Angela J. Sanchez

Analysis of Human Peripheral Blood Samples from Fatal and Nonfatal Cases of Ebola (Sudan) Hemorrhagic Fever: Cellular Responses, Virus Load, and Nitric Oxide Levels

ABSTRACT Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8+ T cells, and activated (HLA-DR+) CD8+ T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (≥150 μM) would have negatively affected vascular tone and contributed to virus-induced shock.

Characterization of the Glycoproteins of Crimean-Congo Hemorrhagic Fever Virus

ABSTRACT Crimean-Congo hemorrhagic fever (CCHF) virus is the cause of an important tick-borne disease of humans throughout regions of Africa, Europe, and Asia. Like other members of the genus Nairovirus, family Bunyaviridae, the CCHF virus M genome RNA segment encodes the virus glycoproteins. Sequence analysis of the CCHF virus (Matin strain) M RNA segment revealed one major open reading frame that potentially encodes a precursor polyprotein 1,689 amino acids (aa) in length. Comparison of the deduced amino acid sequences of the M-encoded polyproteins of Nigerian, Pakistani, and Chinese CCHF virus strains revealed two distinct protein regions. The carboxyl-terminal 1,441 aa are relatively highly conserved (up to 8.4% identity difference), whereas the amino-terminal 243 to 248 aa are highly variable (up to 56.4% identity difference) and have mucin-like features, including a high serine, threonine, and proline content (up to 47.3%) and a potential for extensive O-glycosylation. Analysis of released virus revealed two major structural glycoproteins, G2 (37 kDa) and G1 (75 kDa). Virus protein analysis by various techniques, including pulse-chase analysis and/or reactivity with CCHF virus-specific polyclonal and antipeptide antibodies, demonstrated that the 140-kDa (which contains the mucin-like region) and 85-kDa nonstructural proteins are the precursors of the mature G2 and G1 proteins, respectively. The amino termini of the CCHF virus (Matin strain) G2 and G1 proteins were established by microsequencing to be equivalent to aa 525 and 1046, respectively, of the encoded polyprotein precursor. The tetrapeptides RRLL and RKPL are immediately upstream of the cleavage site for mature G2 and G1, respectively. These are completely conserved among the predicted polyprotein sequences of all the CCHF virus strains and closely resemble the tetrapeptides that represent the major cleavage recognition sites present in the glycoprotein precursors of arenaviruses, such as Lassa fever virus (RRLL) and Pichinde virus (RKLL). These results strongly suggest that CCHF viruses (and other members of the genus Nairovirus) likely utilize the subtilase SKI-1/S1P-like cellular proteases for the major glycoprotein precursor cleavage events, as has recently been demonstrated for the arenaviruses.

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Proteolytic Processing by Subtilase SKI-1

ABSTRACT Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the −4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Precursor Is Cleaved by Furin-Like and SKI-1 Proteases To Generate a Novel 38-Kilodalton Glycoprotein

ABSTRACT Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.

Prognostic Indicators for Ebola Patient Survival

Odds of survival were greatest when first Ebola virus–positive blood sample collected had low viral load.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Genetic and serotypic characterization of Sin Nombre-like viruses in Canadian Peromyscus maniculatus mice.

In Canada, hantavirus infected deer mice (Peromyscus maniculatus) have been collected from British Columbia to Newfoundland. Partial sequencing of G1 and N protein encoding regions from Canadian Peromyscus maniculatus-borne hantaviruses demonstrated the existence of significant genotypic divergence among strains. Phylogenetic analysis showed that Sin Nombre (SN)-like viruses from eastern and western Canadian deer mice can be divided into at least two broad-based genogroups. Sequencing of mitochondrial DNA from infected deer mice originating from various eastern and western provinces showed that SN-like virus genogroups appeared to be associated with distinct haplotypes of mice. Sera from deer mice infected with eastern and western viral genotypes neutralized the Sin Nombre virus strain, Convict Creek 107, but not the New York 1 hantavirus. Despite the genetic heterogeneity of Canadian SN-like strains these hantaviruses do not appear to define unique hantavirus serotypes.