Stephen St. Jeor

Genetic Diversity and Epidemiology of Hantaviruses in Argentina

Phylogenetic analysis of a 292-nucleotide (nt) fragment of the hantavirus M genome segment from 36 rodent and 13 human samples from three known foci of hantavirus infection in Argentina was conducted. A 1654-nt fragment of the M genome segment was analyzed for 1 representative of 7 genetically distinct hantavirus lineages identified. Additionally, the nt sequence of the complete M genome segments of Lechiguanas, Oran, and Hu39694 hantavirus genotypes was determined. nt sequence comparisons reveal that 7 hantavirus lineages from Argentina differ from each other by 11.5%-21.8% and from Sin Nombre, Bayou, and Black Creek Canal viruses by 23.8%-26.5%. Phylogenetic analyses demonstrate that they form a unique, separate branch within the clade containing other New World sigmodontine-borne hantaviruses. Most Oligoryzomys-borne hantavirus genotypes clearly map together. The Oligoryzomys-borne genotypes Lechiguanas, Oran, and Andes appear to be associated with human disease. Oligoryzomys longicaudatus was identified as the likely rodent reservoir for Andes virus.

Hantaviruses: Molecular Biology, Evolution and Pathogenesis

Hantaviruses are tri-segmented negative sense single stranded RNA viruses that belong to the family Bunyaviridae. In nature, hantaviruses are exclusively maintained in the populations of their specific rodent hosts. In their natural host species, hantaviruses usually develop a persistent infection with prolonged virus shedding in excreta. Humans become infected by inhaling virus contaminated aerosol. Unlike asymptomatic infection in rodents, hantaviruses cause two acute febrile diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The mortality rate varies from 0.1% to 40% depending on the virus involved. Hantaviruses are distributed world wide, with over 150,000 HFRS and HPS cases being registered annually. In this review we summarize current knowledge on hantavirus molecular biology, epidemiology, genetic diversity and co-evolution with rodent hosts. In addition, special attention was given in this review to describing clinical manifestation of HFRS and HPS, and advances in our current understanding of the host immune response, treatment, and prevention.

Analysis of Hantavirus Genetic Diversity in Argentina: S Segment-Derived Phylogeny

ABSTRACT Nucleotide sequences were determined for the complete S genome segments of the six distinct hantavirus genotypes from Argentina and for two cell culture-isolated Andes virus strains from Chile. Phylogenetic analysis indicates that, although divergent from each other, all Argentinian hantavirus genotypes group together and form a novel phylogenetic clade with the Andes virus. The previously characterized South American hantaviruses Laguna Negra virus and Rio Mamore virus make up another clade that originates from the same ancestral node as the Argentinian/Chilean viruses. Within the clade of Argentinian/Chilean viruses, three subclades can be defined, although the branching order is somewhat obscure. These are made of (i) “Lechiguanas-like” virus genotypes, (ii) Maciel virus and Pergamino virus genotypes, and (iii) strains of the Andes virus. Two hantavirus genotypes from Brazil, Araraquara and Castello dos Sonhos, were found to group with Maciel virus and Andes virus, respectively. The nucleocapsid protein amino acid sequence variability among the members of the Argentinian/Chilean clade does not exceed 5.8%. It is especially low (3.5%) among oryzomyine species-associated virus genotypes, suggesting recent divergence from the common ancestor. Interestingly, the Maciel and Pergamino viruses fit well with the rest of the clade although their hosts are akodontine rodents. Taken together, these data suggest that under conditions in which potential hosts display a high level of genetic diversity and are sympatric, host switching may play a prominent role in establishing hantavirus genetic diversity. However, cospeciation still remains the dominant factor in the evolution of hantaviruses.

Effects of Tumor Necrosis Factor Alpha on Sin Nombre Virus Infection In Vitro

ABSTRACT Previous data indicate that immune mechanisms may be involved in developing capillary leakage during Sin Nombre virus (SNV) infection. Therefore, we investigated production of tumor necrosis factor alpha (TNF-α) by human alveolar macrophages and human umbilical vein endothelial cells (HUVEC) after infection with SNV. In addition, we examined the effect of TNF-α on HUVEC monolayer leakage. Our results reveal that although TNF-α decreases accumulation of viral nucleoproteins, TNF-α levels do not change in SNV-infected cells. In addition, supernatants from SNV-infected human alveolar macrophages did not cause a significant increase in endothelial monolayer permeability.

Contact heterogeneity in deer mice: implications for Sin Nombre virus transmission

Heterogeneities within disease hosts suggest that not all individuals have the same probability of transmitting disease or becoming infected. This heterogeneity is thought to be due to dissimilarity in susceptibility and exposure among hosts. As such, it has been proposed that many host–pathogen systems follow the general pattern whereby a small fraction of the population accounts for a large fraction of the pathogen transmission. This disparity in transmission dynamics is often referred to as ‘20/80 Rule’, i.e. approximately 20 per cent of the hosts are responsible for 80 per cent of pathogen transmission. We investigated the role of heterogeneity in contact rates among potential hosts of a directly transmitted pathogen by examining Sin Nombre virus (SNV) in deer mice (Peromyscus maniculatus). Using foraging arenas and powder marking, we documented contacts between wild deer mice in Great Basin Desert, central Utah. Our findings demonstrated heterogeneity among deer mice, both in frequency and in duration of contacts with other deer mice. Contact dynamics appear to follow the general pattern that a minority of the population accounts for a majority of the contacts. We found that 20 per cent of individuals in the population were responsible for roughly 80 per cent of the contacts observed. Larger-bodied individuals appear to be the functional group with the greatest SNV transmission potential. Contrary to our predictions, transmission potential was not influenced by breeding condition or sex.

Sin Nombre virus and rodent species diversity: A test of the dilution and amplification hypotheses

Background Species diversity is proposed to greatly impact the prevalence of pathogens. Two predominant hypotheses, the “Dilution Effect” and the “Amplification Effect”, predict divergent outcomes with respect to the impact of species diversity. The Dilution Effect predicts that pathogen prevalence will be negatively correlated with increased species diversity, while the Amplification Effect predicts that pathogen prevalence will be positively correlated with diversity. For many host-pathogen systems, the relationship between diversity and pathogen prevalence has not be empirically examined. Methodology/Principal Findings We tested the Dilution and Amplification Effect hypotheses by examining the prevalence of Sin Nombre virus (SNV) with respect to diversity of the nocturnal rodent community. SNV is directly transmitted primarily between deer mice (Peromyscus maniculatus). Using mark-recapture sampling in the Spring and Fall of 2003–2005, we measured SNV prevalence in deer mice at 16 landscape level sites (3.1 hectares each) that varied in rodent species diversity. We explored several mechanisms by which species diversity may affect SNV prevalence, including reduced host density, reduced host persistence, the presence of secondary reservoirs and community composition. We found a negative relationship between species diversity and SNV prevalence in deer mice, thereby supporting the Dilution Effect hypothesis. Deer mouse density and persistence were lower at sites with greater species diversity; however, only deer mouse persistence was positively correlated with SNV prevalence. Pinyon mice (P. truei) may serve as dilution agents, having a negative effect on prevalence, while kangaroo rats (Dipodomys ordii), may have a positive effect on the prevalence of SNV, perhaps through effects on deer mouse behavior. Conclusions/Significance While previous studies on host-pathogen systems have found patterns of diversity consistent with either the Dilution or Amplification Effects, the mechanisms by which species diversity influences prevalence have not been investigated. Our study indicates that changes in host persistence, coupled with interspecific interactions, are important mechanisms through which diversity may influence patterns of pathogens. Our results reveal the complexity of rodent community interactions with respect to SNV dynamics.

Human cytomegalovirus persists in myeloid progenitors and is passed to the myeloid progeny in a latent form

CD34+ progenitor cells can harbour latent human cytomegalovirus (HCMV); however, the mechanisms of HCMV latency remain unclear. We have investigated the effects of the haematopoietic lineage restriction on the establishment and spread of the latent HCMV to progeny cells. In vitro‐infected and latently‐infected haematopoietic progenitor cells derived from HCMV seropositive donors were studied. The presence of HCMV DNA in bone marrow progenitor (BMP) cells was determined by single colony polymerase chain reaction and fluorescent in situ hybridization (FISH). The presence of CMV DNA was found to be restricted to myeloid progenitors and the percentage of HCMV‐infected cells was lower in naturally‐infected cells than in in vitro‐infected cells. Erythroid differentiation resulted in an abortive infection with persistence of the viral nucleic acids in red cell precursors. In BMP cells from HCMV seronegative donors, HCMV DNA was localized in the nucleus. Bone marrow progenitors in the presence of granulocyte‐macrophage colony stimulating factor (GMCSF) maintained HCMV DNA for extended periods of time. No viral production could be detected throughout the culture but the comparison of the numbers of latently‐infected cells prior to and after the culture suggests that proliferation of haematopoietic progenitor cells may lead to the expansion of latently‐infected cells.

Identification of a putative cell receptor for human cytomegalovirus

Proteins from a variety of cell types were separated using SDS-PAGE, transferred to nitrocellulose filters, and probed with intact human cytomegalovirus (HCMV). Virus bound to cell proteins was detected directly using 125I-labeled HCMV or indirectly using an immunologic assay with a secondary antibody labeled with 125I. Using this approach proteins of molecular weights 32 and 34 kDa were identified that would bind HCMV and were present on T4+ and T8+ lymphocytes, a B lymphoblastoid cell line, and human diploid fibroblasts. Binding of labeled virus to cells could be blocked by the addition of unlabeled homologous virus. Treatment of virus with NP40 to remove the virus envelope blocked binding to cellular proteins. Both the 32- and 34-kDa proteins could be copurified with cellular membranes. HCMV bound equally well to T4+ and T8+ cells as well as to either replicating (PHA or interleukin-2 stimulated) or resting lymphocytes. Interestingly, there appeared to be a single binding site for HCMV on human fibroblasts (34 kDa). The results support the idea that there is a receptor for HCMV present on the surface of human lymphocytes and fibroblasts.

Role of Maternal Antibody in Natural Infection of Peromyscus maniculatus with Sin Nombre Virus

ABSTRACT Data from naturally infected deer mice (Peromyscus maniculatus) were used to investigate vertical transmission of Sin Nombre virus (SNV) and SNV-specific antibody. The antibody prevalence in juvenile mice (14 g or less) was inversely proportional to the mass of the animal, with juvenile deer mice weighing less than 11 g most likely to be antibody positive (26.9%) and juvenile mice weighing between 13 and 14 g least likely to be antibody positive (12.9%). Although a significant sex bias in seropositivity was detected in adult deer mice, no significant sex bias in seropositivity was detected in juvenile animals. Ten juvenile deer mice were identified that had initially tested positive for SNV-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) but had subsequently tested negative when recaptured as adults. SNV RNA was detected by reverse transcriptase PCR (RT-PCR) in the blood of ELISA-positive adult deer mice but not in the blood of ELISA-positive juveniles. One of the juvenile mice initially tested negative for SNV RNA but later tested positive when recaptured as an ELISA-positive adult. The RT-PCR results for that individual correlated with the disappearance and then reappearance of SNV-specific IgG, indicating that the presence of SNV RNA at later time points was due to infection with SNV via horizontal transmission. SNV-specific antibody present in both ELISA-positive juvenile and adult mice was capable of neutralizing SNV. Additionally, our data indicate that SNV is not transmitted vertically.

Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus

ABSTRACT Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected hamsters. No vaccinated animal died, and there were no obvious clinical signs of disease. Further, hantavirus RNA was not detected by sensitive reverse transcription-PCR in tissues and blood of hamsters immunized with both AdGN and AdGC. Cellular immunity appeared to be important for protection because the AdN vector completely protected animals. All three Ad vectors produced strong cytotoxic T-lymphocyte responses directed to hantavirus proteins in mice. Moreover, hamsters vaccinated with AdN, AdGN, or AdGC produced no detectable neutralizing antibodies yet were protected. These Ad vectors represent the first vaccines that prevent lethal hantavirus disease and, in some instances (AdGN and AdGC), provide sterile immunity. These observations set the stage for a more detailed characterization of the types of immunity required to protect humans from hantavirus infections.