Laura K. McMullan

Genetic Detection and Characterization of Lujo Virus, a New Hemorrhagic Fever–Associated Arenavirus from Southern Africa

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever–associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.

A New Phlebovirus Associated with Severe Febrile Illness in Missouri

Two men from northwestern Missouri independently presented to a medical facility with fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, and both had been bitten by ticks 5 to 7 days before the onset of illness. Ehrlichia chaffeensis was suspected as the causal agent but was not found on serologic analysis, polymerase-chain-reaction (PCR) assay, or cell culture. Electron microscopy revealed viruses consistent with members of the Bunyaviridae family. Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the phlebovirus genus. Although Kochs postulates have not been completely fulfilled, we believe that this phlebovirus, which is novel in the Americas, is the cause of this clinical syndrome.

Hantavirus Infection Induces the Expression of RANTES and IP-10 without Causing Increased Permeability in Human Lung Microvascular Endothelial Cells

ABSTRACT Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1β [IL-1β], IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-γ)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-γ for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-κB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.

Evidence for a functional RNA element in the hepatitis C virus core gene

In the core protein-coding region of hepatitis C virus (HCV), evidence exists for both phylogenetically conserved RNA structures and a +1 alternative reading frame (ARF). To investigate its role in HCV infection, we introduced four stop codons into the ARF of a genotype 1a H77 molecular clone. The changes did not alter the core protein sequence, but were predicted to disrupt RNA secondary structures. An attenuated infection was established after inoculation of the mutant HCV RNA into an HCV naïve chimpanzee. The acute infection was atypical with low peak viremia, minimal alanine aminotransferase elevation, and early virus control by a diverse adaptive immune response. Sequencing circulating virus revealed progressive reversions at the third and then fourth stop codon. In cell culture, RNA replication of a genome with four stop codons was severely impaired. In contrast, the revertant genome exhibited only a 5-fold reduction in replication. Genomes harboring only the first two stop codons replicated to WT levels. Similarly, reversions at stop codons 3 and 4, which improved replication, were selected with recombinant, infectious HCV in cell culture. We conclude that ARF-encoded proteins initiating at the polyprotein AUG are not essential for HCV replication in cell culture or in vivo. Rather, our results provide evidence for a functionally important RNA element in the ARF region.

Nosocomial Outbreak of Novel Arenavirus Infection, Southern Africa

This case reinforces the need for strict screening of internationally transferred patients.

Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

ABSTRACT Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.

Molecular evolution of viruses of the family Filoviridae based on 97 whole genome sequences

ABSTRACT Viruses in the Ebolavirus and Marburgvirus genera (family Filoviridae) have been associated with large outbreaks of hemorrhagic fever in human and nonhuman primates. The first documented cases occurred in primates over 45 years ago, but the amount of virus genetic diversity detected within bat populations, which have recently been identified as potential reservoir hosts, suggests that the filoviruses are much older. Here, detailed Bayesian coalescent phylogenetic analyses are performed on 97 whole-genome sequences, 55 of which are newly reported, to comprehensively examine molecular evolutionary rates and estimate dates of common ancestry for viruses within the family Filoviridae. Molecular evolutionary rates for viruses belonging to different species range from 0.46 × 10−4 nucleotide substitutions/site/year for Sudan ebolavirus to 8.21 × 10−4 nucleotide substitutions/site/year for Reston ebolavirus. Most recent common ancestry can be traced back only within the last 50 years for Reston ebolavirus and Zaire ebolavirus species and suggests that viruses within these species may have undergone recent genetic bottlenecks. Viruses within Marburg marburgvirus and Sudan ebolavirus species can be traced back further and share most recent common ancestors approximately 700 and 850 years before the present, respectively. Examination of the whole family suggests that members of the Filoviridae, including the recently described Lloviu virus, shared a most recent common ancestor approximately 10,000 years ago. These data will be valuable for understanding the evolution of filoviruses in the context of natural history as new reservoir hosts are identified and, further, for determining mechanisms of emergence, pathogenicity, and the ongoing threat to public health.

Reemerging Sudan Ebola Virus Disease in Uganda, 2011

Two large outbreaks of Ebola hemorrhagic fever occurred in Uganda in 2000 and 2007. In May 2011, we identified a single case of Sudan Ebola virus disease in Luwero District. The establishment of a permanent in-country laboratory and cooperation between international public health entities facilitated rapid outbreak response and control activities.

High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses.

Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle.

Using next generation sequencing to identify yellow fever virus in Uganda

In October and November 2010, hospitals in northern Uganda reported patients with suspected hemorrhagic fevers. Initial tests for Ebola viruses, Marburg virus, Rift Valley fever virus, and Crimean Congo hemorrhagic fever virus were negative. Unbiased PCR amplification of total RNA extracted directly from patient sera and next generation sequencing resulted in detection of yellow fever virus and generation of 98% of the virus genome sequence. This finding demonstrated the utility of next generation sequencing and a metagenomic approach to identify an etiological agent and direct the response to a disease outbreak.