A Primary Human Liver Cell Culture Model for Hemorrhagic Fever Viruses.

Viral hemorrhagic fevers affect liver functions such as important metabolic processes and the replacement of new blood cells, coagulation factors, and growth factors. Typically, multi-organ diseases such as viral hemorrhagic fevers are studied in an organism, but it is also possible to derive information about the molecular events involved in disease processes by focusing on liver cell culture. Here we describe a multi-cell culture system that is capable of replicating the arenavirus LCMV-WE, a virus that can cause hemorrhagic fever in primates, as a model for liver infection by a hemorrhagic fever virus.

A Primate Model for Viral Hemorrhagic Fever.

Lymphocytic choriomeningitis virus strain WE (LCMV-WE), a Risk Group 3 virus, causes a disease in rhesus monkeys that closely resembles human infection with Lassa fever virus, a Risk Group 4 agent. Three stages of disease progression have been defined and profiled in this model: pre-viremic, viremic, and terminal. The earliest or pre-viremic stage reveals changes in the blood profile predictive of the later stages of disease. In order to identify whether specific changes are pathognomonic, it was necessary to perform a parallel infection with an attenuated virus (LCMV-Armstrong). Here we review the use of nonhuman primates to model viral hemorrhagic fever and offer a step-by-step guide to using a rhesus macaque model for Lassa fever.

Genetic variation in vitro and in vivo of an attenuated Lassa vaccine candidate.

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.

Transcriptome analysis of human peripheral blood mononuclear cells exposed to Lassa virus and to the attenuated Mopeia/Lassa reassortant 29 (ML29), a vaccine candidate.

Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

Lymphocytic choriomeningitis virus (LCMV) infection of macaques: a model for Lassa fever.

Arenaviruses such as Lassa fever virus (LASV) and lymphocytic choriomeningitis virus (LCMV) are benign in their natural reservoir hosts, and can occasionally cause severe viral hemorrhagic fever (VHF) in non-human primates and in human beings. LCMV is considerably more benign for human beings than Lassa virus, however certain strains, like the LCMV-WE strain, can cause severe disease when the virus is delivered as a high-dose inoculum. Here we describe a rhesus macaque model for Lassa fever that employs a virulent strain of LCMV. Since LASV must be studied within Biosafety Level-4 (BSL-4) facilities, the LCMV-infected macaque model has the advantage that it can be used at BSL-3. LCMV-induced disease is rarely as severe as other VHF, but it is similar in cases where vascular leakage leads to lethal systemic failure. The LCMV-infected macaque has been valuable for describing the course of disease with differing viral strains, doses and routes of infection. By monitoring system-wide changes in physiology and gene expression in a controlled experimental setting, it is possible to identify events that are pathognomonic for developing VHF and potential treatment targets.

Circulating natural killer and gammadelta T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus.

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and gammadelta T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and gammadelta T cells, indicating that this is not a pathogenic event. V(3)9 T cells, representing the majority of circulating gammadelta T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Gene expression in primate liver during viral hemorrhagic fever.

BACKGROUND: Rhesus macaques infected with lymphocytic choriomeningitis virus (LCMV) provide a model for human Lassa fever. Disease begins with flu-like symptoms and progresses rapidly with fatal consequences. Previously, we profiled the blood transcriptome of LCMV-infected monkeys (M. Djavani et al J. Virol. 2007) showing distinct pre-viremic and viremic stages that discriminated virulent from benign infections. In the present study, changes in liver gene expression from macaques infected with virulent LCMV-WE were compared to gene expression in uninfected monkeys as well as to monkeys that were infected but not diseased. RESULTS: Based on a functional pathway analysis of differentially expressed genes, virulent LCMV-WE had a broader effect on liver cell function than did infection with non-virulent LCMV-Armstrong. During the first few days after infection, LCMV altered expression of genes associated with energy production, including fatty acid and glucose metabolism. The transcriptome profile resembled that of an organism in starvation: mRNA for acetyl-CoA carboxylase, a key enzyme of fatty acid synthesis was reduced while genes for enzymes in gluconeogenesis were up-regulated. Expression was also altered for genes associated with complement and coagulation cascades, and with signaling pathways involving STAT1 and TGF-beta. CONCLUSION: Most of the 4500 differentially expressed transcripts represented a general response to both virulent and mild infections. However, approximately 250 of these transcripts had significantly different expression in virulent infections as compared to mild infections, with approximately 30 of these being differentially regulated during the pre-viremic stage of infection. The genes that are expressed early and differently in mild and virulent disease are potential biomarkers for prognosis and triage of acute viral disease.

Early blood profiles of virus infection in a monkey model for Lassa fever.

Acute arenavirus disease in primates, like Lassa hemorrhagic fever in humans, begins with flu-like symptoms and leads to death approximately 2 weeks after infection. Our goal was to identify molecular changes in blood that are related to disease progression. Rhesus macaques (Macaca mulatta) infected intravenously with a lethal dose of lymphocytic choriomeningitis virus (LCMV) provide a model for Lassa virus infection of humans. Blood samples taken before and during the course of infection were used to monitor gene expression changes that paralleled disease onset. Changes in blood showed major disruptions in eicosanoid, immune response, and hormone response pathways. Approximately 12% of host genes alter their expression after LCMV infection, and a subset of these genes can discriminate between virulent and non-virulent LCMV infection. Major transcription changes have been given preliminary confirmation by quantitative PCR and protein studies and will be valuable candidates for future validation as biomarkers for arenavirus disease.

Arenavirus Z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds.

Several disulfide-based and azoic compounds have shown antiviral and virucidal properties against arenaviruses in virus yield-inhibition and inactivation assays, respectively. The most effective virucidal agent, the aromatic disulfide NSC20625, was able to inactivate two strains of the prototype arenavirus species Lymphocytic choriomeningitis virus (LCMV). Inactivated viral particles retained the biological functions of the virion envelope glycoproteins in virus binding and uptake, but were unable to perform viral RNA replication. Furthermore, in inactivated virions, the electrophoretic profile of the Z protein was altered when analysed under non-reducing conditions, whereas the patterns of the proteins NP and GP1 remained unaffected. Treatment of a recombinant LCMV Z protein with the virucidal agents induced unfolding and oligomerization of Z to high-molecular-mass aggregates, probably due to metal-ion ejection and the formation of intermolecular disulfide bonds through the cysteine residues of the Z RING finger. NSC20625 also exhibited antiviral properties in LCMV-infected cells without affecting other cellular RING-motif proteins, such as the promyelocytic leukaemia protein PML. Altogether, the investigations described here illustrate the potential of the Z protein as a promising target for therapy and the prospects of the Z-reactive compounds to prevent arenavirus dissemination.

The proline-rich homeodomain (PRH/HEX) protein is down-regulated in liver during infection with lymphocytic choriomeningitis virus.

The proline-rich homeodomain protein, PRH/HEX, participates in the early development of the brain, thyroid, and liver and in the later regenerative processes of damaged liver, vascular endothelial, and hematopoietic cells. A virulent strain of lymphocytic choriomeningitis virus (LCMV-WE) that destroys hematopoietic, vascular, and liver functions also alters the transcription and subcellular localization of PRH. A related virus (LCMV-ARM) that does not cause disease in primates can infect cells without affecting PRH. Biochemical experiments demonstrated the occurrence of binding between the viral RING protein (Z) and PRH, and genetic experiments mapped the PRH-suppressing phenotype to the large (L) segment of the viral genome, which encodes the Z and polymerase genes. The Z protein is clearly involved with PRH, but other viral determinants are needed to relocate PRH and to promote disease. By down-regulating PRH, the arenavirus is able to eliminate the antiproliferative effects of PRH and to promote liver cell division. The interaction of an arenavirus with a homeodomain protein suggests a mechanism for viral teratogenic effects and for the tissue-specific manifestations of arenavirus disease.

Mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever.

Arenaviruses are transmitted from rodents to human beings by blood or mucosal exposure. The most devastating arenavirus in terms of human disease is Lassa fever virus, causing up to 300,000 annual infections in West Africa. We used a model for Lassa fever in which Rhesus macaques were infected with a related virus, lymphocytic choriomeningitis virus (LCMV). Our goals were to determine the outcome of infection after mucosal inoculation and later lethal challenge, to characterize protective immune responses, and to test cross-protection between a virulent (LCMV-WE) and an avirulent (LCMV-ARM) strain of virus. Although intravenous infections in the monkey model were uniformly lethal, intragastric infections recapitulated the spectrum of clinical outcomes seen in human exposure to Lassa fever virus: death, recovery from disease, and most often, subclinical infection. Plaque neutralization, ELISA, lymphocyte proliferation, and chromium-release assays were used to monitor humoral and cellular immune responses. Cross protection between the two strains was observed. The three out of seven monkeys that experienced protection were also the three with the strongest cell-mediated immunity.

Monocytes treated with human immunodeficiency virus Tat kill uninfected CD4(+) cells by a tumor necrosis factor-related apoptosis-induced ligand-mediated mechanism.

The human immunodeficiency virus (HIV) Tat protein has a critical role in viral transcription, but this study focuses on its additional role as an extracellular effector of lymphocyte cell death. It is well known that Tat induces tumor necrosis factor-related apoptosis-induced ligand (TRAIL) in peripheral blood mononuclear cells (PBMC), and we show that the majority of TRAIL is produced by the monocyte subset of PBMC. Human monocytes and U937 monoblastoid cells did not take up soluble HIV Tat-86, as T cells did, yet produced more TRAIL than did T cells. TRAIL secretion was induced by Tat and by a cysteine-rich peptide of Tat but not by sulfhydryl-modified Tat toxoid. Although there was only a slight increase in cell surface expression of TRAIL on monocytes, sufficient TRAIL was secreted to be toxic for T cells. The cytotoxicity of Tat-stimulated monocyte medium could be blocked by a TRAIL-neutralizing antibody. T cells treated with Tat did not secrete enough TRAIL to mediate cell death in our assay. Remarkably, uninfected T cells are more susceptible to TRAIL than are HIV-infected T cells. The production of TRAIL by Tat-stimulated monocytes provides a mechanism by which HIV infection can destroy uninfected bystander cells.

Arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus can cause hemorrhagic fever and liver disease in primates. The WE strain of LCMV (LCMV-WE) causes a fatal Lassa fever-like disease in rhesus macaques and provides a model for arenavirus pathogenesis in humans. LCMV-WE delivered intravenously or intragastrically to rhesus macaques targets hepatocytes and induces high levels of liver enzymes, interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), and soluble tumor necrosis factor receptors (sTNFRI and -II) in plasma during acute infection. Proinflammatory cytokines TNF-alpha and IL-1beta were not detected in plasma of infected animals, but increased plasma gamma interferon was noted in fatally infected animals. Immunohistochemistry of acute liver biopsies revealed that 25 to 40% of nuclei were positive for proliferation antigen Ki-67. The increases in IL-6, sIL-6R, sTNFR, and proliferation antigen that we observe are similar to the profile of incipient liver regeneration after surgical or toxic injury (N. Fausto, Am. J. Physiol. 277:G917-G921, 1999). Although IL-6 was not directly induced by virus infection in vitro, peripheral blood mononuclear cells from acutely infected monkeys produced higher levels of IL-6 upon lipopolysaccharide stimulation than did healthy controls. Our data confirm that acute infection is associated with weak inflammatory responses in tissues and initiates a program of liver regeneration in primates.

Hemorrhagic fever occurs after intravenous, but not after intragastric, inoculation of rhesus macaques with lymphocytic choriomeningitis virus.

Arenaviruses can cause hemorrhagic fever and death in primates and guinea pigs, but these viruses are not highly pathogenic for most rodent carriers. In the United States, arenaviruses precipitated outbreaks of hepatitis in captive monkeys, and they present an emerging health threat in the tropical areas of Africa and South America. We describe infection of rhesus macaques with the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), using the WE strain that has been known to cause both encephalopathy and multifocal hemorrhage. Five macaques were inoculated: two by the intravenous (i.v.) and three by the intragastric (i.g.) route. Whereas the two i.v.-inoculated monkeys developed signs and lesions consistent with fatal hemorrhagic fever, the i.g.-inoculated monkeys had an attenuated infection with no disease. Pathological signs of the primate i.v. infection differ significantly from guinea pig arenavirus infections and make this a superior model for human viral hemorrhagic disease.