Lassa virus origin and evolution

arenavirusI have a soft spot in my heart for Lassa virus: a non-fictional account of its discovery in Africa in 1969 inspired me to become a virologist. Hence papers on this virus always catch my attention, such as one describing its origin and evolution.

Lassa virus, a member of the Arenavirus family, is very different from Ebolavirus (a filovirus), but both are zoonotic pathogens that may cause hemorrhagic fever. It is responsible for tens of thousands of hospitalizations, and thousands of deaths each year, mainly in Sierra Leone, Guinea, Liberia, and Nigeria. Most human Lassa virus outbreaks are caused by multiple exposures to urine or feces from the multimammate mouse, Mastomys natalensis, which is the reservoir of the virus in nature. In contrast, outbreaks of Ebolavirus infection typically originate with a crossover from an animal reservoir, followed by human to human transmission. Despite being studied for nearly 50 years, until recently the nucleotide sequences of only 12 Lassa virus genomes had been determined.

To remedy this lack of Lassa virus genome information, the authors collected clinical samples from patients in Sierra Leone and Nigeria between 2008 and 2013. From these and other sources they determined the sequences of 183 Lassa virus genomes from humans, 11 viral genomes from M. natalensis, and two viral genomes from laboratory stocks. All the data are publicly available at NCBI. Analysis of the data lead to the following conclusions:

  • Lassa virus forms four clades, three in Nigeria and one in Sierra Leona/Liberia (members of a clade evolved from a common ancestor).
  • Most Lassa virus infections are a consequence of multiple, independent transmissions from the rodent reservoir.
  • Modern-day Lassa virus  strains probably originated at least 1,000 years ago in Nigeria, then spread to Sierra Leone as recently as 150 years ago. The lineage is most likely much older, but how much cannot be calculated from the data.
  • The genetic diversity of Lassa virus in individual hosts is an order of magnitude greater than the diversity of Ebolavirus. Furthermore, Lassa virus diversity in the rodent host is greater than in humans, likely a consequence of the longer, persistent infections that take place in the mouse.
  • The gene encoding the Lassa virus glycoprotein is subject to high selection in hosts, leading to variants that interfere with antibody binding.
  • Genetic variants that arise in one rodent are not transmitted to another.

Perhaps the most important result from this work is the establishment of laboratories in Sierra Leone and Nigeria that can safely collect and process samples from patients infected with Lassa virus, a BSL-4 pathogen.

TWiV 242: I want my MMTV

On episode #242 of the science show This Week in Virology, the complete TWiV team talks about how two different viruses shape the evolution of an essential housekeeping protein.

You can find TWiV #242 at www.microbe.tv/twiv.

Dual virus-receptor duel

transferrin receptorViruses are obligate intracellular parasites: they must enter a cell to reproduce. To gain access to the cell interior, a virus must first bind to one or more specific receptor molecules on the cell surface. Cell receptors for viruses do not exist only to serve viruses: they also have cellular functions. An example is the transferrin receptor, which regulates iron uptake and assists in the entry of viruses from three different families. It might appear that such dual-use proteins cannot evolve to block virus entry because their cellular function would then be compromised. A study of two viruses that bind to the same cell surface receptor protein reveals how a cellular protein can change to prevent infection without affecting its role in the cell.

The virus-cell receptor interaction is one of the many arenas where the evolution of host-virus conflict can be studied. Because the virus-receptor interaction is essential for viral replication, host cells with a mutation in the receptor gene that prevents virus infection survive and eventually dominate the population. A virus could overcome this block with an amino acid change allowing binding to the altered receptor. Mutations that alter the interaction to favor the virus or the host are called ‘positively selected’ mutations. Such back-and-forth evolution between viruses and their host cells has been called host-virus arms races. Most have been identified by studying antiviral genes. This study is unusual in that it involves a housekeeping gene that has been usurped for viral attachment.

Evidence for positive selection of host genes can be detected by comparing gene sequences of phylogenetically related species. Nonsynonymous mutations lead to a change in the amino acid sequence, while synonymous mutations do not. The rate at which nonsynonymous mutations occur in the genome is typically much slower than synonymous mutations. The reason for this difference is that most mutations that change the amino acid sequence of a protein are lethal to the host. When genes have been subjected to positive selection by a virus, the ratio of nonsynonymous to synonymous mutations is higher, typically in host amino acids that interact with viral proteins. Computer programs have been designed to scan gene sequences and identify codons which are under positive selection by virtue of a high ratio of nonsynonymous to synonymous mutations.

To determine if the transferrin receptor (TfR1) has evolved to prevent virus attachment, sequences of the protein from seven different rodent species were compared. The analysis revealed that much of the protein is highly conserved, but a small part, comprising six amino acids, is evolving rapidly. Three of these amino acids  are located on the part of TfR1 that binds arenaviruses, and three are at the binding site for the retrovirus mouse mammary tumor virus (MMTV) (see illustration). Changing these three amino acids of TfR1 of the house mouse, which is susceptible to MMTV, to the sequence found in TfR1 of the MMTV-resistant vesper mouse, blocked entry of the virus into cells. In turn, changing these three amino acids of TfR1 of the MMTV-resistant short-tailed zygodont to the sequence of the house mouse enabled virus entry into cells. None of these changes had an effect on ferritin binding by TfR1.

Evidence for positive selection can also be detected in viral genes encoding proteins that interact with the host. The arenavirus glycoprotein, GP, is known to bind to TfR1. Ten GP amino acids were identified that are under positive selection, and four of these directly contact TfR1.

These findings demonstrate that there has been an arms race between TfR1 and both an arenavirus and retrovirus. An interesting question is whether human TfR1 will enter into an arms race with arenaviruses. As these viruses emerge into the human population, it is expected that humans with mutations that make them less susceptible to infection or severe disease will be positively selected. Amino acid 212 of human TfR1, which is near the positively selected resides in murine TfR1, varies in the human population. When this amino acid change (leucine to valine) is introduced into TfR1, it confers some protection against arenavirus entry. Curiously, this polymorphism has only been found in Asian populations, where arenaviruses that bind TfR1 are not found. The polymorphism is probably neutral with respect to TfR1 function, and if TfR1-binding arenaviruses are introduced into Asia, this change could be positively selected.

Because all viruses depend on many host proteins for replication, it will be interesting to use this approach to see how other highly conserved cell proteins balance cell function with the ability to resist virus infections. There are like to be many cell proteins that cannot change to evade viral use without destroying their cell function. Fortunately for cells there are exceptions.

TWiV 240: Virology in Vermont

On episode #240 of the science show This Week in Virology,  Vincent travels to the University of Vermont to talk with Markus and Jason about their work on HIV, influenza virus, arenaviruses and hantaviruses.

You can find TWiV #240 at www.microbe.tv/twiv.

A new rhabdovirus from a patient with hemorrhagic fever

Viral hemorrhagic fevers in AfricaHemorrhagic fevers are among the most graphic viral diseases, inspiring movies, novels, and a general fear of infection. They are characterized by an abrupt onset and a striking clinical course involving bleeding from the nose and mouth, vomiting with blood, and bloody diarrhea. The most famous hemorrhagic fevers are produced by infection with filoviruses like Ebola virus, but members of three other viral families – Arenaviridae, Bunyaviridae, and Flaviviridae – can also cause this syndrome. The isolation of a novel rhabdovirus from an African with hemorrhagic fever suggests that members of a fifth viral family can also cause this disease.

Three cases of hemorrhagic fever that occurred in the spring of 2009 were noteworthy because none of the typical viral suspects could be detected in one patient. Two were young (13, 15 year old) students in the village of Mangala, Bas-Congo province, Democratic Republic of Congo. They lived near each other and went to the same school. Both arrived at the local health center with typical symptoms of hemorrhagic fever, and both died 2-3 days later. The third case was a 32 year old male nurse at the health center who was involved in the care of the other two patients. He developed symptoms of hemorrhagic fever but recovered within a few days.

Deep sequence analysis of RNA extracted from the serum of patient #3 revealed the presence of a novel rhabdovirus, provisionally named Bas-Congo virus (BASV). Phylogenetic analyses reveal that BASV is substantially diverged from the two main human rhabdoviruses, rabies virus and Chandipura virus (ten of the 160 known species of rhabdoviruses have been isolated from humans). BASV is more related to viruses of the Tibrogargan group and the Ephemerovirus genus, which contain arthropod-borne viruses that infect cattle, but clusters separately in an independent branch of the phylogenetic tree.

Antibodies to BASV were detected in the serum of patient #3 and also in the serum of an asymptomatic nurse who had cared for this patient. However, no antibodies to this virus were found in 43 other serum samples from individuals with hemorrhagic fever of unknown origin. These samples came from individuals who lived in 9 of the 11 provinces of the DRC, including Bas-Congo. Nor were antiviral antibodies detected in plasma from 50 random blood donors in one DRC province.

Although the viral genome sequence was determined from RNA extracted from patient serum (where there were 1 million copies per ml of the viral RNA), the virus did not replicate in cell cultures from monkey, rabbit, and mosquito, or in suckling mice. These findings are in contrast to those obtained with a newly discovered coronavirus in humans. It is likely that the samples had not been kept sufficiently cold to maintain viral infectivity. It should be possible to recover virus from a cloned DNA copy of the viral genome.

These data suggest, but do not prove, that BASV caused hemorrhagic fever in the 3 patients. All three cases occurred in a 3 week period within the same small village. BASV nucleic acid and antibodies were detected in the third patient. Given that viruses of the closely related Tibrogargan group and the Ephemerovirus genus are transmitted to cattle by biting midges, it is possible that the initial infections were transmitted by such an arthropod vector. Human to human transmission of the virus could have taken place when the nurse was infected by one or both pediatric patients. However, it should be noted that infection with BASV was not confirmed in either of the first two cases as no clinical samples were available. Other etiologies for this outbreak of hemorrhagic fever should not be ruled out.

Rhabdoviruses are known to cause encephalitis, vesicular stomatitis, or flu-like illness in humans, not hemorrhagic fevers. But these viruses clearly have the potential to cause this disease: members of the Novirhabdovirus genus cause hemorrhagic septicemia in fish. As long as there are viruses to discover, any rules we make about them should be considered breakable.

G Gerard, JN Fair, D Lee, E Silkas, I Steffen, J Muyembe, T Sittler, N Veerarghavan, J Ruby, C Wang, M Makuwa, P Mulembakani, R Tesh, J Mazet, A Rimoin, T Taylor, B Schneider, G Simmons, E Delwart, N Wolfe, C Chiu, E Leroy. 2012. A novel rhabdovirus associated with acute hemorrhagic fever in central Africa. PLoS Pathogens  8.

Behind the scenes: TWiV 200 at the NEIDL

We celebrated the 200th episode of TWiV by visiting the National Emerging Infectious Diseases Laboratories at Boston University Medical Center, where we met with Elke, Paul, and Ron to talk about building and working in a BSL4 facility. It was an amazing visit that will be fully documented in an upcoming video. Here are some behind-the-scenes photographs of two memorable days.

TWiV 200: Threading the NEIDL

TWiV Team in BSL4 suitsOn episode #200 of the science show This Week in Virology, Vincent, Alan, and Rich visit the National Emerging Infectious Diseases Laboratories at Boston University Medical Center, where they meet with Elke, Paul, and Ron to talk about building and working in a BSL4 facility.

You can find TWiV #200 at www.microbe.tv/twiv.

TWiV 196: An arena for snakes

On episode #196 of the science show This Week in Virology, the TWiVites meet with Mark Stenglein and Joseph DeRisi to discuss their discovery of a novel arenavirus in snakes with inclusion body disease.

You can find TWiV #196 at www.microbe.tv/twiv.

A viral mashup in snakes

snake inclusion body diseaseIf you know anything about snakes you might be familiar with snake inclusion body disease, or IBD. This transmissible and fatal disease affects snakes of a variety of species but has been best studied in boas. The name comes from the presence of large masses (inclusions) in the cytoplasm of cells from infected snakes. IBD might be caused by a novel arenavirus.

To identify an etiologic agent of IBD, RNA was extracted from multiple organs of snakes with the disease, and subjected to deep sequencing. This analysis revealed the presence of two distinct arenaviruses. One virus, called CASV (California Academy of Sciences virus) was found in diseased annulated tree boas, and the second, GGV (Golden Gate virus) was detected in boa constrictors. These sequences were found in 6 of 8 IBD snakes but not in 18 disease-free controls.

The finding of arenaviruses in snakes is interesting because these viruses are thought to infect only mammals. Rodents are believed to be the natural host of arenaviruses, which are classified as Old World or New World depending on where they are isolated. In rodents, arenavirus infection is typically asymptomatic. When arenaviruses infect humans, severe disease can result, such as hemorrhagic fever caused by Lassa virus. How CASV and GGV are transmitted to snakes is not known. One possibility is that they are introduced into snakes when they consume mice. The viruses might be transmitted among snakes by contact or via vectors such as blood-sucking mites. The genome sequences of CASV an GGV are very different from those of rodent arenaviruses. If similar viruses circulate in rodents, they have not yet been detected; alternatively, CASV- and GGV-like viruses might have diverged from Old- and New World arenaviruses after many years of transmission among snakes.

Another surprise emerged from analysis of the CASV and GGV viral proteins. Arenaviral genomes encode four main proteins: an RNA polymerase, L; a nucleoprotein, NP; a transmembrane glycoprotein, GPC, and a zinc-binding protein, Z. The amino acid sequences of CASV and GGV L, and NP, but not Z and GPC, resemble those of known arenaviruses. The CASV and GGV glycoproteins are instead related to glycoproteins of filoviruses and retroviruses. This observation suggests that recombination took place between the genomes and arenaviruses and filoviruses or retroviruses, likely a very long time ago.

Whether these novel arenaviruses actually cause snake IBD is not proven by this work. This question is underscored by the observation that no arenaviruses were detected in two of the 8 IBD positive snakes in this study. In addition, two of the virus-positive snakes that were diagnosed with IBD did not have symptoms of the disease. It is possible that the arenaviruses are present but do not cause symptoms. As the authors write,

….sequencing can only ever identify candidate etiologic agents, and demonstration of causality requires significant additional experimental effort.

This additional work would include the demonstration that infectious virus can be consistently recovered from diseased snakes, and that the disease can be induced by inoculation of snakes with the virus. As a first step towards answering these questions, kidney and liver extracts were added to cultured boa constrictor kidney cells. By 5 days post-infection, viral RNA could be detected in the cell supernatant, but it is not known if the viruses produced are infectious.

This work shows convincingly that the host range of arenaviruses is much broader than we thought: they do not just infect mammals. The zoonotic pool continues to grow, and there are now more potential sources of new human arenaviruses. The work also emphasizes that our knowledge of all the viruses on the planet remains miniscule.

Mark D. Stenglein, Chris Sanders, Amy L. Kistler, J. Graham Ruby, Jessica Y. Franco, Drury R. Reavill, Freeland Dunker, and Joseph L. DeRisi. 2012. Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease. mBio 3:e00180-12.

TWiV 35: Much achoo about nothing

twiv-200In episode 35 of the podcast “This Week in Virology”, Vincent, Alan, Dick, and Richard Kessin talk about Lujo virus, a new arenavirus, influenza, WHO rewriting pandemic rules, adjuvants, and a brief history of microbiology.

Click the arrow above to play, or right-click to download TWiV #35 or subscribe in iTunes or by email.