Vaccination with the Crimean-Congo hemorrhagic fever virus viral replicon vaccine induces NP-based T-cell activation and antibodies possessing Fc-mediated effector functions.

Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy.

Complete Genome Sequences of a Hantavirus Isolate from New York.

We report here the complete genome sequences for all three segments of the New York hantavirus (New York 1). This is the first reported L segment sequence for hantaviruses maintained in Peromyscus spp. endemic to the eastern United States and Canada.

Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012.

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.

Impaired hypothalamic endocrine function in neuroacanthocytosis.

Two sisters aged 28 and 26, suffering from neuroacanthocytosis (NA), had hypothyroidism and showed a flat growth hormone (GH) response to the insulin tolerance test. GH-releasing hormone (GHRH) administration resulted in a normal GH response in the older sister and a partial GH response in the younger sister. A pathological process--attributable to NA--can be suggested, leading to impaired hypothalamic function, but sparing the pituitary.

Sin Nombre virus glycoprotein trafficking.

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.

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells.

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 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) 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-gamma)-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-gamma 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-kappaB 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.

Retrospective serological and genetic study of the distribution of hantaviruses in Greece.

A retrospective serological and genetic study of hantaviruses responsible for hemorrhagic fever with renal syndrome (HFRS) in Greece during the last 17 years is presented. Fifty-one serum samples taken from 30 HFRS cases previously diagnosed by immunofluorescence assay were tested by ELISA for IgG (Hantaan, Dobrava, and Puumala) and IgM antibodies (Hantaan and Puumala). Results were compatible with the majority of infections being related to hantaviruses carried by rodents of the subfamily Murinae. RNA was extracted from 26 selected samples and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers specifically designed for the detection of hanta-viruses associated with murine (MS-N-specific, MM-G1-specific primers) or arvicoline rodents (PPT-N-specific primers). In addition, primers previously designed for the detection of the G2 coding region of the Murine-associated hanta-viruses were also used. Sequencing of the PCR products was then performed, followed by phylogenetic analysis of nucleotide sequence differences. Eleven out of the 26 serum samples tested were found to be positive by PCR with the MS-N primers, whereas four were positive with the MM-G1 primers, and only two with the G2 primers. None of the samples was found positive with the PPT primers. The sequence analysis showed that the virus that was responsible for these 11 HFRS cases was the Dobrava virus, which is endemic throughout the Balkans.

Hantavirus pulmonary syndrome: CD8+ and CD4+ cytotoxic T lymphocytes to epitopes on Sin Nombre virus nucleocapsid protein isolated during acute illness.

In 1993 a number of cases of unexplained adult respiratory syndrome occurred in the southwestern United States. The illness was characterized by a prodrome of fever, myalgia, and other symptoms followed by the rapid onset of a capillary leak syndrome with hemoconcentration, thrombocytopenia, and pulmonary edema. Viral RNA sequences in the lungs identified a new member of the hantavirus genus, Sin Nombre virus (SNV), unique to North America. Pulmonary endothelial cells were heavily infected but were not necrotic. We speculated that this capillary leak syndrome was initiated by immune responses to the SNV-infected pulmonary endothelial cells. We isolated a CD8+ cytotoxic T lymphocyte (CTL) clone directly from the blood of a patient with the acute hantavirus pulmonary syndrome (HPS) which recognizes a SNV specific epitope on the virus nucleocapsid protein (aa 234-242) that is restricted by HLA C7 and produces IFN gamma but not IL-4. We identified a second CD8+ CTL epitope located within another site aa 131-139 on the nucleocapsid protein, which is HLA B35 restricted, and a CD4+ CTL epitope located on a third site on nucleocapsid protein aa 372-380 using lymphocytes obtained during HPS from another patient that were stimulated in vitro. Hantavirus specific CD8+ and CD4+ CTL may contribute to the immunopathology and capillary leak syndrome observed in the HPS.

Direct genetic detection of Dobrava virus in Greek and Albanian patients with hemorrhagic fever with renal syndrome.

Blood samples were collected from an Albanian and a Greek patient with hemorrhagic fever with renal syndrome and tested by reverse transcriptase-polymerase chain reaction. The genetic detection assay amplified hantavirus-specific DNA fragments from RNA extracted from the blood of the patients; nucleotide sequence analysis revealed that the causative agent of the disease was Dobrava virus. These findings suggest that Dobrava virus (which was originally isolated from the lungs of an Apodenws flavicollis mouse in Slovenia) is endemic throughout the Balkan States and causes overt human disease.

Complete genetic characterization and analysis of isolation of Sin Nombre virus.

This study reports completion of the genetic characterization of the entire genome of Sin Nombre (SN) virus (NMH10) detected in autopsy tissues from a patient who died of hantavirus pulmonary syndrome (HPS). The large (L) genome segment was found to be 6,562 nucleotides in length and encoded a putative L polymerase that was 2,153 amino acids in length. No evidence of segment reassortment with other well-characterized hantaviruses was obtained. The sequence of the entire S, M, and L genome segments of SN virus (strain NMR11) isolated from a mouse (trapped in the residence of the patient infected with SN virus [NMH10]) by passage two times in Peromyscus maniculatus and then by five passages in E6 Vero cells was determined and compared with that of the virus detected in autopsy tissues. Only 16 nucleotide differences were detected between the virus genomes, and none of these resulted in virus protein amino acid substitutions. Determination of the exact 5'- and 3'-terminal sequences of all genome segments of SN virus and representatives of other serologic groups in the Hantavirus genus, family Bunyaviridae, showed the existence of conserved nucleotide domains that may be involved in important regulatory mechanisms, such as RNA encapsidation, polymerase binding, and control of transcription and replication.

Fatal illness associated with a new hantavirus in Louisiana.

A fatal case of hantaviral illness occurred in Louisiana, outside of the range of P. maniculatus, the rodent reservoir for Sin Nombre virus. Hantavirus RNA and antigens were detected in patient autopsy tissues, and nucleotide sequence analysis of amplified polymerase chain reaction (PCR) products identified a newly recognized unique hantavirus, provisionally named Bayou virus. Prominent features of the clinical illness are compatible with hantavirus pulmonary syndrome (HPS), but several features such as renal insufficiency and intraalveolar hemorrhage are more compatible with hemorrhagic fever with renal syndrome (HFRS), a disease associated with Eurasian hantaviruses.

A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana.

Genetic analysis of virus detected in autopsy tissues of a fatal hantavirus pulmonary syndrome-like case in Louisiana revealed the presence of a previously unrecognized hantavirus. Nucleotide sequence analysis of PCR fragments of the complete S and M segments of the virus amplified from RNA extracted from the tissues showed the virus to be novel, differing from the closest related hantavirus, Sin Nombre virus, by approximately 30%. Both genome segments were unique, and there was no evidence of genetic reassortment with previously characterized hantaviruses. The primary rodent reservoir of Sin Nombre virus, the deer mouse Peromyscus maniculatus, is absent from Louisiana. Thus, the virus detected in Louisiana, referred to here as Bayou virus, must possess a different rodent reservoir.

Identification of a new North American hantavirus that causes acute pulmonary insufficiency.

In May 1993, a pulmonary disease syndrome with novel clinical and epidemiologic features was identified in the southwestern United States. Healthy young adults developed a febrile prodrome followed by the rapid onset of often lethal acute respiratory distress. Although an infectious disease was suspected, intensive investigations initially failed to identify the causative agent. Multiple specialized microbiology laboratories at the National Center for Infectious Diseases (Centers for Disease Control and Prevention) applied classic serologic and culture methods as well as recently developed molecular biological techniques to samples collected from field investigations of the patients. Serologic tests detected the presence of an active immune response to a hantavirus. Reverse transcription and polymerase chain reaction amplification of RNA extracted from human tissues used primers designed from sequences of known hantaviruses to demonstrate genomic sequences of a novel hantavirus. Immunohistochemistry showed the presence of hantavirus antigens in the endothelium of lung tissues from patients and provided the final pathogenetic link to this group of viruses. These methods were concordantly positive in virtually all samples available from 18 patients with compatible clinical histories identified between January and July 1993. Test results of control subjects and searches for other agents in identified cases were negative. This newly recognized hantavirus causes a novel syndrome of acute pulmonary edema and shock; the pathogenesis is related to the presence of virus antigens in the pulmonary capillaries. The virus may be an important cause of severe and fatal disease presenting as adult respiratory distress syndrome in otherwise healthy persons.

Detection of Muerto Canyon virus RNA in peripheral blood mononuclear cells from patients with hantavirus pulmonary syndrome.

To determine if Muerto Canyon Virus (MCV) RNA is present in the peripheral blood of patients with hantavirus pulmonary syndrome, a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for MCV RNA was used on blood samples from 20 seropositive case-patients. RNA was prepared from peripheral blood mononuclear cells (PBMC) or blood clot (or both) from 19 and from plasma from 11 case-patients. All 12 blood clot, all 13 PBMC, and 8 of 11 plasma preparations produced an MCV amplification product after RT-PCR with primers from the G2 gene. All of 5 PBMC RNA preparations tested were positive using unnested primers in S segment. Nucleotide sequences were determined for 16 G2 amplimers and 4 S segment amplimers, verifying that unique MCV cDNA sequences were amplified. Viral RNA became undetectable in 5 of 7 convalescent samples tested but was present up to day 23 of illness in 2 case-patients.

Isolation of the causative agent of hantavirus pulmonary syndrome.

Investigation of a recent outbreak of acute respiratory illness in the southwestern United States resulted in the recognition of a new disease, hantavirus pulmonary syndrome (HPS) with high mortality. Different animals and cell lines were used in attempts to isolate the causative agent. A previously unknown hantavirus was passaged in laboratory-bred deer mice, recovered from lung tissues of a deer mouse, Peromyscus maniculatus, and propagated in the E6 clone of Vero cells. Virus antigen was readily detected in the infected cells by an indirect immunofluorescence assay, using convalescent-phase sera from HPS patients. By electron microscopy, the virus was shown to have the typical morphologic features of members of the genus Hantavirus, family Bunyaviridae. Virus sequences corresponded to those previously detected by a nested reverse transcriptase-polymerase chain reaction assay of hantavirus-infected specimens from rodents and humans. This newly recognized virus, the etiologic agent of HPS, has been tentatively named Muerto Canyon virus.

Serologic and genetic identification of Peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the southwestern United States.

An outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States was etiologically linked to a newly recognized hantavirus. Knowledge that hantaviruses are maintained in rodent reservoirs stimulated a field and laboratory investigation of 1696 small mammals of 31 species. The most commonly captured rodent, the deer mouse (Peromyscus maniculatus), had the highest antibody prevalence (30%) to four hantavirus antigens. Antibody also was detected in 10 other species of rodent and in 1 species of rabbit. Reverse transcriptase-polymerase chain reaction (RT-PCR) products of hantavirus from rodent tissues were indistinguishable from those from human HPS patients. More than 96% of the seropositive P. maniculatus were positive by RT-PCR, suggesting chronic infection. Antibody prevalences were similar among P. maniculatus trapped from Arizona (33%), New Mexico (29%), and Colorado (29%). The numeric dominance of P. maniculatus, the high prevalence of antibody, and the RT-PCR findings implicate this species as the primary rodent reservoir for a new hantavirus in the southwestern United States.

Genome structure and variability of a virus causing hantavirus pulmonary syndrome.

A previously unrecognized hantavirus (family Bunyaviridae) has recently been detected and shown to be associated with a severe respiratory illness with high mortality, termed hantavirus pulmonary syndrome (HPS). This disease has now been identified throughout the western United States. We present nucleotide sequence characterization of the three RNA segments composing the HPS virus genome and address the question of the apparent emergence of this highly lethal virus. No evidence of genetic reassortment with previously recognized hantaviruses was found, each RNA segment being unique and approximately 30% different at the nucleotide level to the segments of the closest relative, Prospect Hill virus. These findings, together with the observed extensive genetic diversity of HPS viruses and examples of geographic clustering of distinct virus genotypes, suggest that HPS and associated virus have likely existed undetected for many years. The virus genome M segment was determined to be 3696 nucleotides in length and encode G1 and G2 proteins, 652 and 488 amino acids in length. The S segment was found to be 2059 nucleotides in length and to encode a nucleocapsid protein, 428 amino acids in length. S segment analysis also revealed an unusually long noncoding region with numerous repeats and evidence for a potential NSS protein encoded in an overlapping frame.

Utilization of autopsy RNA for the synthesis of the nucleocapsid antigen of a newly recognized virus associated with hantavirus pulmonary syndrome.

A newly recognized hantavirus was recently found to be associated with an outbreak of acute respiratory illness in the southwestern United States. The disease, which has become known as hantavirus pulmonary syndrome, has an unusually high mortality (64%). Virus isolation attempts have been unsuccessful thus far, resulting in a lack of homologous antigen for use in diagnostic assays. For this reason, a molecular approach was initiated to produce recombinant homologous antigen. The virus nucleocapsid (N) protein was selected, since N has been shown to be a sensitive antigenic target in other hantavirus systems. The N protein open reading frame of the virus S genome segment was synthesized from frozen autopsy tissue by polymerase chain reaction amplification, followed by cloning and expression in Hela cells (vaccinia-T7 RNA polymerase system) and Escherichia coli. N protein-expressing Hela cells served as excellent antigens for an improved indirect immunofluorescence assay. Use of the E. coli-expressed N protein in an enzyme-linked immunosorbent assay improved the sensitivity and specificity when compared with heterologous antigens used previously. Preliminary analysis also indicates that the higher sensitivity could result in earlier detection of infected persons. These data demonstrate that even in the absence of a virus isolate, the necessary homologous antigen can be produced and can serve to improve the detection and diagnostic capabilities needed to combat this newly recognized fatal respiratory illness in the United States.