Diversity and Ancestry of LCMV | CDC EID

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Volume 16, Number 7–July 2010
Research
High Diversity and Ancient Common Ancestry of Lymphocytic Choriomeningitis Virus
Cesar G. Albariño,1 Gustavo Palacios,1 Marina L. Khristova, Bobbie R. Erickson, Serena A. Carroll, James A. Comer, Jeffrey Hui, Thomas Briese, Kirsten St. George, Thomas G. Ksiazek,2 W. Ian Lipkin, and Stuart T. Nichol
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (C.G. Albariño, M.L. Khristova, B.R. Erickson, S.A. Carroll, J.A. Comer, T.G. Ksiazek, S.T. Nichol); Columbia University, New York, New York, USA (G. Palacios, J. Hui, T. Briese, W.I. Lipkin); and New York State Department of Health, Albany, New York, USA (K. St. George)

Suggested citation for this article

Abstract
Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae. LCMV can be associated with severe disease in humans, and its global distribution reflects the broad dispersion of the primary rodent reservoir, the house mouse (Mus musculus). Recent interest in the natural history of the virus has been stimulated by increasing recognition of LCMV infections during pregnancy, and in clusters of LCMV-associated fatal illness among tissue transplant recipients. Despite its public health importance, little is known regarding the genetic diversity or distribution of virus variants. Genomic analysis of 29 LCMV strains collected from a variety of geographic and temporal sources showed these viruses to be highly diverse. Several distinct lineages exist, but there is little correlation with time or place of isolation. Bayesian analysis estimates the most recent common ancestor to be 1,000–5,000 years old, and this long history is consistent with complex phylogeographic relationships of the extant virus isolates.
The rodent-borne arenaviruses (family Arenaviridae) are enveloped viruses with bisegmented RNA genomes that include several causative agents of hemorrhagic fevers in the New World and Africa (1). The large (L) genome RNA segment encodes the virus polymerase L and the Z protein, whereas the small (S) genome RNA segment encodes the nucleocapsid protein (NP) and glycoprotein precursor (GPC). The prototypic arenavirus, Lymphocytic choriomeningitis virus (LCMV), is distributed worldwide (due to its association with rodents of the species Mus musculus). This virus is typically associated with mild, self-limited, or asymptomatic infections in immunocompetent persons, but infections can lead to aseptic meningitis (1). In immunocompromised persons, LCMV exposure may result in serious systemic infections and death (2). Prenatal infection can cause spontaneous abortion or severe birth defects, including hydrocephalus, chorioretinitis, blindness, or psychomotor retardation (3,4).

Recent clusters of fatal disease in organ transplant recipients have focused new attention on the potential for iatrogenic transmission of LCMV. In December 2003 and April 2005, recipients of solid-organ transplants linked to single donors, died of unexplained infections. LCMV was implicated after the results of viral culture and electron microscopy triggered specific immunohistochemical and molecular tests for arenaviruses (2). In the 2005 cluster, a pet hamster that had been introduced into the donor's household was infected with the same virus that was later detected in the recipients (5). In early 2007, three patients who received visceral transplants on the same day from a single donor died of a febrile illness 4–6 weeks after transplantation. Unbiased high-throughput sequencing yielded sequences that identified a novel LCMV-related arenavirus (6). However, phylogenetic characterization was limited by the paucity of available sequences deposited in public databases. In April 2008, a public health investigation showed evidence of acute LCMV infection in 2 transplant recipients who had received kidneys from a common donor. Both patients died 4 and 10 weeks after transplantation despite intensive supportive care (7).

In spite of the increasing awareness of the public health importance of LCMV, little is known about the genetic diversity or relationships of LCMVs found in various parts of the world. Previous studies have suggested that nucleotide sequence divergence is high, up to 22% between some LCMVs (8–10). In the current study, we investigate the genetic diversity of 29 LCMVs, and infer from those sequences a history reaching back >1,000 years, findings consistent with the existing complex virus phylogeographic patterns.

Materials and Methods
Most of the sequences included in the alignments correspond to complete segment sequences. However, some short sequences, such as those from Kodoko virus, were also included in the analysis. This approach was taken to obtain the best reconstruction of the evolutionary history of the taxa (viruses, in our case) by using the maximum number of informative sites available (11–13). Although the validity of including missing data has been debated in the past, more recent studies have shown that even highly incomplete taxa can be placed accurately within the phylogeny (11,12,14).

The appropriateness of this approach was further examined by running several preliminary analyses. Initially, only full-length segment sequences were analyzed. Once the relationships between taxa and rate estimates were established, partial sequences (e.g., Kodoko virus) were also added to the analyses. No rate shifts were observed nor were any strongly supported phylogenetic relationships obscured. As a result, the tree figures shown in this report were based on the dataset including both whole segment and partial segment sequences.

From virus collections at the Centers for Disease Control and Prevention, the New York State Department of Health, Columbia University, and the World Reference Center of Emerging Viruses and Arboviruses (University of Texas Medical Branch), we selected 12 LCMVs for genetic characterization; origins spanned >70 years with broad geographic distribution (Table 1). Included in the study were representative virus stocks of the classic WE LCMV strain. This strain was originally isolated from a meningoencephalitis patient in New York in 1935 (15). In that era, virus isolation and passage were performed by intracranial inoculation into mice, which resulted in isolates that had multiple passages in mice as part of their passage history. Although the WE strain is used extensively in immunobiology experiments, the exact passage history of these viruses has been poorly documented. We located 2 old stocks of WE, 1 lyophilized in 1950 with a record of 7 passages in mouse brain, and 1 lyophilized in 1960 with a record of 7 passages in mouse brain and virus plaque purification (Table 1). In the 1940s, the WE LCMV strain was transferred to the University of British Columbia from the Rockefeller Institute in New York, only to be returned to the New York State Department of Health some years later. This substrain of WE LCMV became known as UBC (16). The 2 lyophilized vials were both labeled as UBC WE LCMV. Two other early LCMV isolates were also found. The Douglas-4707 and WHI-5107 strains were isolated by intracranial inoculation of suckling mice from the cerebrospinal fluid of patients in New York who had aseptic meningitis in 1947 and 1949, respectively. These viruses were recovered from lyophilized stocks prepared in 1960 and 1950, respectively (17), and represent some of the oldest low passage LCMV stocks still in existence.

The Lyles LCMV strain was isolated in Vero cells from the cerebrospinal fluid (CSF) of a 58-year-old woman from Winder, Georgia, who had nonfatal aseptic meningitis and a history of exposure to mice in her home (18). Similarly, the Michigan 2005 LCMV strain was isolated in 2005 from a mouse captured around the home of a 46-year-old woman with a diagnosis of acute meningitis and mild pancreatitis (19). The California 2003 LCMV was isolated in 2003 from the CSF of a congenitally infected infant with severe neurologic sequelae, including hydrocephalus, chorioretinitis, blindness, and developmental delay (20). The other LCMV isolates were from investigations of clusters of deaths and severe illness in transplant recipients associated with LCMV infection from transplanted organs. Four isolates were obtained during 2003–2008 from infected transplant recipients or rodents suspected of being involved in the exposure of the transplant donor in various locations in the United States (2,5,7). The Dandenong isolate was obtained from the liver of a patient who died after transplantation in Australia; the donor was suspected to have acquired the infection while traveling in the Balkans shortly before death and the harvesting of his organs (6). Finally, the isolate from Bulgaria (1956) is strongly suspected of being the first isolate obtained in Bulgaria from a case-patient with confirmed lymphocytic choriomeningitis (21).

RNA was extracted either directly from virus stocks or from supernatant harvested from infected cell cultures. A 300-μL aliquot of virus stock or cell culture supernatant was mixed with 900 μL of TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and 240 μL of chloroform and extracted according to standard protocols. The nucleic acid obtained was reverse transcribed and amplified by PCR; a total of 12 LCMV S segment sequences and 10 LCMV L segment sequences were amplified and sequenced by dideoxy-sequencing (Applied Biosystems, Foster City, CA, USA). We were unable to amplify by PCR the L segments of LCMV strains WHI-5107 and UBCA337 from the original virus ampoules, and the viruses were found to be no longer viable. The origins of 16 LCMV isolates for which sequences were already available, and that were included in the study, are also shown in Table 1. Multiple sequence alignments were generated using Multiple Alignment with Fast Fourier Transform (22) in SeaView (23) and sequence diversity was calculated by using molecular evolutionary genetics analysis (MEGA) 4 (24). Bayesian phylogenetic analyses of the sequence differences among the S and L segments of LCMV and Kodoko viruses were conducted using the BEAST, BEAUti and Tracer analysis software packages (25). Preliminary analyses were run for 10,000,000 generations with the Hasegawa, Kishino, and Yano + G nucleotide substitution model to select the clock and demographic models most appropriate for the S and L data sets. An analysis of the marginal likelihoods indicated that the relaxed lognormal molecular clock and constant population size model was decisively chosen (log10 Bayes factors = 3.032 for S segment; 13.472 for L segment). Final data analyses included Markov chain Monte Carlo chain lengths of 20,000,000–480,000,000 generations, with sampling every 1,000 states.

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Suggested Citation for this Article
Albariño CG, Palacios G, Khristova ML, Erickson BR, Carroll SA, Corner JA, et al. High diversity and ancient common ancestry of lymphocytic choriomeningitis virus. Emerg Infect Dis [serial on the Internet]. 2010 Jul [date cited]. http://www.cdc.gov/EID/content/16/7/1093.htm
DOI: 10.3201/eid1607.091902