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In 2012, an unprecedented number of four distinct, partially overlapping filovirus-associated viral hemorrhagic fever outbreaks were detected in equatorial Africa. Analysis of complete virus genome sequences confirmed the reemergence of Sudan virus and Marburg virus in Uganda, and the first emergence of Bundibugyo virus in the Democratic Republic of the Congo.

Thermodynamic modeling of Ebola viral RNA predicts the formation of RNA stem-loop structures at the 3′ and 5′ termini and panhandle structures between the termini of the genomic (or antigenomic) RNAs. Sequence analysis showed a high degree of identity among Ebola Zaire, Sudan, Reston, and Cote d’Ivoire subtype viruses in their 3′ and 5′ termini (18 nucleotides in length) and within a second region (internal by approximately 20 nucleotides). While base pairing of the two conserved regions could lead to the formation of the base of the putative stem-loop or panhandle structures, the intervening sequence variation altered the predictions for the rest of the structures. Using an in vivo minigenome replication system, we engineered mutations designed to disrupt potential base pairing in the viral RNA termini. Analysis of these variants by screening for enhanced green fluorescent protein reporter expression and by quantitation of minigenomic RNA levels demonstrated that the upper portions of the putative panhandle and 3′ genomic structures can be destabilized without affecting virus replication.

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.

Sin Nombre virus (SNV) is a major representative of the New World hantaviruses and the most common cause of hantavirus pulmonary syndrome (HPS) with high mortality in North America. Unlike other members of the family Bunyaviridae which mature in the Golgi complex, New World hantaviruses have been previously reported to mature at the cell surface. For family Bunyaviridae viruses, retention of the viral glycoproteins at the Golgi complex is thought to be responsible for their Golgi maturation. In our studies, the majority of SNV glycoproteins, G1 and G2, was localized in the Golgi complex when expressed from a full-length GPC clone or in SNV-infected cells, in agreement with data for other members of the family Bunyaviridae, including the Old World hantaviruses. However, the SNV glycoproteins could also be detected at the cell surface at advanced posttransfection or postinfection time points. G1 expressed in the absence of G2 did not accumulate in the Golgi, but remained predominantly associated with the endoplasmic reticulum (ER). Overexpressed amounts of apparently misfolded G1 were aggregated in a subcellular compartment likely to represent the aggresome. Unexpectedly, an additional major pool of G1 was detected intracellularly in SNV-infected and GPC-expressing transfected cells, by using a SNV G1-specific Fab antibody. This pool of G1 is predominantly localized in late endosomes–lysosomes.