Robert W. Cross

Genetic Diversity and Histo-Blood Group Antigen Interactions of Rhesus Enteric Caliciviruses

ABSTRACT Recently, we reported the discovery and characterization of Tulane virus (TV), a novel rhesus calicivirus (CV) (T. Farkas, K. Sestak, C. Wei, and X. Jiang, J. Virol. 82:5408-5416, 2008). TV grows well in tissue culture, and it represents a new genus within Caliciviridae, with the proposed name of Recovirus. We also reported a high prevalence of CV antibodies in macaques of the Tulane National Primate Research Center (TNPRC) colony, including anti-norovirus (NoV), anti-sapovirus (SaV), and anti-TV (T. Farkas, J. Dufour, X. Jiang, and K. Sestak, J. Gen. Virol. 91:734-738, 2010). To broaden our knowledge about CV infections in captive nonhuman primates (NHP), 500 rhesus macaque stool samples collected from breeding colony TNPRC macaques were tested for CVs. Fifty-seven (11%) samples contained recovirus isolates. In addition, one NoV was detected. Phylogenetic analysis classified the recovirus isolates into two genogroups and at least four genetic types. The rhesus NoV isolate was closely related to GII human NoVs. TV-neutralizing antibodies were detected in 88% of serum samples obtained from primate caretakers. Binding and plaque reduction assays revealed the involvement of type A and B histo-blood group antigens (HBGA) in TV infection. Taken together, these findings indicate the zoonotic potential of primate CVs. The discovery of a genetically diverse and prevalent group of primate CVs and remarkable similarities between rhesus enteric CVs and human NoVs opens new possibilities for research involving in vitro and in vivo models of human NoV gastroenteritis.

Lassa Fever in Post-Conflict Sierra Leone

Background Lassa fever (LF), an often-fatal hemorrhagic disease caused by Lassa virus (LASV), is a major public health threat in West Africa. When the violent civil conflict in Sierra Leone (1991 to 2002) ended, an international consortium assisted in restoration of the LF program at Kenema Government Hospital (KGH) in an area with the worlds highest incidence of the disease. Methodology/Principal Findings Clinical and laboratory records of patients presenting to the KGH Lassa Ward in the post-conflict period were organized electronically. Recombinant antigen-based LF immunoassays were used to assess LASV antigenemia and LASV-specific antibodies in patients who met criteria for suspected LF. KGH has been reestablished as a center for LF treatment and research, with over 500 suspected cases now presenting yearly. Higher case fatality rates (CFRs) in LF patients were observed compared to studies conducted prior to the civil conflict. Different criteria for defining LF stages and differences in sensitivity of assays likely account for these differences. The highest incidence of LF in Sierra Leone was observed during the dry season. LF cases were observed in ten of Sierra Leones thirteen districts, with numerous cases from outside the traditional endemic zone. Deaths in patients presenting with LASV antigenemia were skewed towards individuals less than 29 years of age. Women self-reporting as pregnant were significantly overrepresented among LASV antigenemic patients. The CFR of ribavirin-treated patients presenting early in acute infection was lower than in untreated subjects. Conclusions/Significance Lassa fever remains a major public health threat in Sierra Leone. Outreach activities should expand because LF may be more widespread in Sierra Leone than previously recognized. Enhanced case finding to ensure rapid diagnosis and treatment is imperative to reduce mortality. Even with ribavirin treatment, there was a high rate of fatalities underscoring the need to develop more effective and/or supplemental treatments for LF.

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design.

The Domestic Ferret (Mustela putorius furo) as a Lethal Infection Model for 3 Species of Ebolavirus

Small-animal models have been developed for several Filoviridae species; however, serial adaptation was required to produce lethal infection. These adapted viruses have sequence changes in several genes, including those that modulate the host immune response. Nonhuman primate models do not require adaptation of filoviruses. Here, we describe lethal models of disease for Bundibugyo, Sudan, and Zaire species of Ebolavirus in the domestic ferret, using wild-type nonadapted viruses. Pathologic features were consistent with disease in primates. Of particular importance, this is the only known small-animal model developed for Bundibugyo and the only uniformly lethal animal model for Bundibugyo.

Multiple Circulating Infections Can Mimic the Early Stages of Viral Hemorrhagic Fevers and Possible Human Exposure to Filoviruses in Sierra Leone Prior to the 2014 Outbreak

Lassa fever (LF) is a severe viral hemorrhagic fever caused by Lassa virus (LASV). The LF program at the Kenema Government Hospital (KGH) in Eastern Sierra Leone currently provides diagnostic services and clinical care for more than 500 suspected LF cases per year. Nearly two-thirds of suspected LF patients presenting to the LF Ward test negative for either LASV antigen or anti-LASV immunoglobulin M (IgM), and therefore are considered to have a non-Lassa febrile illness (NLFI). The NLFI patients in this study were generally severely ill, which accounts for their high case fatality rate of 36%. The current studies were aimed at determining possible causes of severe febrile illnesses in non-LF cases presenting to the KGH, including possible involvement of filoviruses. A seroprevalence survey employing commercial enzyme-linked immunosorbent assay tests revealed significant IgM and IgG reactivity against dengue virus, chikungunya virus, West Nile virus (WNV), Leptospira, and typhus. A polymerase chain reaction-based survey using sera from subjects with acute LF, evidence of prior LASV exposure, or NLFI revealed widespread infection with Plasmodium falciparum malaria in febrile patients. WNV RNA was detected in a subset of patients, and a 419 nt amplicon specific to filoviral L segment RNA was detected at low levels in a single patient. However, 22% of the patients presenting at the KGH between 2011 and 2014 who were included in this survey registered anti-Ebola virus (EBOV) IgG or IgM, suggesting prior exposure to this agent. The 2014 Ebola virus disease (EVD) outbreak is already the deadliest and most widely dispersed outbreak of its kind on record. Serological evidence reported here for possible human exposure to filoviruses in Sierra Leone prior to the current EVD outbreak supports genetic analysis that EBOV may have been present in West Africa for some time prior to the 2014 outbreak.

Modeling the Disease Course of Zaire ebolavirus Infection in the Outbred Guinea Pig

BACKGROUND Rodent models that accurately reflect human filovirus infection are needed as early screens for medical countermeasures. Prior work in rodents with the Zaire species of Ebola virus (ZEBOV) primarily used inbred mice and guinea pigs to model disease. However, these inbred species do not show some of the important features of primate ZEBOV infection, most notably, coagulation abnormalities. METHODS Thirty-six outbred guinea pigs were infected with guinea pig-adapted ZEBOV and examined sequentially over an 8-day period to investigate the pathologic events that lead to death. RESULTS Features of disease in ZEBOV-infected outbred guinea pigs were largely consistent with disease in humans and nonhuman primates and included early infection of macrophages and dendritiform cells, apoptosis of bystander lymphocytes, and increases in levels of proinflammatory cytokines. Most importantly, dysregulation of circulating levels of fibrinogen, protein C activity, and antifibrinolytic proteins and deposition of fibrin in tissues demonstrated both biochemical and microscopic evidence of disseminated intravascular coagulation. CONCLUSIONS These findings suggest that the outbred guinea pig model recapitulates ZEBOV infection of primates better than inbred rodent models, is useful for dissecting key events in the pathogenesis of ZEBOV, and is useful for evaluating candidate interventions prior to assessment in primates.

Comparison of the Pathogenesis of the Angola and Ravn Strains of Marburg Virus in the Outbred Guinea Pig Model

BACKGROUND Phylogenetic comparisons of known Marburg virus (MARV) strains reveal 2 distinct genetic lineages: Ravn and the Lake Victoria Marburg complex (eg, Musoke, Popp, and Angola strains). Nucleotide variances of >20% between Ravn and other MARV genomes suggest that differing virulence between lineages may accompany this genetic divergence. To date, there exists limited systematic experimental evidence of pathogenic differences between MARV strains. METHODS Uniformly lethal outbred guinea pig models of MARV-Angola (MARV-Ang) and MARV-Ravn (MARV-Rav) were developed by serial adaptation. Changes in genomic sequence, weight, temperature, histopathologic findings, immunohistochemical findings, hematologic profiles, circulating biochemical enzyme levels, coagulation parameters, viremia levels, cytokine levels, eicanosoid levels, and nitric oxide production were compared between strains. RESULTS MARV-Rav infection resulted in delayed increases in circulating inflammatory and prothrombotic elements, notably lower viremia levels, less severe histologic alterations, and a delay in mean time to death, compared with MARV-Ang infection. Both strains produced more marked coagulation abnormalities than previously seen in MARV-infected mice or inbred guinea pigs. CONCLUSIONS Although both strains exhibit great similarity to pathogenic markers of human and nonhuman primate MARV infection, these data highlight several key differences in pathogenicity that may serve to guide the choice of strain and model used for development of vaccines or therapeutics for Marburg hemorrhagic fever.

The immunomodulating V and W proteins of Nipah virus determine disease course

The viral determinants that contribute to Nipah virus (NiV)-mediated disease are poorly understood compared with other paramyxoviruses. Here we use recombinant NiVs (rNiVs) to examine the contributions of the NiV V and W proteins to NiV pathogenesis in a ferret model. We show that a V-deficient rNiV is susceptible to the innate immune response in vitro and behaves as a replicating non-lethal virus in vivo. Remarkably, rNiV lacking W expression results in a delayed and altered disease course with decreased respiratory disease and increased terminal neurological disease associated with altered in vitro inflammatory cytokine production. This study confirms the V protein as the major determinant of pathogenesis, also being the first in vivo study to show that the W protein modulates the inflammatory host immune response in a manner that determines the disease course.

Pathogenic Differences between Nipah Virus Bangladesh and Malaysia Strains in Primates: Implications for Antibody Therapy

Nipah virus (NiV) is a paramyxovirus that causes severe disease in humans and animals. There are two distinct strains of NiV, Malaysia (NiVM) and Bangladesh (NiVB). Differences in transmission patterns and mortality rates suggest that NiVB may be more pathogenic than NiVM. To investigate pathogenic differences between strains, 4 African green monkeys (AGM) were exposed to NiVM and 4 AGMs were exposed to NiVB. While NiVB was uniformly lethal, only 50% of NiVM-infected animals succumbed to infection. Histopathology of lungs and spleens from NiVB-infected AGMs was significantly more severe than NiVM-infected animals. Importantly, a second study utilizing 11 AGMs showed that the therapeutic window for human monoclonal antibody m102.4, previously shown to rescue AGMs from NiVM infection, was much shorter in NiVB-infected AGMs. Together, these data show that NiVB is more pathogenic in AGMs under identical experimental conditions and suggests that postexposure treatments may need to be NiV strain specific for optimal efficacy.

Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever

There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.