VIROLOGY 242, 99±106 (1998) ARTICLE NO. VY978990

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provided by Elsevier - Publisher Connector Genetic Reassortment among Causing Hantavirus Pulmonary Syndrome

Luis L. Rodriguez, Jessica H. Owens, Clarence J. Peters, and Stuart T. Nichol1

Special Pathogens Branch, Centers for Disease Control and Prevention, Mailstop G-14, 1600 Clifton Road NE, Atlanta GA 30333 Received October 22, 1997; returned to author for revision November 18, 1997; accepted December 8, 1997

In order to determine the frequency and characteristics of reassortment among viruses causing hantavirus pulmonary syndrome (HPS), mixed were initiated in tissue culture by using two closely related strains of Sin Nombre , CC107 (from eastern California) and NMR11 (from New Mexico), which share the same species of rodent host in nature, the deer mouse (Peromyscus maniculatus). Potential reassortant virus plaques were screened by multiplex RT±PCR, using primers specific for individual genome segments of each strain. Reassortant viruses involving the M and S segments and, to a lesser extent, the L segment were detected in 8.5% of 294 progeny plaques tested. In addition, approximately 30% of the progeny virus plaques appeared to contain S or M segments originating from both parental virus strains, i.e., they were diploid. Most of these diploid virus genotypes were not stable, becoming either reassortant or parental virus strains upon plaque-to-plaque virus passage. In contrast to the results above, only one virus reassortant and four diploids were observed among 163 progeny virus plaques from mixed infections between Sin Nombre virus NMR11 and the genetically more distant Black Creek Canal virus, an HPS-causing virus from Florida, which has the cotton rat (Sigmodon hispidus) as its natural host.

INTRODUCTION pulmonary syndrome (HPS), an often fatal disease trans- mitted to humans by inhalation of infectious aerosols RNA viruses with segmented genomes have the ca- generated from urine and excreta of naturally infected pacity to reassort their RNA segments upon dual infec- rodents hosts (Nichol et al., 1993; Childs et al., 1994). Like tions of target cells. This ability is believed to play a key other members of the family Bunyaviridae, hantaviruses role in the evolution, pathogenesis, and epidemiology of possess a genome consisting of three segments of sin- important pathogens such as influenza viruses, rotavi- gle-stranded, negative-sense RNA. The large (L) seg- ruses, and arthropod-borne orbiviruses (Peng et al., ment encodes a protein with replicase, transcriptase, 1996; Gouvea and Brantly, 1995; Beaty et al., 1996). Ge- and endonuclease activity; the medium (M) segment netic reassortment has been demonstrated both in na- encodes a glycoprotein precursor that is processed to ture and in the laboratory among arthropod-borne vi- yield the two surface glycoproteins G1 and G2; and the ruses of the family Bunyaviridae, such as California En- small (S) segment encodes a nucleocapsid protein (Ply- cephalitis serogroup members La Crosse, Snowshoe usnin et al., 1996). Hantaviruses are rodent-borne vi- Hare, Tahyna, and Trivittatus; and Bunyamwera sero- ruses, unlike other members of the Bunyaviridae, which group members Bunyamwera, Batai and Maguari (Prin- gle, 1996). A high frequency of reassortment has been are arthropod-borne. The phylogenetic relatedness be- observed, both in dually infected cells and in arthropod tween these hantaviruses generally mirrors that of their vectors, within members of each of the Bunyamwera, natural rodent hosts, indicating cospeciation of the vi- California , and Simbu serogroups, but not ruses and their rodent hosts (Spiropoulou et al., 1994; between members of different serogroups (Pringle,1996). Antic et al., 1992; Xiao et al., 1994). Little is known about Genetic reassortment has not been well documented in the ability of hantaviruses to undergo genetic reassort- rodent-borne members of the Bunyaviridae (Hantavirus) ment. In two recently published studies, genetic analysis and it is believed to be more restricted in these viruses of SNV RNA sequences from human cases and rodents than in arthropod-borne members of this family (Beaty et collected in Nevada and eastern California suggested al., 1996). that RNA segment reassortment has taken place in na- Sin Nombre virus (SNV) and several other recently ture between SNV genetic variants (Li et al., 1995; Hend- recognized hantaviruses are the cause of the hantavirus erson et al., 1995). Our study was aimed at determining the ability of HPS-causing hantaviruses to undergo genetic reassort- ment in vitro and to compare these results with those 1 To whom correspondence and reprint requests should be ad- observed under natural conditions. To more precisely dressed at Special Pathogens Branch, Mailstop G14, DVRD/NCID/CDC, 1600 Clifton Rd. N.E., Atlanta, GA 30329-4018. Fax: (404) 639 1118. define the reassortment process between closely related E-mail: [email protected]. SNV strains, we performed genetic analysis of virus

0042-6822/98 99 100 RODRIGUEZ ET AL.

TABLE 1 Description of Viral Strains

Place and year Virus strain of isolation Rodent host Passage history Reference

Sin Nombre New Mexico (1993) Peromyscus maniculatus Isolated from P. maniculatus, 2ϫ P. Elliott et al. (1994) (NMR11) maniculatus mice, 3ϫ plaque purified, and 14ϫ Vero E6 Chizhikov et al. (1995) Sin Nombre California (1993) Peromyscus maniculatus Isolated from P. maniculatus, 1ϫ plaque Schmaljohn et al. (1995) (CC107) purified and 4ϫ Vero E6 Li et al. (1995) Black Creek Canal Florida (1994) Sigmodon hispidus Isolated from S. hispidus, 3ϫ plaque Rollin et al. (1995) (BCC) purified, 9ϫ Vero E6 Ravkov et al. (1995)

progeny from mixed infections of tissue culture cells, type. Reassortant genotypes were observed in 25 prog- using SNV isolates CC107 (from eastern California) and eny plaques (8.5%). Twelve were M-segment reas-

NMR11 (from New Mexico). Both of these viruses natu- sortants (NLCMNS), 12 were S-segment reassortants rally infect Peromyscus maniculatus rodents. In addition, (NLNMCS), and 1 was an L-segment reassortant (NLCMCS). to investigate the potential for reassortment between Unexpectedly, despite the selection of individual well- more distantly related hantaviruses, we analyzed the separated plaques, over 29% of the progeny plaques progeny from mixed infections of SNV (NMR11) and appeared to contain S or M segments originating Black Creek Canal virus (BCCV), a newly recognized from both parental strains, i.e., they were diploid for hantavirus isolated in Florida from the cotton rat (Sigmo- these genome segments. Most involved the M segment don hispidus) (Ravkov et al., 1995). (NLCNMNS) or the S segment (NLNMCNS) or both M and S segments (NLCNMCNS) (Table 3). Four (1.4%) progeny RESULTS plaques had each of all three segments from both par- ents. In order to determine the genetic stability of diploid- Reassortment between SNV strains NMR11 and CC107 TABLE 2 The methodology used here allowed us to directly Description of Primers Used for Multiplex RT±PCR screen the genetic constellation of progeny virus present in plaques from mixed infections without the need for Primer a Sequence 5Ј to 3Ј Sizeb further virus amplification in tissue culture (Tables 1 and 2). Therefore, we were able to study the first-generation NML2156F tctagagttgtttataagcactatcgtagt progeny from mixed infections. Figure 1 shows examples NML2449R tgcttgtaaatacttgttgacttct 293 of the screening of progeny plaques from a mixed infec- NMM1414F caatgcatttatacattcactagtta NMM1560R cttcagtataattaaggtgactgcc 146 tion between SNV strains NMR11 (N) and CC107 (C), by NMS1586F gatagtattaagtcatatgtatggggg multiplex RT±PCR using primers specific for CC107 (Fig. NMS1775R ataaaccctgattaatgatgagaga 189 1A) or NMR11 (Fig. 1B). Primers were specific and only CCL2230F cttacatggaaatttaaacgaa yielded a product with homologous RNA template (Fig. 1, CCL2515R cgtttgactaaaataaggtttgct 285 lanes 1 and 2). Three types of reassortant genomes were CCM1412F gtcaatgtatctacacatttactagtctg CCM1926R cacctgctcttgtagcgg 514 detected, NLNMCS,NLCMNS, and NLCMCS (Fig. 1, lanes CCS412F ctttacatcttgagttttgccc 3±5, respectively). In addition, we detected progeny CCS800R ggtcttttgttcaggcaag 388 plaques that yielded RT±PCR bands for the same seg- BCL140F gcaactacctgtttctttct BCL377R gatttggcatgtaagtattt 257 ment of both parental strains, such as NLNMCNS, N CN N , and N CN CN (shown in Fig. 1, lanes 6±8, BCM1552F tggttacttattcctaccaca L M S L M S BCM1852R aagcctcatgaaagcgccc 319 respectively). A few plaques had mixed genotypes that BCS1762F atgtgatttatactgtcggat yielded RT±PCR products for each of the three segments BCS1905R gggttgagggtttagga 160 for both parental strains. The description and frequency a of the genotypes observed are summarized in Table 3. Primer name composed of the virus (NMR11 ϭ NM, CC107 ϭ CC, Black Creek Canal BC), segment (L, M, or S), 5 nucleotide position Most of the progeny plaques collected (60.9%) had the ϭ Ј of beginning of the primer, and, sense: forward ϭ F or reverse ϭ R. parental genotype of SNV strain NMR11; none of 294 b RT±PCR product size in base pairs with the two primers specific for progeny plaques tested had the CC107 parental geno- each segment. GENETIC REASSORTMENT AMONG HPS-CAUSING HANTAVIRUSES 101

TABLE 4 Analysis of Diploids by Plaque-to-Plaque Passage

Genotype Genotypes found Plaque LMSNo. tested (No.)

97 N CN N 3 N, CN, N (3) 125 N CN N 2 N, CN, N (1); N, N, N (1) 128 N CN N 3 N, CN, N (1); N, C, N (2) 44 N N CN 1 N, N, N (1) 147 CN N CN 3 N, N, N (3) 166 N N CN 3 N, N, N (3) 171 N CN N 3 N, N, N (3)

genotype. Three of 24 diploid-genome plaques passaged once on Vero E6 cells prior to replaquing yielded some progeny plaques that retained their diploid genotype (Table 5). The remainder produced viruses that lost their diploid status, yielding either parental or reassortant genotype viruses only (Table 5). Of the 11 progeny FIG. 1. Agarose gel of multiplex RT±PCR products from individual plaques that maintained their diploid genotype for one plaques, tested with primers specific for SNV strains CC107 (A) or passage, none yielded infectious virus with diploid ge-

NMR11 (B). Lanes 1 and 2 are parental strains NMR11 (NLNMNS) and notypes on subsequent passage. CC107 (CLCMCS), respectively. Lanes 3±5 show examples of reassor- tants NLNMCS,NLCMNS, and NLCMCS, respectively. Lanes 6±8 show Reassortment between SNV and BCCV examples of ``diploid genomes'' NLNMCNS, and NLCNMCNS, respec- tively. Lane 9 are size markers ␾X174 digested with ␭ HindIII. To test if more distantly related HPS-associated han- taviruses that infect different rodent hosts in nature were capable of genetic reassortment, we performed mixed genome viruses, several of them were replaqued either infections of E6 Vero cells with SNV strain NMR11 and directly or following an additional passage in tissue cul- BCCV and tested progeny plaques by RT±PCR as de- ture. Three of 7 diploid-genome viruses replaqued di- scribed above. Of 163 progeny plaques screened, 72 rectly (97, 125, and 128) yielded some progeny plaques (44.2%) and 34 (20.8%) had the NMR11 (NLNMNS)or that retained their diploid genotype (Table 4). The other 4 BCCV (BLBMBS) parental genotype, respectively (Table 6). (44, 147, 166, and 171) produced virus that lost the het- One plaque was an M-segment reassortant (BLNMBS). erologous segment, yielding only the parental NMR11 This plaque, when tested by RT±PCR on three separate experiments, was always negative for SNV-specific L and S primers and BCCV-specific M primers, but was always TABLE 3 positive for BCCV L and S segments and SNV M seg- Genotype of Progeny Plaques Resulting from Mixed Infections ment. In addition, 3 plaques (1.8%) were diploid for the L of SNV Strains NMR11 and CC107 segment (BNLNMNS) and 1 (0.6%) for L and M segments (BNLNMBNS) (Table 6). Despite our efforts to pick individ- Genotype ual well-separated plaques, 52 (31.9%) of the progeny LMSNumber Percent plaques contained all three segments from both parental strains (Table 6). One possible explanation for this would N N N 179 60.9 be that these were mixed plaques, originating from wells C C C 0 0.0 with a higher plaque density, hence making it difficult to N C N 12 4.1 collect individual ones. However, this was not the case in N N C 12 4.1 N C C 1 0.3 at least 14 of 52 plaques with mixed genomes that N CN N 42 14.3 originated from wells with as few as 2 and not more than N CN CN 22 7.5 10 individual plaques. Another possible explanation is N N CN 19 6.5 that there was a ``background'' of poorly visible virus C CN C 1 0.3 plaques in the cell monolayer between the visible CN CN N 1 0.3 CN N CN 1 0.3 plaques. However, this trivial explanation was ruled out CN CN CN 4 1.4 when 10 random samples between visible plaques from selected wells were collected and found to be negative Total 294 100 to both viruses by RT±PCR. 102 RODRIGUEZ ET AL.

TABLE 5 Analysis of Diploid Plaques Passaged Once on Vero E6 Cells

Genotype Plaque No. Genotypes found (passage 1) LM Stested (No.)

128 N CN N 19 N,CN,N (3); N,C,N (16) 166 N N CN 2 N,N,CN (2) 175 N CN CN 2 N,CN,N (1); N,N,N (1) 44 N N CN 3 N,N,N (3) 53 N CN N 6 N,N,N (6) 71 N CN N 3 N,N,N (3) 97 N CN N 16 N,N,N (4); N,C,N (12) 122 N CN N 3 N,N,N (3) 125 N CN N 17 N,N,N (16); N,C,N (1) 135 N CN N 4 N,N,N (3); N,C,N (1) 175 N CN CN 7 N,N,N (7) 199 N N CN 7 N,N,N (5); N,N,C (2) 201 N CN N 2 N,C,N (2) 218 N CN CN 10 N,N,N (10) 219 N CN CN 3 N,N,N (2); N,C,N (1) 226 N CN N 15 N,N,N (15) 233 N CN N 9 N,N,N (9) 240 N N CN 10 N,N,N (10) 251 N CN N 9 N,N,N (9) 260 N CN N 10 N,N,N (10) 267 N CN N 10 N,N,N (10) 272 N CN N 9 N,N,N (9) 475 N CN N 3 N,C,N (3) 476 N N CN 8 N,N,C (8) 478 N N CN 3 N,N,N (3) 479 N CN CN 3 N,N,N (3)

DISCUSSION these viruses (Bishop, 1985; Klimas et al., 1981). Genetic reassortment has been detected in dual infections of Several lines of evidence have suggested that reas- arthropods or tissue culture cells with viruses belonging sortment could play an important role in the evolution, to the same serogroup. For instance, the frequency of pathogenesis, and epidemiology of the arthropod-borne reassortants observed in the progeny of dual infections members of the Bunyaviridae (Beaty et al., 1985; Bishop in mammalian cells with La Crosse encephalitis and and Beaty, 1988; Janssen et al., 1986; Endres et al., 1991; Snowshoe Hare viruses was 51% (Urquidi and Bishop, Griot et al., 1993). A number of studies have shown that 1992). In addition, a 64% reassortant ratio has been high-frequency genetic reassortment can occur between observed in virus progeny from mosquitoes dually in- fected with two different strains of virus (Turell et al., 1990). However, reassortment has not been TABLE 6 observed between members of different serogroups Genotype of Progeny Plaques of Mixed Infections within the same genus (Bishop, 1985; Urquidi and of SNV-NMR11 and BCCV Bishop, 1992). Little information is available on genetic reassortment Genotype of the rodent-borne hantaviruses, particularly the newly recognized HPS-related viruses, such as SNV. Genetic LMSNumber Percentage analysis of SNV RNA sequences obtained from infected N NN 72 44.2 wild rodents or human HPS cases from Nevada and B BB 34 20.8 eastern California suggested that RNA segment reas- B NB 1 0.6 sortment had taken place in nature between SNV genetic BN N N 3 1.8 variants (Li et al., 1995; Henderson et al., 1995). Among BN N BN 1 0.6 BN BN BN 52 32.0 35 SNV viruses analyzed, 17 (48.6%) appeared to be reassortants based on phylogenetic analysis of each of Total 163 100.0 the virus genome segments (Henderson et al., 1995). The GENETIC REASSORTMENT AMONG HPS-CAUSING HANTAVIRUSES 103 largest number of reassortants (14/17) detected ap- has greater fitness to grow on these cells than does peared to be M-segment reassortants. Only 2/35 and CC107, which has been passaged only 6 times in these 1/35 appeared to be S- or L-segment reassortants cells. The alternative explanation is that NMR11 inter- (Henderson et al., 1995). One explanation for these data fered with CC107 growth, despite the fact that for the is that virus M segments reassort more readily than the mixed infections both viruses were inoculated at the other two segments. The purpose of our study was to same time. better document and characterize the occurrence of ge- Despite our efforts to pick only well-separated individ- netic reassortment between hantaviruses involved in ual plaques, we found that a substantial percentage of causing HPS. our progeny plaques had individual segments from more Our current study represents the first report of labora- than one parent. There are several possible explanations tory-induced reassortment between rodent-borne hanta- for this observation. It is possible that these are a mixture viruses. Among 294 progeny plaques from mixed infec- of both parental strains or virus aggregates. However, tions of Vero E6 cells with SNV strains NMR11 and this is unlikely, since usually only one segment from both CC107, we found 25 (8.5%) reassortant plaques. This parents is detected. It is also possible that these plaques proportion is lower than what has been observed for are a mixture of one of the parental viruses and a reas- arthropod-borne members of the Bunyaviridae. Genetic sortant involving the duplicated segment. To test these reassortment appeared to be nonrandom, with only three possibilities, we subcloned the suspected diploid of six possible reassortant genotypes being observed. plaques either by plaque-to-plaque passage or by pla- These all included the L segment of the NMR11 parent. quing the progeny after amplification of the original Nonrandom reassortment has been reported earlier plaque in tissue culture. From the ``diploids'' tested, most among the progeny of mixed infections of bunyaviruses, of the subcloned progeny plaques were of the NLNMNS with a preference for homologous L±M and M±S being parental genotype, with a small proportion of plaques observed in one study (Urquidi and Bishop, 1992) and with reassortant genotype. Two examples are plaque 135

L±S in another (Pringle et al., 1984). The field data con- (NLCNMNS), which yielded three plaques of parental ge- cerning SNV reassortants indicated that the largest num- notype (NLNMNS) and one reassortant (NLCMNS), and ber of reassortants that accumulated in the virus popu- plaque 199 (NLNMCNS), which yielded five plaques of lation had homologous L and S segments (14/17), and parental NLNMNS and two of reassortant NLNMCS geno- only a few had heterologous L and S segments (3/17) type (Table 5). However, three diploids yielded several (Henderson et al., 1995). In contrast, homologous L±M plaques with diploid genomes, either after a direct and L±S combinations were found in equal numbers with plaque-to-plaque passage [e.g., plaques 97, 125, and 128

SNV reassortants generated in our tissue culture mixed yielded (NLCNMNS) progeny plaques] or after one pas- infections. These observed differences may reflect dis- sage on Vero E6 cells [e.g., plaques 128 and 175 yielded tinct selective pressures acting on different viruses and (NLCNMNS) and 166 (NLNMCNS) progeny plaques] (Table in natural versus tissue culture infections. However, with 5). These results suggest that the partial diploidy ob- the field data, it is not possible to determine the actual served might represent an intermediate state, before the frequency of reassortment events because the investi- virus segregates into reassortant or parental genotypes. gator is merely recording the number of reassortant It could be argued that this is an artifact detected only viruses that have accumulated in the virus population. within infected cells, but the fact that some of the dip- Unexpectedly, of the two SNV parent viruses initiating loids maintained their diploid genotype upon one pas- the mixed infections, only the NMR11 parental genotype sage argues against this possibility. While the detection was found among the progeny plaques. This result sug- of diploid genotype viruses was surprisingly high (approx gests that NMR11 outcompetes CC107 in a mixed infec- 30% of progeny plaques), it should be noted that the tion of Vero E6 cells. In addition, there was a clear method of PCR analysis would fail to detect diploid predominance of the NMR11 L segment in the progeny viruses which contain two copies of a genome segment virus, with this segment being absent from only one originating from the same parental strain. Thus, the over- progeny plaque. This predominance was observed even all degree of diploidy may be even greater. when ratios of input CC107 to NMR11 virus used to Others have reported the presence of diploids in the initiate the mixed were as high as 3:1 (data not progeny of mixed infections of bunyaviruses. Hampson shown). One possible explanation is that viruses con- described a virus clone obtained from a Maguari- taining the NMR11 L segment plaque more readily than Bunyamwera mixed infection, which contained S seg- those with CC107 L segment. However, this seems not to ments from both parents, and maintained its diploid be the case since both viruses tested individually pro- genotype upon subcloning in tissue culture (Hampson, duced plaques between 12 and 14 days after infection, 1987). Urquidi and Bishop (1992) also reported progeny when progeny plaques were collected. Another possible plaques with diploid genotypes from mixed infections of explanation is that NMR11, since it has been plaque La Crosse and Snowshoe Hare viruses, but did not purified 3 times and passaged 14 times on Vero E6 cells, characterize them further. It is believed that hantaviruses 104 RODRIGUEZ ET AL. package within the virus glycoprotein-containing lipid TABLE 7 membrane an average of three apparently circular nu- Genetic Divergence of Some Bunyaviruses for Which Genetic cleocapsids, each containing a RNA segment (Bishop, Reassortment Has Been Observed 1996). However, it is structurally possible that more than one copy of the same nucleocapsid subunit could be Approx nucleotide divergence packaged. Furthermore, the variable size of virions as Virus reassortants (%) in N coding sequence seen by electron microscopy (Talmon et al., 1987), and NOR ϫ GER 25 the nonequimolar ratios of L, M, and S observed in La BAT ϫ MAG 14 Crosse virus (Bishop and Shope, 1979) indicate that the BUN ϫ NOR 14 number of nucleocapsid subunits in virus particles is BAT ϫ BUN 16 variable. What these observations mean in terms of the BAT ϫ GER 23 NOR ϫ MAG 10 mechanism of packaging of hantaviruses is not clear, but LAC ϫ SSH 11 the subject deserves further investigation. Similar find- LAC ϫ JC 21 ings have been reported for the . For in- NMR11 ϫ CC107 15 stance, lymphocytic choriomeningitis viruses have be NMR ϫ BCC 34 described which are diploid for the virus S RNA seg- Note. NOR, Northway; GER, Germiston; BAT, Batai; MAG, Maguari; ments (Romanowski and Bishop, 1983). It is possible that BUN, Bunyamwera; LAC, La Crosse; SSH, Snowshoe Hare; JC, James- diploidy among segmented RNA viruses may be more town Canyon; NMR11, SNV-NMR11; CC107, SNV-CC107; and BCC, common than it is currently thought. Black Creek Canal. Table adapted from Pringle (1996), Bowen et al. In addition to the two strains of SNV that infect the (1995), and our data. same rodent host (P. maniculatus), we also tested the capacity for genetic reassortment between SNV NMR11 strain and the more distantly related BCCV, which has a In conclusion, this is the first direct evidence that different rodent host: the cotton rat (Sigmodon hispidus). reassortment can occur between rodent-borne hantavi- In this case, the frequency of reassortment observed ruses. Reassortants were frequently observed between was significantly lower than that observed between SNV closely related SNV strains NMR11 and CC107 that infect strains. Furthermore, in contrast to our mixed infections the same rodent host. However, they were rarely found with SNV NMR11 and CC107 strains, we did not notice a between SNV-NMR11 and BCCV, which infect different marked predominance of one of the parental genotypes. rodent hosts. The fact that viruses which use different rodent hosts are capable (albeit with low frequency) of Only one reassortant, with genotype NLBMNS, was de- tected among more than 163 plaques screened. This low genetic reassortment highlights the relevance that this frequency of reassortment might be explained by the fact genetic mechanism might have in the emergence of new that SNV-NMR11 and BCCV differ by over 30% in their hantaviruses in nature. nucleotide sequence for each of their three segments and by 16, 22, 16, and 15% in the amino acid sequences MATERIALS AND METHODS of N, G1, G2, and partial L proteins, respectively (Ravkov Virus and cells et al., 1995). This divergence is greater than that ob- served among insect-borne bunyaviruses that are capa- Three virus strains were used during this study; SNV ble of genetic reassortment (Table 7). The fact that the strains NMR11 and Convict Creek 107 (CC107) and only reassortant detected involved the M segment could BCCV. The origin and passage history of the viruses are be explained by the need for the intimate interaction of N shown in Table 1. Mixed infections were done by inocu- and L proteins (encoded by the S and L segments) for lating Vero E6 cells at a multiplicity of infection of 1.0±1.5 efficient replication of the virus, with heterologous L and for each of NMR11 and CC107 or NMR11 and BCCV. N proteins leading to less fit or nonviable viruses. Progeny virus from these mixed infections was collected It has been proposed that the ability of viruses to at 8 to 12 days after inoculation, divided into aliquots, and undergo genetic reassortment be included as part of the kept frozen at Ϫ70 C until it was used in plaque assays definition of virus species, i.e., if two viruses can reassort as described below. segments, then they should be considered members of Plaque assay and RNA extraction the same virus species (VanRegenmortel et al., 1997). If this were applied here, then BCCV and SNV would be Hantavirus plaques in tissue culture are difficult to considered one species, despite being clearly distinct visualize, and successful outcomes depend on factors viruses, as indicated by the differences in natural host such as cell type, passage, age of the cells, incubator reservoir, geographic distribution, and sequence diver- temperature, and concentration of CO2, among others. sity. Perhaps the use of reassortment as a species def- We optimized the following protocol for plaque assays inition criterion should be limited to those cases in which using SNV strains NMR11 and CC107. Monolayers of reassortment is observed under natural conditions. Vero E6 cells (between passages 15 and 30) in 6-well GENETIC REASSORTMENT AMONG HPS-CAUSING HANTAVIRUSES 105 tissue culture dishes were inoculated with 200 ␮l of virus REFERENCES dilutions and incubated for2hinaCO2incubator (37°C and a high-humidity atmosphere containing 5% CO ). 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The genetic basis for describing viruses as ents plus 0.12% neutral red was added on top of the species. Intervirology 24, 79±93. existing overlays at day 12 postinoculation. Individual Bishop, D. H. (1996). Biology and molecular biology of bunyaviruses. In plaques were collected when they became visible at ``The Bunyaviridae'' (R. M. Elliott, Ed.), pp. 19±61. Plenum, New York. Bishop, D. H., and Beaty, B. J. (1988). Molecular and biochemical days 14 to 16 postinoculation. Agarose plugs from each studies of the evolution, infection and transmission of insect bunya- plaque were resuspended in 300 ␮l of MEM containing viruses. Philos. Trans. R. Soc. London Ser. B: Biol. Sci. 321, 463±483. 5% fetal bovine serum (Hyclone). One-third of this Bishop, D. H., and Shope, R. E. (1979). Bunyaviridae. In ``Newly Char- suspension was mixed with 500 ␮l of guanidine thiocya- acterized Vertebrate Viruses'', Vol. 14, ``Comprehensive Virology'' (H. nate±phenol (Tri-pure reagent, Boehringer-Manheim, In- Fraenkel-Conrat and R. R. Wagner, Eds.), pp. 1±156. Plenum, New York. dianapolis, IN) and RNA was extracted according to the Bowen, M. D., Jackson, A. O., Bruns, T. D., Hacker, D. L., and Hardy, J. L. manufacturer's instructions. (1995). Determination and comparative analysis of the small RNA genomic sequences of California encephalitis, Jamestown Canyon, Primer design and multiplex RT±PCR Jerry Slough, Melao, Keystone and Trivittatus viruses (Bunyaviridae, genus Bunyavirus, California serogroup). J. Gen. Virol. 76, 559±572. Complete or partial sequences of the L, M, and S Childs, J. E., Ksiazek, T. G., Spiropoulou, C. F., Krebs, J. W., Morzunov, S., segments of SNV strains NMR11 and CC107 and from Maupin, G. O., Gage, K. L., Rollin, P. E., Sarisky, J., Enscore, R. E., Frey, J. K., Peters, C. J., and Nichol. S. T. (1994). Serologic and genetic BCCV were obtained from GenBank (Accession numbers identification of Peromyscus maniculatus as the primary rodent for L, M, and S segments, respectively, are for NMR11: reservoir for a new hantavirus in the southwestern United States. L37902-4; for CC107: L33474, L33683, and L35008; and J. Infect. Dis. 169, 1271±1280. for BCCV: L39949-51). Sequences were aligned using the Chizhikov, V. E., Spiropoulou, C. F., Morzunov, S. P., Monroe, M. C., Wisconsin Group Software (GCG). Primer pairs were Peters, C. J., and Nichol, S. T. (1995). Complete genetic characteriza- tion and analysis of isolation of Sin Nombre virus. J. Virol. 69, chosen that recognized specifically each virus individual 8132±8136. L, M, or S genome segment and yielded PCR products Elliott, L. H., Ksiazek, T. G., Rollin, P. E., Spiropoulou, C. F., Morzunov, S., discernible by their size on agarose gel electrophoresis. Monroe, M., Goldsmith, C. S., Humphrey, C. D., Zaki, S. R., and Krebs, Table 2 shows the sequences of each of the primers. J. W. (1994). Isolation of the causative agent of hantavirus pulmonary RNA extracted from each individual plaque was tested in syndrome. Am. J. Trop. Med. Hyg. 51, 102±108. Endres, M. J., Griot, C., Gonzalez-Scarano, F., and Nathanson, N. (1991). two separate multiplex reverse transcription±PCR (RT± Neuroattenuation of an avirulent bunyavirus variant maps to the L PCR) reactions, each containing the six specific primers RNA segment. J. Virol. 65, 5465±5470. for each parental strain. A one-tube RT±PCR was per- Gouvea, V., and Brantly, M. (1995). Is rotavirus a population of reassor- formed using a thermostable DNA polymerase from tants?. Trends Microbiol. 3, 159±162. Thermus thermophilus (GeneAmp EZ rTth RNA PCR kit, Griot, C., Pekosz, A., Lukac, D., Scherer, S. S., Stillmock, K., Schmeidler, D., Endres, M. J., Gonzalez-Scarano, F., and Nathanson, N. (1993). Perkin±Elmer, NJ). This enzyme performs both reverse Polygenic control of neuroinvasiveness in California serogroup transcription and DNA amplification. Amplification was bunyaviruses. J. Virol. 67, 3861±3867. done using a 9600 Perkin±Elmer thermocycler, with a Hampson, J. (1987). ``A Study of the Genetics and Growth of a Tripartite temperature profile of 50°C for 30 min (reverse transcrip- RNA Virus Using ts Mutants.'' Ph.D. thesis/dissertation, Department tion step), followed by 94°C for 2 min, and 35 cycles of of Biological Sciences, University of Warwick, UK. Henderson, W. W., Monroe, M. C., St. Jeor, S. C., Thayer, W. P., Rowe, 94°C for 15 s, 50°C for 60 s, and a final elongation step J. E., Peters, C. J., and Nichol, S. T. (1995). Naturally occurring Sin at 60°C for 10 min. Products were analyzed by electro- Nombre virus genetic reassortants. Virology 214, 602±610. phoresis on 2.5% agarose gels and visualized by Janssen, R. S., Nathanson, N., Endres, M. J., and Gonzalez-Scarano, F. ethidium bromide staining. (1986). Virulence of La Crosse virus is under polygenic control. J. Virol. 59, 1±7. Klimas, R. A., Thompson, W. H., Calisher, C. H., Clark, G. G., Grimstad, ACKNOWLEDGMENTS P. R., and Bishop, D. H. (1981). Genotypic varieties of La Crosse virus isolated from different geographic regions of the continental United We thank Dr. Connie Schmaljohn for providing SNV strain CC107, States and evidence for a naturally occurring intertypic recombinant Suyu Ruo and Chuck Fullhorst for providing initial plaque assay proto- La Crosse virus. Am. J. Epidemiol. 114, 112±131. cols, and Phillipe Calain for valuable discussions. J. H. Owens was Li, D., Schmaljohn, A. L., Anderson, K., and Schmaljohn, C. S. (1995). supported by the Summer Employment Program, Centers for Disease Complete nucleotide sequences of the M and S segments of two Control and Prevention. hantavirus isolates from California: Evidence for reassortment in 106 RODRIGUEZ ET AL.

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