INTRODUCTION

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Hantaan virus is the prototype of the genus Hantavirus, which belongs to the family Bunyaviridae (44). Hantavirus includes the etiologic agents of two distinct disease syndromes in humans, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which are transmitted from rodents to humans (40). Old World hantaviruses, such as the Hantaan (HTN), Seoul, Dobrava, Puumala, and related viruses cause HFRS, which is characterized by fever, renal failure, and, in severe cases, hemorrhagic manifestations. The severity of HFRS is dependent on the serotype of the causative virus. New World hantaviruses, Sin Nombre and related viruses, cause HPS, which is characterized by severe acute respiratory dysfunction and a mortality rate of 50% (10, 34, 62). Each hantavirus is primarily associated with a specific rodent species. Many field observations and epizootiological studies have shown that hantaviruses cause chronic infections without signs of disease in their reservoirs (40).

HTN virus causes a severe form of HFRS in humans. It is an acute prostrating febrile illness with renal failure, in which one-third of patients develop hemorrhagic manifestations and 10 to 15% develop shock. In addition, the mortality rate of severe HFRS is 5 to 10% (37, 49). The pathology of HFRS is multisystemic. The spread and tropism of the virus in a patient's body, and immune-related pathogenesis and responses, are thought to be important factors in an HFRS crisis (7, 18, 24, 54, 55). Despite many investigations, little is known about the pathogenesis of HTN virus infection in humans (HFRS), since a suitable animal model for the disease in humans has not been reported. In animal experiments, only neonatal animals and immunologically deficient animals, such as nude mice and severe combined immune deficient (SCID) mice, die after inoculation with HTN virus (32, 59). Moreover, in contrast to Puumala virus and Prospect Hill virus (15, 56), attempts to induce HFRS manifestations in monkeys using HTN virus have been unsuccessful. Previously, several groups studied the pathogenesis of HTN virus in newborn mice (23, 26, 29). Although newborn mice develop fatal illness, resistance to the disease increases with age (33). In newborn mice experimentally infected by any inoculation route, the virus causes a widespread infection with high titers of virus in almost all organs. Moreover, the site of viral multiplication is endothelial cells and monocytes/macrophages, just as in human infection (16, 26, 31, 36, 49, 57, 62). Similar to fatal and severe cases of human HFRS (1, 2, 6), infected mice develop inflammatory and destructive lesions in various organs and die within 2 to 4 weeks, with wasting and neurological signs (23, 26, 33). Several studies suggest that these lesions are immune mediated (29, 32, 59). These investigations suggest that a newborn mice model may be useful for studying the spread of HTN virus in a host body and the pathogenesis of acute systemic infection.

Similar to other bunyaviruses, the hantavirus genome consists of three negative-strand RNA segments, the large (L), medium (M), and small (S) segments, which encode RNA-dependent RNA polymerase (L protein), two surface glycoproteins (G1 and G2), and nucleocapsid protein (NP), respectively (43, 45, 46). The 3' and 5' termini of the hantavirus genome segments are complementary to each other and highly conserved. The complementary sequences at the 3' and 5' termini of each segment are capable of forming stable base-paired panhandle structures and probably are the basis of the noncovalently closed circular RNA structures. The panhandles of hantaviruses are thought to play a role in regulating viral transcription and replication and to serve as recognition sites for viral RNA polymerase (12, 41, 42).

Unlike other viruses that cause disease in animals, there are few studies of the molecular basis of HTN virus virulence. Tamura et al. reported two plaque-purified variant clones derived from strain HTN virus 76-118: a highly virulent clone 1 (cl-1) against newborn mice and an avirulent clone 2 (cl-2) (47). Sequencing analysis of these viruses suggested that a single amino acid substitution in the G2 glycoprotein determined virulence in mice (20). Several similar studies of other members of the Bunyaviridae (LaCrosse and Rift Valley fever virus) involving genetic reassortants between virulent and avirulent strains have been reported (11, 14, 21, 52). In segmented-RNA viruses, study of the genetic reassortment between different virulence strains can be used to determine the genome segment associated with their virulence. However, further characterization of the genetic determinants relating to pathogenesis is difficult when there are numerous nucleotide differences between the virulent and avirulent strains. Recently, reverse genetics systems were established for negative-stranded RNA viruses, allowing characterization of the molecular basis of viral pathogenesis and of their biological properties. However, no such system has yet been established for bunyaviruses except for Bunyamwera virus (5). Therefore, reassortment between monoclonal antibody (MAb)-resistant variants is also effective for genetic mapping of virulence. Recently, we generated a mutant virus (designated mu11E10) from HTN virus strain cl-1 by selection with neutralizing MAb 11E10 to a glycoprotein G2 (3). mu11E10 was highly attenuated against newborn mice. Sequencing of the M segment of mu11E10 showed that this involved a single amino acid substitution: isoleucine (cl-1) to threonine (mu11E10) in position 515 of the G1 glycoprotein (22). However, except for the coding region of the mutant M segment, the entire nucleotide sequences of the L, M, and S genome segments have yet to be determined. Therefore, the significance of the single amino acid change in glycoprotein G1 in virulence is unclear.

In this study, we compared the pathogenesis of HTN virus in newborn mice infected with virulent (cl-1) and attenuated (mu11E10) strains. In a recent study, Rodriguez et al. successfully generated genetic reassortment among viruses causing HPS (38), although no study of the generation of HTN virus reassortants in vitro has been reported. Therefore, this is the first report of the generation of genetic reassortant viruses between the two viruses, and it identifies the genetic determinant related to their virulence.

	MATERIALS AND METHODS

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Infectivity titration and cell culture of viruses. Two strains of HTN virus that differ in virulence to neonatal mice, cl-1 (virulent) (47) and mu11E10 (22) (attenuated), were used in this study. Cl-1, which is derived from strain 76-118, was provided by K. Yamanishi, Osaka University Medical School, Osaka, Japan. Cl-1 was passaged two times in Vero E6 cells cultured in our laboratory and used as the stock virus. The entire RNA genome sequence of cl-1 used in this study was determined (Table 1) and compared with the genome sequence of cl-1 published by Isegawa et al., since the passage history of strain cl-1 propagated in our laboratory differed from the published one (20). There were seven and six nucleotide differences in the L and M segment sequences between the published cl-1 and the cl-1 used in this study, respectively. An amino acid substitution was observed in the L segment (data not shown). Strain mu11E10 was generated as a neutralizing MAb escape mutant from the cl-1 used in this study, using MAb to envelope protein G2 (11E10) (3), as described previously (22). Working stocks of both strains were prepared from the culture supernatants of Vero E6 cells infected with each virus.

Mice and animal experiments. Specific-pathogen-free pregnant inbred BALB/c/slc mice were obtained from SLC (Hamamatsu, Japan). Within 24 h after birth, neonatal mice were inoculated subcutaneously (s.c.) or intracerebrally (i.c.) with 1,000 FFU of cl-1, mu11E10, or genetic reassortants between the two parental viruses. The mortality, clinical signs, and body weight of each of the 5 to 41 mice in each group inoculated were recorded for days 4 to 35 after inoculation.

Histological studies and immunohistochemistry. The organs were collected from two mice at 2- or 4-day intervals from 4 to 22 days after inoculation. The mice were anesthetized and killed by exsanguination. Brains, lungs, spleens, and kidneys were excised aseptically. Whole organ tissue was fixed in Bouin's solution for 24 h at 4°C; the specimens were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin.

Virus titration in organs of mice infected with cl-1 and mu11E10. Two mice were sacrificed at 2- to 4-day intervals from days 4 to 22 after inoculation. The mice were anesthetized and killed by exsanguination, and blood samples were collected at the same time. Brains, lungs, spleens, and kidneys were excised aseptically. The organs were frozen and thawed once, and 10% homogenates were prepared with PBS. The 10% homogenate was centrifuged at 6,000 rpm for 5 min at 4°C, and the supernatant was stored at 80°C until assayed. The infectivity titers of the supernatants were assayed by measuring focus formation (see above) in serial 10-fold dilutions on Vero E6 cell monolayers in 96-well plastic plates.

Detection of serum viremia in infected mice by semiquantitative RT-PCR. To optimize the conditions of reverse transcription-PCR (RT-PCR) for semiquantitation of the virus titer in the serum of infected mice, we first determined the numbers of cycles of PCR required to amplify viral RNA (vRNA) corresponding to the virus titers. RNA was extracted from serially diluted 250-µl culture supernatants including 10⁵ to 10 FFU of viruses (cl-1) using Isogen-LS (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions, and the extracted RNA was resuspended in 5 µl of diethylpyrocarbonate treated water. To synthesize the cDNA of the S segment genome RNA, we used a specific oligonucleotide primer (cm-S459F; 5'-GACAACAAGGGGGAGGCAAACTACCAAGG-3') complementary to the HTN virus S segment vRNA. vRNA isolated from culture supernatants (virus titer, 10⁵ to 10 FFU) and 2.5 µM cm-S459F in 20 µl of solution, containing First-Strand Buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl₂; Gibco BRL), 20 mM dithiothreitol, and 0.5 mM deoxynucleoside triphosphates, was incubated with 200 U of Superscript II RNase H reverse transcriptase (Gibco BRL) at 55°C for 1 h, 70°C for 10 min, and then at 99°C for 1 h. Specific oligonucleotide primers for S segment cm-S459F and cm-S675R (5'-CCGTGCCTTAATCTGTGCAGGGTAGAGCCC-3') were used to amplify a 216-bp PCR product of the S segment. PCR amplification was carried out with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Branchburg, N.J.) using a RoboCycler gradient 96 temperature cycler (Stratagene, La Jolla, Calif.) for 40 to 18 cycles in one-cycle steps, each consisting of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The reaction mixture was made up of PCR buffer II (Perkin-Elmer), 1.6 mM MgCl₂, 1 mM deoxynucleoside triphosphates, and 0.5 U of AmpliTaq Gold. The PCR products were electrophoresed on 2% agarose gels containing ethidium bromide, and the optimal number of PCR cycles for detecting PCR products corresponding to each virus titer was determined. The optimum numbers of PCR cycles for detecting vRNA were 35, 33, 31, 29, and 23 cycles for 10, 10², 10³, 10⁴, and 10⁵ FFU, respectively. The relationship between the number of PCR cycles and the viral titer did not change in two independent experiments. To confirm whether these conditions for RT-PCR would detect vRNA in mouse sera, we performed the same RT-PCR study using a 1:1 mixture of culture supernatants (viral titers, 10 to 10⁵ FFU) and normal mouse serum. RNA was extracted from untreated mixtures and mixtures that were heat inactivated at 56°C for 30 min. No change in the relationship between the number of PCR cycles and the virus titers of the mixtures was observed with the different conditions. Thus, we were able to detect serum viremia in infected mice using a semiquantitative RT-PCR method.

Detection of serum IFA antibodies in infected mice. IFA was carried out using acetone-fixed smears of Vero E6 cells infected with HTN virus as antigen. Fluorescein isothiocyanate-conjugated goat anti-mouse IgG [IgA + IgG + IgM (H + L); Zymed] was used as the second antibody. The IFA titers were expressed as the reciprocal of the highest dilution of the antisera that resulted in a specific immunofluorescence in the cytoplasm of the infected cells.

Sequencing analysis of cl-1 and mu11E10. Total cellular RNA was extracted from Vero E6 cells infected with each stock virus (cl-1 and mu11E10) using Isogen (Nippon Gene) according to the manufacturer's instructions. cDNA representing the complete HTN virus L, M, and S segments was synthesized using a synthetic oligonucleotide complementary to 14 bases of the 3' end of the vRNA sequence conserved in all segments (14-Primer; 5-TAGTAGTAGACTTC-3') (46). The reverse transcription procedure was as described above. Primers designed from the published sequence of cl-1 were used to amplify several overlapping cDNA fragments of the L, M, and S genome segments by PCR. The PCR conditions were those described above. The PCR products were separated on 1% agarose gels, and the fragments were excised and purified using a GeneClean III kit (Bio 101, La Jolla, Calif.) according to the manufacturer's instructions. Direct sequencing of the purified PCR products was then performed using the same or newly synthesized primers used to amplify the fragments. Purified PCR products were sequenced by the dideoxy-chain termination method using an ABI PRISM dye terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.). The samples were sequenced on a model 373A or 377 DNA sequencing system (Perkin-Elmer, Applied Biosystems Division). Sequence data were analyzed using GENETYX-MAC sequence analysis software (Software Development Co., Ltd.) on a Macintosh computer. Two independent sequencing analyses were done.

Generation and recovery of genetic reassortants between cl-1 and mu11E10. Genetic reassortants between cl-1 and mu11E10 were generated by coinfection of Vero E6 cells (2.5 × 10⁶ cells/T-25 flask) at multiplicties of infection (MOIs) of 0.004 for cl-1 and 0.012 for mu11E10. At 72 h after coinfection, the supernatants were harvested and plaque purified on CV-7 cells. Briefly, the supernatants were adsorbed on a monolayer of CV-7 cells grown in six-well plastic plates (2 × 10⁵ cells) for 1 h at 37°C. After adsorption, cells were overlaid with 2 ml of growth medium supplemented with 10% FBS, 2 mM L-glutamine, and 0.1 mM MEM nonessential amino acids solution (Gibco BRL) containing 0.75% SeaKem ME agarose (FMC BioProducts, Rockland, Maine) and incubated for 9 days at 37°C in 5% CO₂. After the first overlay incubation, the monolayers were stained by the addition of a 2-ml layer of the same agarose-medium solution containing 5% neutral red solution (Gibco BRL). Plaques were visualized 2 to 3 days later, and well-separated plaques were harvested by plunging a pipette directly into the agarose. The viruses were inoculated on Vero E6 cells grown in a 96-well plastic plate for 7 days and then passaged to Vero E6 cells in 24-well plates. Since the titers of plaque-purified viruses were low, we used soybean agglutinin (SBA), which is an N-acetylgalactosamine-specific lectin, to enhance virus adsorption to cells, as previously described (35). The mixture of culture supernatant harvested from Vero E6 cells in the 24-well plates and 500 µg of SBA (Vector Laboratories) per ml was incubated for 30 min and adsorbed for 60 min on Vero E6 cells in T-25 flasks at 37°C in 5% CO₂. After incubation for 7 days on Vero E6 cells in T-25 flasks, the culture supernatants were harvested, treated with SBA, and passaged into Vero E6 cells in T-150 flasks. The culture supernatants of Vero E6 cells in the T-150 flasks were used as the working stock virus.

Viral growth kinetics in putative target cells in vitro. (i) Mouse brain microvascular endothelial cells (MBMEC; Cell Applications, Inc.) were grown in MBMEC growth medium with growth supplement (Cell Applications). MBMEC (3.0 × 10⁶ cells in T-75 flasks) were infected at an MOI of 0.03. Culture supernatants were harvested at 1- to 2-day intervals on days 1 to 8 after infection. The growth medium was exchanged at 4-day intervals.

Statistics. The significance of differences in mortality and mean day of death was determined using Fisher's exact probability test and Student's t test.

	RESULTS

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Comparison of virulence of cl-1 and mu11E10. To compare the virulence of cl-1 and mu11E10, newborn BALB/c mice were inoculated s.c. or i.c. with 1,000 FFU of cl-1 or mu11E10 within 24 h after birth. The mortality, clinical signs, and body weight of each of the inoculated mice were recorded from days 4 to 35 after inoculation (Fig. 1). The survival rates at 35 days for cl-1 and mu11E10 after s.c. inoculation (Fig. 1A) were 11 and 100%, respectively (P < 0.001). Thus, we confirmed the significant difference in the virulence of cl-1 and mu11E10 described by Kikuchi et al. (22). The clinical features of mice infected with cl-1 first became apparent 2 weeks after s.c. inoculation. By days 13 to 14, the coats of the mice began to appear ruffled, and the animals were hyperexcitable. Paralysis of the hind limbs, hunched posture, and progressively diminishing mobility appeared by days 15 to 18. The first deaths occurred 3 weeks after inoculation. In contrast, mice inoculated with mu11E10 did not exhibit clinical signs, except for the appearance of ruffled fur by days 13 to 14. The mice recovered from their illness by day 20 to 25. Furthermore, the respective body weights of mice infected with cl-1 and mu11E10 were 50 and 80% that of control mice (Fig. 1C). Therefore, the difference in body weights of the mice infected with each virus was also consistent with their clinical course and mortality.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Survival and body weights of newborn mice infected s.c. and i.c. with cl-1 or mu11E10 and of uninfected (control) mice. Newborn BALB/c mice were inoculated s.c. or i.c. with 1,000 FFU of cl-1 (n = 18 [s.c.] and 7 [i.c.]) or mu11E10 (n = 7 [s.c. and i.c.]) within 24 h of birth. Mortality (A and B) and body weight (C and D) were recorded for days 4 to 35 (body weight, until day 30) after inoculation. Crosses indicate that all mice infected with HTN virus died. Each point is the mean weight of two mice. Vertical bars indicate the range of values. The range of values for 4 to 22 days after inoculation is ±0.3 g (C and D).

Relationship between histopathological changes and pathogenicity. To explain the difference in the pathogenesis of mice infected with cl-1 and mu11E10, we examined the pathological changes in the organs of mice infected s.c. with either virus. Necrosis of neurons, infiltration of inflammatory cells in the cerebral cortex, and meningoencephalitis were observed to be more intense in the brains of mice infected with cl-1 (Fig. 2A) than in those of mu11E10-infected mice (Fig. 2B). Necrosis of neural cells was evident after day 14 in cl-1- and mu11E10-infected mice. These histopathological changes in the brain seemed to correlate to the clinical course of mice infected with HTN virus. No marked histopathological changes were observed in the lung, kidney, or spleen of cl-1- and mu11E10-infected mice (data not shown).

View larger version (91K): [in this window] [in a new window]   FIG. 2.   Histopathological changes of mouse brain after s.c. inoculation of cl-1 (A) or mu11E10 (B) and normal control mice (C). Brains were removed, fixed, sectioned, and stained with hematoxylin and eosin as described in Materials and Methods. (A) Cl-1-infected mouse brain showing intense necrosis of neurons and infiltration of inflammatory cells in the cerebral cortex and meningoencephalitis. (B) Mutant 11E10-infected mouse brain showing mild sclerotic lesions and necrosis of neural cells in the brain, which was less intense than that observed in cl-1-infected mice. (C) No changes were observed in the normal mouse brain. These photographs were taken at an original magnification of ×50.

Difference in the in vivo level of viral multiplication and tropism in tissues between cl-1 and mu11E10. To characterize the growth kinetics and tropism of cl-1 and mu11E10, we compared the time of detection and the level of viral antigen in several organs of mice inoculated s.c. with either virus (Fig. 3). In addition, the virus titers were determined in tissues of mice infected s.c. and i.c. (Fig. 4).

View larger version (126K): [in this window] [in a new window]   FIG. 3.   Distribution of NP in the tissues of mice infected with cl-1 or mu11E10. The brains, lungs, kidneys, and spleens were removed from two mice infected s.c. with each virus, fixed, sectioned, and immunostained with anti-HTN virus MAbs and serum, using the PAP or ABC method as described in Materials and Methods. The brain and lung sections obtained on day 14 after inoculation and the kidney and spleen sections obtained on day 10 after inoculation are shown. The antigen-positive foci rapidly spread widely in every tissue of cl-1-infected mice compared with the tissues of mu11E10-infected mice. Viral antigen was detected in the neurons and glial cells from the brains of infected mice (A and B). In the lung, viral antigen was detected in alveolar epithelial cells and endothelial cells (C and D). In the kidney, viral antigen was detected in tubulointerstitial endothelial cells, fibroblasts (E and F), and mesangial cells (F). In the spleen, viral antigen was detected in splenic sinus endothelial cells and macrophage-like cells (G and H). Organs from mock-infected mice showed no positive staining for viral antigen (data not shown). The magnification is shown in each photograph.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Virus titers in the tissues of newborn mice infected s.c. and i.c. with cl-1 and mu11E10. Newborn mice were inoculated s.c. or i.c. with 1,000 FFU of cl-1 or mu11E10. Two mice were sacrificed at 2- to 4-day intervals from days 4 to 22 after inoculation. The brain (A) lung (B), kidney (C), and spleen (D) were collected and stored at 80°C. Crosses show that all mice infected with HTN virus died. Each point is the mean titer for two samples. Vertical bars indicate the range of values.

Relationship between serum virus titer and antibody response with stage of infection. To further characterize the mechanism of viral spread in vivo, we measured vRNA in the sera as a viral titer, using a semiquantitative RT-PCR system (described in Materials and Methods) and IFA antibody titers, in mice infected either s.c. or i.c. In the late stage of infection, direct measurement of serum virus titers in infected mice by neutralization of virus with increasing serum antibody is difficult. Therefore, we used the level of vRNA measured by semiquantitative RT-PCR system as an indicator of the virus titer.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   vRNA and IFA antibody titers in sera of mice infected s.c. (A and C) or i.c. (B and D) with cl-1 (circles) or mu11E10 (squares). The serum was collected from the infected mice described for Fig. 4. (A and B) Correlation of the amount of viral RNA to virus titer; (C and D) IFA antibody titers. Optimization of the RT-PCR conditions for semiquantitation of virus titers in infected mice sera is described in Materials and Methods. IFA antibody was measured by assay using acetone-fixed smears of Vero E6 cells infected with HTN virus as antigen.

Molecular differences between cl-1 and mu11E10. To further ascertain the possible molecular basis for the difference in the virulence of the two viruses, we determined the complete nucleotide sequences of all the genome segments (L, M, and S) of both cl-1 and mu11E10. Table 1 shows the nucleotide and deduced amino acid substitutions for cl-1 and mu11E10. Nucleotides are indicated by reference to the cDNA sense sequence. There are three nucleotide differences in the L segment and one in the M segment. There were no nucleotide or amino acid substitutions in the S segment. In the L segment, two of three nucleotide changes located in the open reading frame were synonymous substitutions (position 2038, G in cl-1 and C in mu11E10; position 3373, T in cl-1 and C in mu11E10). Another nucleotide substitution was located in the noncoding region in the 5' termini of the vRNA (position 6497, T in cl-1 and C in mu11E10). In the M segment, there was only one nucleotide substitution (position 1584, T in cl-1 and C in mu11E10); this resulted in an amino acid substitution at position 515 in glycoprotein G1 (isoleucine in cl-1 and threonine in mu11E10).

Generation of genetic reassortants between cl-1 and mu11E10. Since there were molecular differences between cl-1 and mu11E10 in the L and M segments, we generated genetic reassortants between the two viruses to identify the segment associated with virulence in newborn mice.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   Determination of the genotype of reassortants between cl-1 and mu11E10 by restriction endonuclease digestion (A) and a schematic diagram of genotype screening of reassortants by restriction endonuclease digestion (B). (A) L and M, L and M RNA segments, respectively; c and m, genome segments derived from cl-1 and mu11E10, respectively. Lanes 1 through 4 show the restriction enzyme digestion patterns of the parent viruses (DdeI, SspI, DdeI, and SspI, respectively). Lanes 5 through 8 show genetic crosses between cl-1 and mu11E10. Lanes 5 (DdeI digested) and 6 (SspI digested) show the patterns for RC-08, which is a representative clone of genotype m c reassortants. Lanes 7 (DdeI digested) and 8 (SspI digested) show the patterns for RC-37, which is a clone of a genotype c m reassortant. Lane M, marker DNA fragments with sizes as indicated. (B) Genotypes of reassortants were determined by restriction endonuclease digestion of the RT-PCR products (L [220 bp] and M [619 bp]). Restriction endonucleases DdeI and SspI recognized strain-specific mutations on the L (2,038 nt) and M (1,584 nt) segments, respectively.

Determination of the genome segment associated with virulence. To identify the genome segment associated with virulence in mice, the reassortants described above were inoculated s.c. and i.c. into newborn mice. The virulence of reassortants was determined using the same procedure described above. The survival rates at 35 days for RC-08 (m c) and RC-37 (c m) after s.c. inoculation (Fig. 7A) were 15 and 78%, respectively (P < 0.001). The mortality rate and clinical course of mice infected with RC-08 (m c) were similar to those of mice infected with parental cl-1 (Fig. 1A and 7A). RC-37 (c m) showed a significantly attenuated phenotype against mice compared with RC-08 (m c) but was slightly more virulent than parental mu11E10. These results indicate that the M genome segment is the most related to virulence, and the L segment is partly related in peripheral route infection.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   Survival curve of newborn mice infected with reassortants (A) and viral multiplication in MBMEC (B). (A) Newborn BALB/c mice were inoculated s.c. or i.c. with 1,000 FFU of RC-08 (n = 41 [s.c.] and 6 [i.c.]) or RC-37 (n = 34 [s.c.] and 5 [i.c.]) within 24 h after birth, and mortality was recorded from days 4 to 35 after inoculation. (B) MBMEC were infected at an MOI of 0.03 with cl-1, mu11E10, RC-08, or RC-37. Culture supernatants were harvested 1, 2, 3, 4, 6, and 8 days after infection, and the virus titer was determined by focus-forming assay on Vero E6 cells. Points are the mean titer for two samples. Vertical bars indicate the range of values.

Relationship between the genetic determinant of virulence in vivo and viral multiplication in putative target cells in vitro. The in vivo analysis of virulence suggested that the M segment from mu11E10 contributed to the attenuation of virulence in newborn mice inoculated s.c. To investigate the mechanism for viral spread from peripheral tissues to the central nervous system (CNS), MBMEC (Fig. 7B), PEC, and neuroblastoma (NA) cells (data not shown) were infected with the reassortants and parental viruses, and their patterns of growth were compared. In MBMEC, the titer of viruses possessing the M segment from cl-1 (cl-1 and RC-08) was approximately 10-fold (10³ FFU) higher than that of viruses containing the M segment from mu11E10 (mu11E10 and RC-37) (Fig. 7B). In PEC collected from BALB/c mice, cl-1 and RC-08 grew with titers 4- to 10-fold higher than those of mu11E10 and RC-37 in the early period of growth. From day 4 after inoculation, however, only cl-1 maintained a higher titer (data not shown). In NA cells, the highest virus titers of all the viruses were too low to compare (<10² FFU) (data not shown). These results indicate that viruses that possessed the M segment from cl-1 grew with higher titers in MBMEC than attenuated viruses containing the M segment from mu11E10. Therefore, there was a correlation between viral multiplication in peripheral target cells and virulence associated with the M genome segment in vitro and in vivo.

	DISCUSSION

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In this study, newborn mice infected with virulent and attenuated HTN viruses were compared to examine the mechanism of pathogenesis in the mouse model. In addition, we generated genetically reassorted viruses from virulent and attenuated viruses to examine the genetic determinants related to virulence.

Pathogenesis of strains cl-1 and mu11E10 in newborn mice. Several groups have reported that HTN virus causes neurological disease in newborn mice and that the brain is the major target organ (26, 29). Our study confirmed these findings by comparing the pathogenesis of a virulent virus and its attenuated mutant. When the viruses were inoculated via a peripheral route (s.c.), only the virulent cl-1 virus caused lethal infection, while all the mice infected with mu11E10 survived. The brains of cl-1-infected mice showed more severe histopathological changes than did those of mu11E10-infected mice. No apparent histopathological difference was observed in other organs. On the other hand, after i.c. inoculation, both viruses produced severe histopathological changes (data not shown) and all mice died. Therefore, the severity of lesions in the brain is thought to be the critical factor determining mortality in mice.

Genetic determinants of HTN virus virulence. In this study, we produced genetically reassorted viruses from the parental virulent virus and its attenuated mutant, to examine the genetic determinants related to the virulence of HTN virus in the mouse. The survival rates after peripheral inoculation (s.c.) showed the M segment to be the most related to virulence and the L segment to be partly related. A single deduced amino acid substitution was detected at amino acid 515 of G1 protein, changing Ile in cl-1 to Thr in mu11E10. This amino acid is thought to be a major determinant of the greater viral multiplication in peripheral tissues in newborn mice. Since the survival of mice infected via the i.c. route with reassortant virus that possessed the mu11E10-derived L segment was significantly longer than that of the parental cl-1 (Table 1, P < 0.001), the L segment is thought to be a determinant of neurovirulence. Since the only nucleotide substitution found was in the 5' noncoding region, the difference in neurovirulence might be related to the efficiency of L RNA synthesis rather than to altered function of the L protein.

Genetic reassortment of HTN virus. Recently, Rodriguez et al. reported genetic reassortment among viruses causing HPS in an in vitro system (38). Several sets of genetic epidemiological data also suggest naturally occurring genetic reassortment in the field (17, 27). There are no reports of genetic reassortment of HTN virus in nature or in vitro. Our paper is the first report of artificial genetic reassortment by coinfection in vitro.