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-␣ and IL-1␤ 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.

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 choriome-ningitis 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.

Since the India and Indian Ocean outbreaks of 2005 and 2006, the global distribution of chikungunya virus (CHIKV) and the locations of epidemics have dramatically shifted. First, the Indian Ocean lineage (IOL) caused sustained epidemics in India and has radiated to many other countries. Second, the Asian lineage has caused frequent outbreaks in the Pacific islands and in 2013 was introduced into the Caribbean, followed by rapid spread to nearly all of the neotropics. Further, CHIKV epidemics, as well as exported cases, have been reported in central Africa after a long period of perceived silence. To understand these changes and to anticipate the future of the virus, the exact distribution, genetic diversity, transmission routes, and future epidemic potential of CHIKV require further assessment. To do so, we conducted the most comprehensive phylogenetic analysis to date, examined CHIKV evolution and transmission, and explored distinct genetic factors associated with the emergence of the East/Central/ South African (ECSA) lineage, the IOL, and the Asian lineage. Our results reveal contrasting evolutionary patterns among the lineages, with growing genetic diversities observed in each, and suggest that CHIKV will continue to be a major public health threat with the potential for further emergence and spread. IMPORTANCE Chikungunya fever is a reemerging infectious disease that is transmitted by

Lymphocytic chorimeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), causative agent of Lassa hemorrhagic fever (LHF), belong to the Old World group of the family Arenaviridae. Both viruses have extensive strain diversity and significant variations in lethality and pathogenicity for man and experimental animals. We have shown that the LHF-like infection of rhesus macaques with the WE strain of LCMV affects liver functions, induces hepatocyte proliferation, and causes a rise in IL-6 and soluble TNF receptors (sTNFR) concomitant with a rise in viremia. The levels of IL-6 and sTNFR can serve as an additional diagnostic tool for liver involvement in pathogenesis of arenavirus infection. Mucosal inoculation of rhesus macaques with LCMV-WE can result in attenuated infection with a transient viremia and liver enzyme abnormalities. The ARM strain of LCMV shares 88% amino acid homology with WE. In contrast to LCMV-WE, ARM strain does not induce manifested disease in monkeys, does not affect liver functions, and does not induce hepatocyte proliferation. Previously we demonstrated that LCMV-ARM infection protected rhesus macaques challenged with LCMV-WE. Here we have shown that the protected animals have no signs of hepatitis and hepatocyte proliferation.

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Lymphocytic choriomeningitis virus (LCMV) induces type I interferon (alpha and beta interferon [IFN-␣ and IFN-␤]) upon infection and yet is sensitive to the addition of type II interferon (gamma interferon[Arenaviruses can replicate without significantly impacting the host or causing cytopathic effects. The arenavirus replication complex contains the viral genomic single-stranded RNA segments, nucleocapsid protein (NP), an RNA-dependent RNA polymerase (RdRp or L protein), and a small zinc-binding protein (Z) (17). Cellular proteins are also involved in viral replication (3,4,12). Here we describe the inhibitory influence of the promyelocytic leukemia protein (PML) that coprecipitates and colocalizes with cell-associated arenavirus complexes (2). PML is an oncoprotein that is expressed primarily in myeloid, epithelial, and endothelial cells, all infectable by arenaviruses and important in the pathogenesis of arenaviral hemorrhagic fevers. PML is induced by the alpha/beta interferons (IFN-␣/␤) acting on the ISRE and GAS promoter response elements (5,13,20). Interferons IFN-␣ and IFN-␤ are produced by many cell types upon viral infection, and IFN-␥ is produced in T lymphocytes or natural killer cells in response to antigens (16). IFNs are known for their inhibitory effects on cellular proliferation, and PML, as an effector of this function, is capable of suppressing cell proliferation (11,22,24).IFNs are also known for their antiviral effects. There are 50 to 100 IFN-inducible genes and several of them have antiviral activity, e.g., the p68 protein kinase, the 2Ј,5Ј-oligoadenylate synthetase (OAS), and certain Mx family proteins (19,20,23). The IFN-inducible PML has also recently been shown to have antiviral activity. In the absence of IFN, overexpression of PML diminishes infection by vesicular stomatitis virus (VSV) and influenza A virus, without affecting infection by encephalomyocarditis virus (EMCV), a virus known to be IFN resistant (6).Coimmunoprecipitation studies show specific interaction between PML and Z proteins of LCMV and Lassa fever virus, a related arenavirus. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML, and this domain of PML, unlike the PML RING or the nuclear localization signal, is essential for colocalization of Z and PML (2). The work presented here demonstrates that PML expression diminishes LCMV expression, possibly through its interaction with the LCMV Z protein.PML and LCMV affect proliferation of MEF. The effects of PML expression on cell proliferation were examined in earlypassage mouse embryonic fibroblasts (MEFs) (22; this study). Fibroblasts lacking PML (PML Ϫ/Ϫ) grew faster and achieved higher cell densities than wild-type (PML ϩ/ϩ) cells and yet their cultures were morphologically indistinguishable. IFN treatment, which increases PML expression, reduces cell growth rates even more in both PML ϩ/ϩ and Ϫ/Ϫ fibroblasts. Infection with LCMV shortens the life of both MEF cultures approximately twofold (P Ͻ 0.05) (Fig. 1)...

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 cellmediated immunity.

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.