Development of Infectious Clones for Virulent and Avirulent Pichinde Viruses: a Model Virus To Study Arenavirus-Induced Hemorrhagic Fevers

Abstract

Several arenaviruses can cause hemorrhagic fever diseases (VHFs) in humans, the pathogenic mechanism of which is poorly understood due to their virulent nature and the lack of molecular clones. A safe, convenient, and economical small animal model of arenavirus hemorrhagic fever is based on guinea pigs infected by the arenavirus Pichinde (PICV). PICV does not cause disease in humans, but an adapted strain of PICV (P18) causes a disease in guinea pigs that mimics arenavirus hemorrhagic fever in humans in many aspects, while a low-passaged strain (P2) remains avirulent in infected animals. In order to identify the virulence determinants within the PICV genome, we developed the molecular clones for both the avirulent P2 and virulent P18 viruses. Recombinant viruses were generated by transfecting plasmids that contain the antigenomic L and S RNA segments of PICV under the control of the T7 promoter into BSRT7-5 cells, which constitutively express T7 RNA polymerase. By analyzing viral growth kinetics in vitro and virulence in vivo, we show that the recombinant viruses accurately recapitulate the replication and virulence natures of their respective parental viruses. Both parental and recombinant virulent viruses led to high levels of viremia and titers in different organs of the infected animals, whereas the avirulent viruses were effectively controlled and cleared by the hosts. These novel infectious clones for the PICV provide essential tools to identify the virulence factors that are responsible for the severe VHF-like disease in infected animals.

Arenaviruses are enveloped RNA viruses with single-stranded ambisense RNA genomes (6). The genomes of these viruses consist of two RNA segments, the large (L) segment of ∼7.2 kb and the small (S) segment of ∼3.4 kb (6). The L RNA segment encodes the viral polymerase L protein in a negative orientation and a small multifunctional Z protein in a positive sense. The S RNA segment encodes the nucleoprotein NP in a negative orientation and the envelope glycoprotein precursor GPC in a positive sense (6). The terminal 19 nucleotides (nt) from both ends of the segments are imperfectly complementary to each other and are predicted to form the panhandle structures that serve as the cis-acting elements required for viral RNA transcription and replication (15, 23, 24, 33). A unique feature of the arenavirus genomic RNAs is the noncoding intergenic regions located between the two open reading frames (6). They range from 59 to 217 nt in length and are predicted to form one to three energetically stable stem-loop structures (6, 40) that are proposed to contribute to transcriptional termination. Currently, the only available molecular clone of arenavirus was developed for the prototype lymphocytic choriomeningitis virus (LCMV) (13, 38). This system has served as an invaluable tool to study the biological functions of arenavirus proteins and RNA elements.

Several arenaviruses have been known to cause deadly hemorrhagic fever diseases (VHFs) in humans, including Lassa fever that is endemic in West Africa (12, 27). The disease is caused by an infection of Lassa fever virus, an Old World arenavirus that is usually found in the rodent Mastomys natalensis (29). The consequences of human Lassa fever virus infection may range from asymptomatic to severe multisystem disease that is clinically indistinguishable from other febrile illnesses, such as Ebola fever and dengue (28). Besides Lassa virus, several other arenaviruses (i.e., Junin, Machupo, Guanarito, Sabia, and Chapare viruses) are known to cause VHFs in humans (10, 14). Currently, there are no effective vaccines, except one for Junin virus, and only limited treatment options for these pathogenic arenaviruses. Therefore, these highly pathogenic arenaviruses must be handled in biosafety level 4 (BSL-4) facilities.

The pathogenesis of arenavirus-induced VHFs is poorly understood. Despite the presence of high virus titers in a wide range of tissues and organs, the pathological lesions are generally not severe enough to explain the cause of death (39). Nevertheless, high viremia level is closely associated with poor prognosis (20). Several animal models for Lassa fever have been developed in rodents and nonhuman primates, which have provided invaluable information on disease pathogenesis and have been used as important tools to evaluate the efficacies of vaccines and antiviral treatments (5, 7, 8, 17, 19, 26, 30, 34, 36). Experiments done on these animals, especially on nonhuman primates, are expensive and restricted to BSL-4 laboratories. A safe, convenient, and economical small animal model for Lassa fever has been developed by Jahrling and colleagues, which is based on guinea pigs infected by a nonpathogenic arenavirus, the Pichinde virus (PICV) (1, 18). PICV is a BSL-2 agent that does not cause disease in humans. Guinea pigs infected with a long-term spleen-passaged PICV strain developed a severe disease mimicking Lassa fever in many respects (18, 25, 37). Both types of infection resulted in a fulminating disease course with terminal vascular leak syndrome (21). Viral distribution in infected tissues appears to be identical in both Lassa virus and PICV infections (18, 39). The viremia level is closely associated with the disease outcome (1, 20). Like those found in fatal Lassa virus infections, histopathological findings in lethally infected animals by PICV generally could not explain the main cause of death of the animals (9, 18, 25, 35, 39). Generalized immune suppression is also seen in both types of infection (2, 4, 11).

In contrast to the highly virulent strain P18 of PICV that has been passaged in a guinea pig spleen 18 times, the low-spleen-passage strain P2 causes a limited febrile illness in the infected animals (18, 41). We have recently reported the complete genomic sequences of both the P2 and P18 strains, which differ by 48 nt and 9 amino acids that are localized to the GPC, NP, and L genes (22). In order to determine the molecular determinants of virulent PICV infection, we have decided to build reverse genetics systems for both the P2 and P18 strains. These novel molecular clones have allowed for the generation of recombinant P2 and P18 (rP2 and rP18, respectively) viruses that can recapitulate the parental viruses in terms of viral growth kinetics in vitro and virulence in vivo. The availability of these systems allows for an opportunity to characterize viral virulence determinants in vivo in order to understand the molecular mechanisms involved in VHF pathogenesis.

MATERIALS AND METHODS

Cells, viruses, and antibodies.

BSRT7-5 cells, which stably express the T7 RNA polymerase, were obtained from Conzelmann (Ludwig-Maximilians-Universität, Germany) and cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1 mg Geneticin per ml, and 50 mg penicillin and streptomycin per ml. We obtained the avirulent P2 and virulent P18 PICV from Aronson (UTMB) (41). Both the P2 and P18 viruses originated from the Pichinde Munchique strain CoAn4763 with different passage numbers. The P2 strain was the spleen stock of CoAn4763 after passaging twice in guinea pigs. The P18 strain was derived from Jahrling's original passage-adapted CoAn4763 adPIC virus with a total of 18 successive passages in inbred guinea pigs (18).

Generation of infectious PICV from plasmid transfection.

Using Lipofectamine 2000 reagent (Invitrogen), BSRT7-5 cells seeded in six-well plates were transfected with plasmids bearing the L and S RNA segments (the construction of these plasmids is described in the supplemental material). The supernatant was replaced with fresh medium at 4 h posttransfection, harvested at different time points posttransfection, and used for plaque assaying on Vero cells.

PICV plaque assay.

Vero cells were seeded into six-well plates at 90 to 100% confluence and infected with 0.5 ml of serial 10-fold dilutions of viruses in MEM for 1 h at 37°C. After removing the medium, the cells were incubated in fresh MEM supplemented with 0.5% agar and 10% FBS and cultured for 4 days at 37°C. Plaques were stained overnight with diluted neutral red solution (1:50) in 0.5% agar-MEM-10% FBS.

Growth curve analysis.

Cells were seeded in six-well plates at 90 to 100% confluence and infected (in triplicate) with viruses at a multiplicity of infection (MOI) of 0.01 for 1 h at 37°C. After the cells were washed with PBS, a fresh aliquot of medium was added to the culture. At different times postinfection, aliquots of the supernatant were harvested for plaque assaying on Vero cells.

Isolation and culture of primary guinea pig peritoneal macrophages.

All animal experiments were conducted according to the guidelines and approved protocol of the Emory University Institutional Animal Care and Use Committee (IACUC). A healthy outbred Hartley guinea pig was injected with 3 ml of mineral oil (Sigma) 1 week prior to peritoneal macrophage isolation. After the guinea pig was euthanized, macrophages were obtained from intraperitoneal lavage fluid in a buffer that contains 5% FBS and 1% penicillin-streptomycin. After being resuspended in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin, the macrophages were allowed to settle into the culture dish overnight. After removing the suspended cells, the attached cell population was determined to be >90% macrophages by flow cytometric analysis using a macrophage-specific antibody, MCA518S (Serotec).

Guinea pig experiments.

We followed the experimental procedures described originally by Aronson and colleagues (38, 42). Briefly, healthy 350- to 400-g male outbred Hartley guinea pigs were housed for 5 to 7 days for acclimatization, after which they were randomly divided into different groups and injected intraperitoneally with 10,000 PFU of each of the virus strains or PBS as a mock infection. Rectal temperature and body weight were measured daily for 18 days. Blood was drawn from the saphenous vein at different days postinfection (dpi) and used for virus titer determination by plaque assay. Guinea pigs were declared moribund and euthanized when body weight decreased by 25% and rectal temperature fell below 39.8°C.

RESULTS AND DISCUSSION

The avirulent P2 and virulent P18 strains of PICV, a nonpathogenic member of the Arenaviridae family, cause distinct disease phenomena in guinea pigs, the molecular mechanism of which is not understood. We report here the development of two separate reverse genetics systems for the P2 and P18 PICV strains, as the first step to identify the viral virulence factors.

We have previously provided the complete genomic sequences for both the P2 and P18 strains (22). In the current work, we assembled the full-length cDNA sequences of the L and S segments of both viruses into expression vectors as outlined in the supplemental material. Briefly, the full-length viral RNA segments, in an antigenomic orientation, are individually placed immediately downstream of the T7 promoter. The T7 promoter produces viral RNA transcripts with the correct viral 5′ termini that include a nontemplated G residue as has previously been shown to exist in arenaviral genomic and antigenomic RNAs (38). The hepatitis delta virus ribozyme sequence was engineered into the 3′ end of the primary RNA transcripts in order to mediate a self-cleavage reaction to generate the authentic arenaviral 3′ ends. A nonviral cytosine nucleotide was added immediately upstream of the hepatitis delta virus ribozyme sequence, as this has previously been shown to enhance the self-cleavage of LCMV RNA without affecting viral RNA replication activity (32, 38). Additionally, the unique NcoI restriction enzyme site was introduced into the GPC gene (at nucleotide position 807) without changing its coding sequence in order to distinguish the recombinant viral S segment from the parental one.

To generate recombinant viruses, BSRT7-5 cells, a stable cell line that constitutively expresses the T7 RNA polymerase, were transfected with plasmids containing the T7 promoter-driven antigenomic RNA strands of the L and S segments. Virus in the supernatant was collected at 48-h posttransfection and used in a plaque assay on Vero cells. Infectious viruses were recovered from both the P2 and P18 molecular clones, as evidenced by plaque formation on Vero cells (Fig. 1A). To distinguish the recombinant viruses from the original stock viruses, we amplified a fragment of the S segment that contains the engineered silent NcoI site and showed that this restriction enzyme site was present only in the recombinant viruses (designated rP2 and rP18) (Fig. 1B). Taken together, these data suggest that we have successfully generated high-titer infectious PICV viruses purely from plasmid transfection, highlighting the functionality of the novel reverse genetics systems for both the virulent P18 and avirulent P2 strains.

FIG. 1.

Generation of the recombinant viruses by plasmid transfection. The BSRT7-5 cells were transfected with two plasmids bearing the antigenomic strands of the L and S segments under the control of the T7 promoter, as described in Materials and Methods. (A) Plaques formed on Vero cells by the recombinant viruses rP2 or rP18, which were collected from the supernatants of the transfected BSRT7-5 cells. (B) The engineered genetic marker NcoI site was present in the genomes of the recombinant viruses rP2 (lanes 1 to 2) and rP18 (lanes 3 to 4) but was absent in that of the parental virus P18 (lane 6). Lane 5, DNA molecular size markers.

The rP18 viruses appeared to grow faster than the rP2 viruses in Vero cells, as judged by the differences in the plaque sizes generated from these viruses (Fig. 1A). In order to validate this observation, we conducted a growth curve analysis of the recombinant rP2 and rP18 viruses, and of the parental P2 and P18 viruses, in Vero cells using an MOI of 0.01. As shown in Fig. 2A, similar growth kinetics were observed for both the virulent viruses P18 and rP18 and for both the avirulent viruses P2 and rP2, suggesting that the recombinant viruses can accurately recapitulate the growth capacity of their respective parental viruses in cell cultures. As predicted, the virulent P18 and rP18 viruses grew to higher titers than the avirulent P2 and rP2 by ∼0.5 to 1 log at 48 h postinfection (hpi). The avirulent viruses peaked at 72 hpi, while the virulent viruses peaked at 48 hpi (Fig. 2A). These results were replicated in a variety of established cell lines, including the guinea pig cell line JH4 (data not shown). In addition, we compared virus growth kinetics in primary guinea pig cells. As it has been shown that monocytes/macrophages are the early target cells of Lassa fever virus and PICV infections in vivo (9, 31), we infected primary peritoneal macrophages isolated from healthy guinea pigs with P2, P18, rP2, and rP18 viruses at an MOI of 0.01. Consistent with results obtained from established cell lines, the virulent P18 and rP18 viruses grew to higher titers than the avirulent P2 and rP2 viruses by ∼1 log (Fig. 2B). Taken together, our data strongly suggest that the virulent P18 and rP18 viruses appear to exhibit an inherent growth advantage, which may contribute to a higher degree of virulence caused by these viruses in infected animals.

FIG. 2.

Comparison of the growth kinetics between the parental viruses (P2 and P18) and recombinant viruses (rP2 and rP18) in Vero cells (A) and in the primary peritoneal macrophages of guinea pigs (B).

To determine the degree of virulence generated by the recombinant viruses, we injected guinea pigs intraperitoneally with 10,000 PFU of the P2, rP2, P18, or rP18 virus or PBS as a control. The degree of virulence was determined by mortality rate, febrile reaction, duration of the fever, and body weight loss. The mortality rate in animals infected with the rP18 virus was 100% (six out of six animals died) and with the P18 virus was ∼90% (five out of six animals died), whereas none of the six animals died due to either P2 or rP2 virus infection or PBS injection. The P18 and rP18 viruses led to an early onset of disease (as early as 4 dpi) and prolonged fever (an average of 8 days), whereas the P2 and rP2 viruses caused a late-onset and brief (1- to 2-day) fever (Fig. 3A and B). Accordingly, guinea pigs infected with the P18 or rP18 virus suffered from greater body weight loss than those infected with the P2 or rP2 virus (Fig. 3C). The euthanization of some moribund animals at day 13 resulted in a slight increase in the average rectal temperature and body weight for the groups of animals infected with virulent viruses (Fig. 3A and C). Similarly, the seemingly higher average body weight for the virulent P18-infected animals from days 15 to 18 postinfection is due to the recovery of one of the animals in this cohort at around day 15 (Fig. 3C). Regardless, our data demonstrate that the P18 and rP18 viruses are distinctly more virulent than the P2 and rP2 viruses in outbred guinea pigs and that the recombinant viruses can accurately recapitulate phenotypes generated by their respective parental viruses in vivo.

FIG. 3.

Comparison of the degrees of virulence in animals infected with either the parental viruses (P2 and P18) or recombinant viruses (rP2 and rP18). (A) Daily rectal temperature of animals injected with different PICV strains or with PBS as a control. (B) Average duration of fever (temperature of >39.5°C). (C) Daily body weight as a percentage of the original body weight at day 0.

We next quantified the viremia levels in infected animals at every 3 dpi. Figure 4 shows the viremia levels in animals infected by rP18 (two animals), P18 (three animals), P2 (three animals), and rP2 (three animals). The viremia levels differ drastically in animals infected with either the virulent or avirulent viruses over the course of the infection. In the P18- or rP18-infected animals, viremia levels rose above the detection threshold (200 PFU/ml) at 6 dpi and quickly increased by more than 3 × 10⁴ PFU/ml by 12 dpi, after which four out of five virulent virus-infected animals approached the terminal point (Fig. 4A). In contrast, viremia levels were below the detection level (i.e., <200 PFU/ml) in those animals infected with the avirulent P2 or rP2 viruses throughout the course of the infection. Taken together, these results reiterate the close association between viremia levels and disease severity, which is an important feature of arenavirus-induced hemorrhagic fevers in humans.

FIG. 4.

(A) Comparison of viremia levels (PFU/ml) in animals infected with P18 (○), rP18 (▪), or P2 and rP2 (×) throughout the course of the infections. (B) Comparison of the virus titers in the spleens of animals infected with rP2 (×) and rP18 (▪) throughout the course of the infections.

Similarly to the postmortem findings on fatal human Lassa virus infections in which the viruses were pantropical (39), the rP18 virulent viruses were found at high levels in almost all organs at terminal points, ranging from 3.1 × 10⁶ PFU/g in the lymph nodes to 3.2 × 10⁸ PFU/g in the adrenal glands (Table 1). The highest levels of virus were found in the livers, lungs, stomachs, and adrenal glands, and the lowest levels were in the brains (8.8 × 10⁴ PFU/g). In contrast, the levels of avirulent rP2 viruses in the infected animals, which were sacrificed at the same time postinfection as the rP18-infected animals, were below the limit of detection by the plaque assaying method. Since we failed to detect any viruses in the blood or in the different organs of animals infected by the avirulent viruses (P2 or rP2) (Fig. 4A and data not shown), we asked whether these viruses could indeed replicate in vivo or were effectively cleared by the host. To address these issues, we collected the spleens from animals infected with the rP2 or rP18 viruses at the early stages of infection (0, 4, 6, and 8 dpi) and quantified the virus titers by plaque assay. As shown in Fig. 4B, the rP2 and rP18 levels in the spleen increased sharply from 0 to 6 dpi. The virulent rP18 virus reached a titer of ∼1 × 10⁷ PFU/g at 4 dpi, peaked at 6 dpi with a level of 5 × 10⁷ PFU/g, and remained high thereafter (Fig. 4B). In contrast, the avirulent rP2 virus replicated to a level of ∼1 × 10⁶ PFU/g at 4 to 6 dpi, after which the virus titer decreased sharply to below the detection level at day 18. These data strongly suggest that rP2 avirulent viruses are indeed replication competent in vivo, but they are effectively cleared by host immune responses. In contrast, host immune responses failed to control the rP18 virulent viruses, which eventually resulted in the fatality of the infected animals.

TABLE 1.

Virus titers in different organs of rP18-infected animals^a

Organ	rP18 virus titer (PFU/g)
Heart	6.5E+06
Lung	6.7E+07
Liver	1.9E+08
Spleen	3.0E+07
Stomach	5.6E+08
Pancreas	6.5E+06
Intestine	1.4E+07
Adrenal gland	3.2E+08
Kidney	3.9E+06
Lymph node	3.1E+06
Brain	8.8E+04

^aOrgans were collected at terminal points of infected animals. Organs were weighed and homogenized in PBS. Virus titers in organ homogenates were determined by plaque assay on Vero cells and shown as PFU per g. Results shown are the averages of the results from three independent experiments.

In summary, we have successfully developed two novel infectious cDNA clones for the avirulent P2 and virulent P18 PICV strains and have shown that these recombinant viruses can recapitulate the parental viruses in terms of virus growth in vitro and virulence in vivo. The infectious cDNA clones for PICV and LCMV (13, 38) provide convenient systems to study the basic mechanism of arenavirus replication. Furthermore, the ability to generate both recombinant virulent and avirulent PICV from plasmid DNAs will allow us to characterize the molecular determinants of virulent PICV infection in guinea pigs, which could potentially shed important light on human infection by other deadly arenaviruses (14). For example, why only a few arenaviruses cause VHF upon human infection and what determines the degrees of heterogeneity in clinical manifestations in Lassa fever virus infection (27) remain unresolved. One hypothesis is that molecular changes in the pathogenic arenaviruses lead to the acquired virulence in infected hosts. Indeed, there is a significant sequence heterogeneity in circulating Lassa fever virus strains in the area of West Africa where Lassa fever is endemic with some being more lethal in animal models than others (3, 16). However, there are no systematic studies to link Lassa fever virus variants to degrees of disease severity. Using an established small animal model of arenaviral VHF—guinea pigs infected with PICV—we and other investigators have shown in the current work and elsewhere (22, 41, 42) that the genetic differences between two PICV strains (P2 and P18) can partly account for the distinct degrees of virulence in infected animals. Our newly developed reverse genetics systems for both of these PICV strains provide a unique opportunity to directly test the hypothesis that molecular determinants existing in the genomes of these viruses can contribute to the acquired virulence in infected animals by altering the efficiency of virus replication and/or by modulating the host immune responses to viral infection. These studies are currently under way in our laboratory and will be discussed in future reports.