culture supernatant of insect cells infected with homogenates of Uranotaenia mashonaensis mosquitoes. This is the first time that this mosquito genus has become relevant as a possible vector for arboviruses. The virus was tentatively termed nounané virus (NOUV), which means "having its own way" in the local Oubi language. Here we describe the full coding sequence of two isolates (NOUV B3 and B31). The genome organization of the entire ORF was analyzed and its phylogenetic relationship investigated. The isolated flavivirus is a completely novel strain which shows little homology to other known flaviviruses and takes a unique position within the mosquito-transmitted flaviviruses.

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Virus isolation. The virus was isolated from adult Uranotaenia mashonaensis mosquitoes collected in 2004 in the Taï National Park, Côte d'Ivoire. Details on trapping locations, collected mosquito species, and other viruses isolated will be described elsewhere. Two pools of Uranotaenia mashonaensis mosquitoes were generated: one collected 1 m above the forest floor, containing 20 mosquitoes (B3), and a second one, of 10 mosquitoes, collected 24 m above the forest floor (B31). Mosquitoes were divided into head and abdomen, and the former were used for cell culture infection. Mosquito heads were homogenized with a mortar in 300 µl of chilled Dulbecco's modified Eagle medium (DMEM) without additives. A 1.2-ml amount of DMEM was added, and suspensions were centrifuged at 3,000 x g for 10 min. The supernatant was filtrated through a 0.45-µm filter and used for culture inoculation in Aedes albopictus (C6/36) cells (3 x 10⁵ cells per well in a 24-well plate). For infection, medium was removed, and 100 µl of cleared mosquito suspension and 200 µl medium without additives were combined. After 1 h of incubation, 800 µl medium with additives (5% fetal calf serum and 1% Glu) was added. The cells were observed daily for signs of a cytopathic effect (CPE). On day eight postinfection, 200 µl of the supernatant was transferred to fresh cell cultures. The remaining supernatant was centrifuged at 1,000 x g for 5 min and stored at –80°C. This was repeated twice. Cell cultures of the second passage showed clear signs of CPEs after 5 days and were harvested when about 80% of the cells showed a CPE. From the third passage of both pools, viral stocks were generated and aliquots were used for further investigation.

Infection of different cell lines. African green monkey cells (Vero), baby hamster kidney cells (BHK), three human cell lines (293, A549, and Hep2), porcine stable equine kidney cells, and primary chicken embryo fibroblasts were infected at a multiplicity of infection of 0.1 with the cell culture supernatant of C6/36 cells and passaged two times on fresh cells. The supernatant of each cell line of the second passage was titrated on C6/36 cells, and real-time reverse transcription-PCRs with NOUV-specific primer sets (NOUV_7148_F, NOUV_7338_R, and NOUV_TM; see Table S1 in the supplemental material) were performed on RNA extracts from the cell culture supernatants of all infected cell lines.

EM of ultrathin sections. Cell culture supernatant was cleared from debris by low-speed centrifugation at 1,000 x g for 5 min followed by ultracentrifugation through a 36%-sucrose cushion at 100,000 x g for 60 min (5). Pellets were resuspended in phosphate-buffered saline, fixed with 2% paraformaldehyde, and processed for direct negative staining electron microscopy (1, 18). Infected cells were fixed with 2.5% glutaraldehyde, enclosed in low-melting-point agar, embedded in resin, and evaluated by transmission EM after ultrathin sectioning, as described elsewhere (1).

Sequencing of the complete viral genome. In the first step, viral sequence fragments were generated based on the method described by Stang et al. (32). Briefly, a 175-cm² flask of C6/36 cells was infected and harvested when 80% of the cells showed a CPE. Cells were frozen and thawed, and cell debris was pelleted at 3,220 x g for 10 min. Supernatant was ultracentrifuged through a cushion of 5 ml 36% sucrose at 28,000 rpm for 4 h (Rotor SW35; Beckman, Krefeld, Germany). The pellet was dried for 10 min and resuspended overnight in 150 µl phosphate-buffered saline without additives at 4°C. RNA was extracted from this virus pellet using the Qiagen viral RNA kit according to the manufacturer's instructions. Double-stranded cDNA synthesis was performed using a Promega double-strand synthesis kit. Random primers with attached short 5' anchor sequences were annealed overnight to the double-stranded cDNA, and PCR (Platinum Taq; Invitrogen) was completed the following day, using the anchor part of the random primer as PCR primers. PCR products were purified with the Qiagen PCR purification kit (Qiagen, Hilden, Germany) and cloned into the pCR2.1 Topo vector (Invitrogen, Karlsruhe, Germany). Colonies were analyzed by colony PCR, and inserts were sequenced using the ABI BigDye termination kit (Applied Biosystems, Weiterstadt, Germany) and the ABI Prism genetic analyzer (Applied Biosystems). Sequences were compared to the GenBank database using BLAST (NCBI).

Based on these sequences, specific internal primers were designed in order to combine the sequence fragments and fill gaps in the sequence (see Table S1 in the supplemental material). PCR products were sequenced directly on both strands, and internal primers were used for further sequencing of the amplicons. The 5' and 3' ends of the genome were amplified using a 5'- or 3'-rapid amplification of cDNA ends (RACE) kit, according to the manufacturer's instructions (Roche, Mannheim, Germany). The RACE PCR products were cloned and sequenced as described above.

Assembling of overlapping sequences for full-length genome and phylogeny. Generated forward and reverse sequences were first analyzed for quality; consensus sequences of all obtained sequences were then created using SeqMan II (LaserGene software program; DNAStar). Overlapping consensus sequences were assembled using the same software to generate a contiguous full-genome sequence.

For phylogenetic analyses, the entire ORF was aligned with other flavivirus sequences available at the public database (NCBI) using the ClustalW software program in BioEdit (software version 7.0.9). Phylogenetic analyses were performed using the PHYLIP (phylogenetic inference program) package (version 3.57c), using the neighbor-joining (NJ) and maximum-likelihood (ML) methods (14). Bootstrap resampling with 1,000 replicates was employed to place approximate confidence limits on individual branches.

Genetic characterization. The entire nucleotide sequence of the NOUV genome was analyzed to determine the ORF, and nucleotide sequences were translated into amino acid sequences using SeqBuilder (LaserGene program; DNAStar). Potential cleavage sites were identified according to the proteolytic processing cascade pattern for the flavivirus ORF (as developed by Chambers et al. [3]) and using the software program SignalP 3.0 (www.cbs.dtu.dk). Identified cleavage sites were compared to those of other flaviviruses. Relationships of NOUV protein sequences to those of other flavivirus proteins were compared using the protein-protein BLAST program (http://www.ncbi.nlm.nih.gov/). Predicted glycosylation sites and cysteine residues were determined using NetNGlyc 1.0 (www.cbs.dtu.dk) and Protean (v. 5.03) of the LaserGene program (DNAStar).

Nucleotide sequence accession numbers. The ORF sequences of the two NOUV isolates obtained in this work have been submitted to GenBank under accession numbers EU159426 and FJ711167.

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Full-genome sequencing. For full-genome sequencing of the NOUV genome, the isolate from pool B3 was used. Through particle-associated nucleic acid PCR (32), 25 clones were obtained, of which 21 had inserts larger than 500 bp. These were sequenced, and five contigs with lengths between 529 nucleotides (nt) and 1,500 nt were generated and compared to the public database (GenBank; NCBI). Genetic relationships to different flaviviruses and to different regions along the entire flavivirus genome were found, allowing the genome fragments to be placed in a putative order. With fragment-specific primers, gaps between the fragments were amplified and sequenced. The generated genome fragment was 7,256 bp in length. The full NS5 gene and parts of the 3' NCR were amplified using conserved primer pairs published by Kuno et al. (21). Parts of the 5' NCR of the flavivirus genome were amplified by RACE PCR, cloned, and sequenced. The full-length genome sequence of the coding region was obtained, but parts of the NCR seemed to be missing. From the 5' NCR, 79 bp were generated, and from the 3' NCR, 347 bp. According to the literature, these regions are from 95 to 132 bp and from 385 to 585 bp in length, respectively, and terminate with conserved dinucleotides (3). None of these conserved dinucleotides were found. Multiple approaches in trying to amplify the rest of the NRC were not successful. Within the obtained 10,755 kb of the NOUV genome, the ORF encodes a total length of 3,442 amino acids (aa) (Table 1).

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TABLE 1. Genome organization of NOUV

The same genome sequence of a second isolate, detected in another pool (B31) of Uranotaenia mashonaensis mosquitoes, was determined using the NOUV-specific primers for PCR amplification and sequencing. The ORF sequences of the two isolates displayed 97% nucleotide and 99% amino acid identities and indicated that these are two slightly different strains of NOUV.

Genetic characterization. To investigate the flavivirus polyprotein processing, the entire ORFs of both genomes were translated into amino acid sequences and investigated for putative cleavage sites. All 12 cleavage sites for the flavivirus polyproteins were identified, and NOUV shows the same genome organization as other flaviviruses, with three structural and seven nonstructural proteins encoded (Table 1). No differences in protein residues flanking the cleavage sites were found between the isolates B3 and B31. Whereas the first or second protein residues directly flanking the protein cleavage sites were mostly conserved among all flaviviruses and NOUV, differences were found in amino acid residues not directly flanking the cleavage site. All sites cleaved by the viral serine protease (VirC/AnchC, NS2a/NS2b, NS2b/NS3, NS3/NS4a, NS4a/2K, and NS4b/NS5) occurred after two C-terminal basic residues, such as KR, RR, or QR. The residues flanking the sites that are cleaved by the host protease (AnchC/Pr, Pr/M, M/E, E/NS1, and 2K/NS4B) were more similar among NOUV and other flaviviruses (Table 2).

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TABLE 2. Predicted cleavage sites in the polyproteins of NOUV and other flavivirusesa

The polyprotein length of 3,442 aa was the same for NOUV B3 and for NOUV B31 (Table 3). This is the longest ORF within the Flaviviridae to date (see Fig. S1 in the supplemental material). Regarding the sizes of proteins, great differences were found between NOUV and other flaviviruses. The E, NS1, NS3, and NS4b proteins of NOUV have about the same length as the proteins of mosquito-borne flaviviruses, such as Rocio virus (ROCV), St. Louis encephalitis virus (SLEV), JEV, and WNV, but are up to 30 aa longer, respectively. The highly conserved NS5 protein of NOUV was found to be about 30 aa shorter than the NS5 protein of other flaviviruses. No significant difference was found in comparing the sizes of the other proteins.

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TABLE 3. Lengths of proteins of NOUV and other flavivirusesa

Typical flavivirus cysteine residues were found for NOUV: 6 in the PrM protein, 12 in the E protein, and 12 in the NS1 protein. As in the work of Chambers et al. (3), N-glycosylation sites were found: two in the PrM protein, two in the E protein, and two in the NS1 protein. Two putative N-glycosylation sites were detected in the NS3 protein, which have not been described previously.

Phylogenetic analyses. To understand the phylogeny of NOUV, the entire ORF of NOUV and those of all flaviviruses with available sequence data were aligned. An NJ and ML tree was generated, and bootstrap resampling with 1,000 replicates was employed to place approximate confidence limits on individual branches (35). The insect-borne CFAV was used as an outgroup. The tree topologies generated from the NJ and ML methods were similar, with differences confined to terminal nodes (data not shown). A close relationship between the two NOUV isolates was found (Fig. 1A). The novel isolates clustered as an outgroup of mosquito-borne flaviviruses with 100% bootstrap support, creating a novel subcluster, with Uranotaenia mosquitoes as a host. No close relationship to other flaviviruses was found, and NOUV may belong to a new lineage within the Flaviviridae. To support this finding, another phylogenetic tree based on the NS3 helicase protein was generated as described above. Again NOUV clustered within the mosquito-borne flaviviruses but showed no close relationship to other flaviviruses (Fig. 1B).

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FIG. 1. Phylogenetic trees of NOUV and other flaviviruses showing the association of virus groups with their invertebrate vectors and vertebrate hosts and associated human diseases. The trees are based on the amino acid sequences of the entire ORF (A) or on the NS3 protein (B). Trees were constructed using the NJ method. CFAV was used as an outgroup. Bootstrap values were calculated with 1,000 replicates. ALF, Alfuy virus; ALK, Alkhurma hemorrhagic fever virus; APOI, Apoi virus; DEN, dengue virus; ENT, Entebbe bat virus; ILH, Ilheus virus; JE, Japanese encephalitis virus; KR, Kamiti River virus; KOK, Kokobera virus; LI, louping ill virus; MML, Montana myotis leukoencephalitis virus; MVE, Murray Valley encephalitis virus; NOU, nounané virus; OHF, Omsk hemorrhagic fever virus; POW, Powassan virus; RB, Rio Bravo virus; ROC, Rocio virus; SEP, Sepik virus; SLE, St. Louis encephalitis virus; TBE, tick-borne encephalitis virus; USU, Usutu virus; WN, West Nile virus; YF, yellow fever virus; ZIK, Zika virus.

Investigation of host spectrum in vitro. The phylogenetic analyzes showed that NOUV could be grouped with other mosquito-borne arboviruses pathogenic for humans. In order to test the possible host spectrum of NOUV and to investigate if NOUV may infect human and nonhuman primates, virus replication of NOUV on different vertebrate cell lines was tested, but none of them showed signs of CPE. Also, titration of inoculated cell culture supernatants and viral RNA detection by real-time reverse transcription-PCRs with NOUV-specific primer sets were negative, indicating NOUV did not replicate on the tested vertebrate cell lines.

Virus maturation. To investigate virus maturation in infected cells, ultrathin sections of C6/36 cells were screened by EM for viral particles. Flavivirus-like particles were detected 3 days postinfection in infected cells, revealing an electron-dense core of about 30 nm and surrounded by a lipid envelop of about 40 nm (Fig. 2A). The enveloped, spherical particles seem to mature in the endoplasmic reticulum and to be transported in inclusion bodies, as described for flaviviruses (20). During their maturation process, large cytoplasmic replication complexes containing still-uncondensed material were visualized (Fig. 2B), suggesting structures involved in early RNA synthesis.

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FIG. 2. Transmission electron micrograph of ultrathin sections of C6/36 cells infected with NOUV. Enveloped virions are located in the endoplasmatic reticulum (A) and in vesicles (B), as indicated by arrows. Scale bar, 100 nm.

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We have described the isolation and genetic characterization of a novel flavivirus, termed NOUV, from mature Uranotaenia (Pseudoficalbia) mashonaensis (34) mosquitoes collected 1 m and 24 m above the forest floor in the primary rain forest of Côte d'Ivoire. Investigation of the polyprotein processing and phylogenetic relationships of the entire ORFs of two isolates demonstrates the unique genetic composition of NOUV, indicating that this virus belongs to a new lineage within the Flaviviridae.

The two NOUV isolates showed high sequence identities of 97% on the nucleotide level and 99% on the amino acid level. The 3% nucleotide difference between the strains is comparable to that described for diverse genotypes of other flaviviruses (23), indicating that the two strains are closely related and seem to belong to one genotype. Investigation of protein cleavage sites, glycosylation sites, and cysteine residues showed that the polyprotein of NOUV is processed as described for flaviviruses in general (3). NOUV has the longest ORF of all previously described flaviviruses, which is most similar to those of YFV, JEV, and WNV (see Fig. S1 in the supplemental material). Differences from other flaviviruses were found in respect to amino acid length in the E, NS4b, and NS5 proteins. The E protein, as the major structural protein, plays a role in virion assembly, receptor binding, and membrane fusion. NS4b is poorly conserved among flaviviruses and may form membrane components of the replication complexes. The NS5 protein is the most highly conserved protein among flaviviruses (24) and codes for the viral RNA polymerase. Thus, differences in these proteins may have a direct impact on the viral replication cycle and infectivity. The amino acid lengths of the NOUV proteins and the protein cleavage sites were most similar to those of YFV, JEV, and WNV and showed differences from those of the insect viruses CFAV and KRV. In addition, NOUV showed the conserved cysteine residues encoded by mosquito-borne flaviviruses in the PrM, E, and NS1 proteins (28), indicating that NOUV may be related to mosquito-borne flaviviruses.

Mosquito-borne flaviviruses form phylogenetically separated groups, mirroring the relationship of their primary transmission vectors (21). NOUV clusters significantly with this group of mosquito-borne flaviviruses but forms a subgroup on its own and adds the mosquito genus Uranotaenia to the list of possible flavivirus vectors. Uranotaenia is a large, widely distributed genus of culicine mosquitoes, with 165 described species (29, 30). Of the 28 representatives of Uranotaenia in Saharan Africa, eight species are described for Côte d'Ivoire (11). Uranotaenia species prefer breeding places that are moist and not exposed to the sun (12), and adult mosquitoes are found mainly in the forest (11). In our study, Uranotaenia mosquitoes were found almost exclusively in the primary rain forest and not in surrounding areas, which are warmer and drier habitats (e.g., the secondary forest, plantations, and villages; details will be published elsewhere). Less is known about the host range of this mosquito genus, but among the few anthropophilic species, none are known to transmit pathogens (29, 30). The only virus described is a cypovirus (Reoviridae) isolated from Uranotaenia sapphirina (31). Thus, the isolation of a novel flavivirus that clusters within the mosquito-borne viruses from Uranotaenia mashonaensis indicates that mosquitoes of this genus may also be involved in pathogen transmission. However, more information is needed on the feeding preferences of this mosquito species, potential for transmitting NOUV, and identification of possible amplification hosts.

Despite the grouping with other mosquito-borne flaviviruses, NOUV grew successfully on insect cells only. This may indicate that this virus is only able to infect insects. But primary isolation of viruses can be difficult, and even arboviruses that are isolated from infected humans often do not replicate on mammalian cell cultures (4, 10). Serological studies connected to clinical observations will be needed to shed light on the pathopotential of NOUV for humans and animals.

YFV has shown that through human encroachment into pristine ecosystems and introduction into new habitats or host populations, devastating epidemics can occur (25). We described the isolation of a novel flavivirus with an unknown host spectrum from a vector with unknown feeding preferences from a primary rain forest. Given the emergence of YFV and other mosquito-borne viruses, future surveillance studies should include Uranotaenia mashonaensis mosquitoes and concomitant NOUV infections in these and other mosquito species in order to detect the potential spread of NOUV.

 
We thank the Ivorian authorities for long-term support, especially the Ministry of the Environment and Forest, as well as the Ministry of Research, the directorship of the Taï National Park, and the Swiss Research Center in Abidjan. We are grateful for logistic support by the Taï chimpanzee project and C. Boesch and for mosquito traps received from S. Rich. For help during field work, we thank G. Gagné, and for help with DNA sequencing, we thank J. Tesch and A. Kus. For helpful discussion and copyediting, we gratefully acknowledge D. Williams.

This work was supported by the Robert Koch-Institute and the Max-Planck-Society.

 
* Corresponding author. Mailing address: Research Group "Emerging Zoonoses," Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany. Phone: 49 30 18754 2592. Fax: 49 30 18754 2181. E-mail: JunglenS{at}rki.de

Published ahead of print on 18 February 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, May 2009, p. 4462-4468, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.00014-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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