Departmento de Virología, Instituto Nacional de Enfermedades Infecciosas, ANLIS Dr Carlos G. Malbrán, Av. Velez Sarsfield 563, 1281 Buenos Aires, Argentina1
Special Pathogens Branch, Division of Viral and Rickettsial Disease, Center for Disease Control and Prevention, 1600 Clifton Road, Atlanta, Georgia 30333, USA2
Author for correspondence: Paula Padula. Fax +54 11 4301 3146. e-mail ppadula{at}cvtci.com.ar
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Abstract |
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Introduction |
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In common with other members of the genus, Andes virus possesses a genome consisting of three single-stranded, negative-sense RNA segments, designated large (L), medium (M) and small (S), which encode the virus polymerase, glycoproteins (G1 and G2) and the nucleocapsid (N) protein, respectively. The 5' and 3' termini of hantavirus genome RNA segments are highly conserved and complementary to each other (Schmaljohn, 1996 ). To date, the termini of RNA genomes of viruses of the family Bunyaviridae are exactly complementary for 8 or 9 nt, whereas the complementarity extends to at least 17 nt among the hantaviruses. The 5' and 3' termini of virus RNAs are thought to form a double-stranded promoter regulating RNA transcription and replication, similar to that seen for other segmented, negative-stranded RNA viruses.
Knowledge of the genetic characteristics of Andes virus strains detected in patient samples is limited, as only S and partial M segment sequences are available. More recently, additional sequence information has been determined from the first Andes virus (AH1) case reported (López et al., 1996 ), from the southern Chilean cases (López et al., 1997
) and from a variety of Andes virus lineages detected in cases or rodents in distinct geographical regions in Argentina (Levis et al., 1998
; Padula et al., 2000
). Here, we report the complete Andes virus M segment sequence, its phylogenetic relationship with other HPS-associated viruses and a partial Andes virus L segment sequence. We present data on the variability of the RNA termini of the S, M and L segments. In addition, we report the successful isolation of Andes virus from one Oligoryzomys longicaudatus rodent captured in the vicinity of the AH1 HPS case.
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Methods |
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DNA sequence analysis.
PCR amplification products were separated on agarose gels, gel-purified and manually sequenced using the dideoxy-cycle sequencing technique (fmol DNA Sequencing System, Promega) or the fluorescent sequencing technique (dRhodamine Terminator Cycle Sequencing kit, Applied Biosystems) with an ABI 377 automatic sequencer. GenBank accession numbers of hantavirus M and L segments used in analyses are listed in the legend for Fig. 3. Multiple sequence alignment and comparisons of deduced amino acid sequences were performed using the CLUSTAL V program of the PCGENE software, version 6.8 (Intelligenetics).
Virus isolation.
Virus isolation was attempted using lung tissues obtained from three Andes virus-positive O. longicaudatus rodents (as determined by RTPCR). Briefly, Vero E6 cell monolayers (ATCC, CRL 1586) were inoculated with approximately 50 mg of tissue suspension, cultivated in T-12·5 flasks and, after 1 h adsorption, the tissue suspension inoculum was removed and maintenance medium was added to the cells. Cells were maintained at 37 °C under 5% CO2. On day 14, cells were suspended and half of them were used to infect another flask. After 14 days, a small amount of cells was scraped off for RNA extraction.
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Results and Discussion |
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The Andes virus G1 glycoprotein extends from aa 1 to 651 (nt 522004), including the putative cleavage site, and the G2 glycoprotein extends from aa 652 to 1138 (nt 20053465). Three of the four putative potential glycosylation sites in the Andes virus G1 protein are conserved in all HPS-associated hantaviruses (positions 138140, 350352 and 402404). However, the site at positions 524526 is conserved among the G1 proteins of the Argentinean hantaviruses and Prospect Hill (PH) virus (Parrington et al., 1991 ) but is absent from LN virus. The one putative potential glycosylation site in the Andes virus G2 protein (position 930932) is conserved in all hantaviruses. Interestingly, hantaviruses associated with subfamily Murinae rodents have more G1 putative potential glycosylation sites (five to seven), than subfamily Arvicolinae or Sigmodontinae-associated viruses. Such glycosylation differences, which can alter the charge on the virus surface glycoproteins, may be relevant to the pathogenic differences seen among these viruses. This could include the differences seen between HPS-associated and non HPS-associated viruses, and also more minor differences, such as renal insufficiency, which is often a feature of Andes virus-associate HPS but which is lacking for SN virus-associated HPS.
The positions of the cysteine residues in the predicted G1 and G2 proteins are very highly conserved, as are many of the proline residues, indicating a conserved three-dimensional structure for the protein. Hydrophilicity plots of the glycoproteins of the subfamily Sigmodontinae-associated hantaviruses were very similar (data not shown). Although the virus complete M segment sequences were divergent at the nucleotide level (approximately 79% identity) and the deduced amino acid sequences were highly conserved (93% identity) when Andes virus was compared with related viruses such as LEC, Oran or Hu39694 viruses. When comparing with North American HPS hantaviruses, the identity was 70 and 7678% [Bayou (BAY), Black Creek Canal (BCC), SN and New York (NY) viruses] at the nucleotide and amino acid levels, respectively.
The sequence of a 606 nt fragment of the Andes virus L segment was determined for the region 28323437 (numbered relative to SN virus). Nucleotide and amino acid sequence comparison revealed 79·5 and 90·7% identity, respectively, with LN virus (521 nt compared), the most closely related available virus, while SN virus showed 72·7% nt and 87·1% aa identity.
Andes virus phylogenetic analysis
Analyses of M and partial L segment nucleotide sequence differences using the maximum-parsimony method with a 2:1 weighting of transversions to transitions indicated that Andes virus represents a distinct phylogenetic lineage that is closely related to hantaviruses of other subfamily Sigmodontinae rodents (Fig. 3). Phylogenetic analyses of both S and M segment sequences indicate that Andes virus, together with the more related hantaviruses (Oran, LEC and Hu39694 viruses) and LN viruses, represent a distinct lineage of South American HPS-associated viruses. Some support is found for the North American BAY and BCC viruses forming a clade with the other viruses associated with sigmodontine rodents (which are predominantly distributed throughout South America), suggestive of a common ancient ancestor. Our data further indicate that Andes virus sequences could have been diverged into at least four branches and these branches contain antigenically similar but genetically distinct viruses.
Determination of the nucleotide sequences of the termini of Andes virus S, M and L RNA genomic segments
The terminal sequences of Andes virus S and M genome RNA segments were determined for virus RNA extracted from the lung of the AH1 case patient. The S-terminal nucleotides were conserved relative to other hantaviruses associated with subfamily Sigmodontinae rodents (Schmaljohn, 1996 ) (Fig. 1
). Prior to this analysis, the published terminal sequences of Andes virus S genome segments had been deduced from PCR primers (López et al., 1997
) and two base changes were thought to exist relative to the sequence consensus for the HPS-associated hantaviruses.
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The 5' and 3' termini of the virus L RNA segment had sequences similar to those reported for other hantaviruses. A panhandle structure at least 18 bp in length could be potentially formed by complementary regions of the 5' and 3' termini. When compared with SN virus, few exceptions to the panhandle structure were found. Differences included a single nucleotide at position 16 (for SN virus, UA and for Andes virus, CG), one each at positions 20 and 24 at the 5' end, which result in complementary nucleotides for Andes virus, and another one at position 23 at the 3' end. To determine if the termini were full-length or deleted, the PCR products were cloned and 12 clones were sequenced. All 12 L clones had 5'- or 3'-terminal deletions. Three clones had only two bases deleted but the exact position could not be determined because both bases could belong either to the 3' or to the 5' end. However, based on earlier studies (Meyer & Schmaljohn, 2000 ), the deletions would appear to belong to the 3' end.
It has to be noticed that L segment terminal sequences were recovered from the virus particles obtained from infected cells together with culture supernatant, while the sources for the S and M segment terminal sequences were obtained from virus RNA extracted from lung tissue of patient AH1. Although differences in the panhandle structure between Andes virus and the rest of the hantaviruses were observed, it has not been proven that those were connected to the unique properties of Andes virus.
The finding that the Andes virus M and L RNA segment termini had short deletions in the region believed to contain the sequence and/or structural features necessary for initiation of replication and transcription is intriguing. According to a proposed prime-and-realign model for Hantaan virus initiation, virus RNAs with very short 3' deletions could be expected potentially to replicate (Garcin et al., 1995 ). The Andes virus M virus RNAs with 3' deletions that were longer than a few nucleotides are probably not replication competent, because deletions were very rare (5 of 24) at the 5' termini. Terminally deleted RNAs were proposed previously to have a role in downregulating virus gene expression for a persistent virus in the Arenaviridae family, lymphocytic choriomeningitis virus (Meyer & Southern, 1994
, 1997
). Several possible explanations of why the 3' termini of genome RNAs are more heterogeneous than the 5' termini were also discussed previously for Tula virus (Kukkonen et al., 1998
). In addition, it has also been proposed that accumulation of terminally deleted RNAs may play a role in Seoul virus persistence, as the percentage of 3'-deleted virus RNAs increases in the population, they could potentially compete with standard virus and downregulate virus replication (Meyer & Schmaljohn, 2000
). It is possible that the differences in the relative abundance of G1G2 and N proteins seen in virus-infected cells could reflect deletions found in the Andes virus M segment that are not present in the S segment termini and which could downregulate G1G2 synthesis relative to N synthesis.
Clinical manifestations of human infections with Andes virus range from fulminant respiratory disease with high lethality to mild course without sequelae. It is generally assumed that humans usually become infected with hantaviruses by close contact with infected rodents or infectious rodent secretions or excretions. In addition, Andes virus has been associated with person-to-person transmission. Accumulation of Andes virus genetic data will be essential for understanding the factors that regulate virus gene expression and transmission and to determine HPS pathogenesis.
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Footnotes |
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References |
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Received 14 February 2002;
accepted 18 April 2002.