Isolation of a novel adenovirus from Rousettus leschenaultii bats from India.

Surveillance work was initiated to study the presence of highly infectious diseases like Ebola-Reston, Marburg, Nipah and other possible viruses that are known to be found in the bat species and responsible for causing diseases in humans. A novel adenovirus was isolated from a common species of fruit bat (Rousettus leschenaultii) captured in Maharashtra State, India. Partial sequence analysis of the DNA polymerase gene shows this isolate to be a newly recognized member of the genus Mastadenovirus (family Adenoviridae), approximately 20% divergent at the nucleotide level from Japanese BatAdV, its closest known relative.

Immunization for Ebola virus infection.

Infection by Ebola virus causes rapidly progressive, often fatal, symptoms of fever, hemorrhage and hypotension. Previous attempts to elicit protective immunity for this disease have not met with success. We report here that protection against the lethal effects of Ebola virus can be achieved in an animal model by immunizing with plasmids encoding viral proteins. We analyzed immune responses to the viral nucleoprotein (NP) and the secreted or transmembrane forms of the glycoprotein (sGP or GP) and their ability to protect against infection in a guinea pig infection model analogous to the human disease. Protection was achieved and correlated with antibody titer and antigen-specific T-cell responses to sGP or GP. Immunity to Ebola virus can therefore be developed through genetic vaccination and may facilitate efforts to limit the spread of this disease.

Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus.

A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be alpha-dystroglycan (alpha-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified alpha-DG protein. Soluble alpha-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, alpha-DG is a cellular receptor for both LCMV and LFV.

Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness.

A mysterious respiratory illness with high mortality was recently reported in the southwestern United States. Serologic studies implicated the hantaviruses, rodent-borne RNA viruses usually associated elsewhere in the world with hemorrhagic fever with renal syndrome. A genetic detection assay amplified hantavirus-specific DNA fragments from RNA extracted from the tissues of patients and deer mice (Peromyscus maniculatus) caught at or near patient residences. Nucleotide sequence analysis revealed the associated virus to be a new hantavirus and provided a direct genetic link between infection in patients and rodents.

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.

Care of Ebola Survivors and Factors Associated With Clinical Sequelae-Monrovia, Liberia.

BACKGROUND: The Eternal Love Winning Africa (ELWA) Clinic was the first clinic to provide free, comprehensive care to Ebola virus disease (EVD) survivors in Liberia. The objectives of this analysis were to describe the demographics and symptoms of EVD survivors at ELWA from January 2015 through March 2017 and to identify risk factors for development of sequelae. METHODS: Patients' demographic and clinical information was collected by chart review in June 2016 and March 2017. Associations with clinical sequelae were analyzed using the chi-square test, t test, and multivariate logistic regression. RESULTS: From January 2015 to March 2017, 329 EVD survivors were evaluated at ELWA. Most survivors experienced myalgia/arthralgia (73%; n = 239) and headache (53%; n = 173). The length of time from Ebola Treatment Unit (ETU) discharge to first clinic visit ranged from 0 to 30 months. Many visits (30%) occurred 24 or more months after ETU discharge. The proportion of visits for headache, weight loss, joint pain, visual problems, insomnia, fatigue, memory loss, decreased libido, depression, and uveitis decreased over time. More men than women had visits for depression; however, these differences were not significant. Symptom prevalence differed in adults and children; significantly more adults experienced myalgia/arthralgia (77% vs 44%), visual problems (41% vs 12%), post-EVD-related musculoskeletal pain (42% vs 15%), and insomnia (17% vs 2%). CONCLUSIONS: EVD survivors frequented ELWA for EVD-related symptoms many months after ETU discharge, indicating a long-term need for care. Reported symptoms changed over time, which may reflect eventual resolution of some sequelae.

Hantavirus pulmonary syndrome in Florida: association with the newly identified Black Creek Canal virus.

Hantavirus pulmonary syndrome (HPS) is a recently recognized viral zoonosis. The first recognized cases were caused by a newly described hantavirus. Sin Nombre virus (previously known as Muerto Canyon virus), isolated from Peromyscus maniculatus (deer mouse). We describe a 33-year-old Floridian man who resided outside the ecologic range of P maniculatus but was found to have serologic evidence of a hantavirus infection during evaluation of azotemia associated with adult respiratory distress syndrome. Small mammal trapping conducted around this patient's residence demonstrated the presence of antihantaviral antibodies in 13% of Sigmodon hispidus [cotton rat). Serologic testing using antigen derived from the Black Creek Canal hantavirus subsequently isolated from this rodent established that this patient was acutely infected with this new pathogenic American hantavirus. HPS is not confined to the geographical distribution of P maniculatus and should be suspected in individuals with febrile respiratory syndromes, perhaps associated with azotemia, throughout the continental United States.

RNA genome stability of Toscana virus during serial transovarial transmission in the sandfly Phlebotomus perniciosus.

We have carried out a T1 ribonuclease fingerprinting analysis of the RNA genomes of Toscana virus isolates from successive generations of an experimentally virus-infected laboratory colony of Phlebotomus perniciosus sandflies. This analysis detected no virus RNA genome changes during transovarial transmission of the virus over 12 sandfly generations (a period of almost 2 years). These results demonstrate that although RNA viruses can exhibit high rates of mutational change under a variety of conditions, Toscana virus RNA genomes can be maintained in a stable manner during repeated transovarial virus transmission in the natural insect host. The implications of these results for insect RNA virus evolution are discussed.

Structure, expression and phylogenetic analysis of the glycoprotein gene of Cocal virus.

A cDNA copy of the mRNA of the glycoprotein G of Cocal virus, a rhabdovirus, has been cloned, sequenced and expressed in mammalian cells. The deduced amino acid sequence shows a typical transmembrane glycoprotein, 512 amino acids in length, containing two potential N-linked glycosylation sites. The amino acid sequence showed a high degree of identity with that of the prototype vesicular stomatitis virus serotype Indiana [VSV (IND)] G protein. In addition, phylogenetic analysis of amino acid sequence differences among the G proteins of vesiculoviruses indicated that Cocal virus represents a distinct lineage within the VSV (IND) serotype. Expression of the cloned Cocal G gene in mammalian cells produced a glycoprotein of mol.wt 71000 which was not palmitylated but induced cell fusion at acid pH.

Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus.

Infectious hematopoietic necrosis virus (IHNV) causes a highly lethal, economically important disease of salmon and trout. The virus is enzootic throughout western North America, and has been spread to Asia and Europe. The nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes of 12 diverse IHNV isolates were determined in order to examine the molecular epizootiology of IHN, the primary structure and conservation of NV, and the evolution of the virus. The G and NV genes and their encoded proteins were highly conserved, with a maximum pairwise nucleotide divergence of 3.6 and 4.4%, and amino acid divergence of 3.7 and 6.2%, respectively. Conservation of NV protein sequence (111 amino acids in length) confirms that the protein is functional and plays an important role in virus replication. The phylogenetic relationship of viruses was found to correlate with the geographic origin of virus isolates rather than with host species or time of isolation. These data are consistent with stable maintenance of virus in enzootic foci. Two main IHNV genetic lineages were identified; one in the Columbia River Basin (Oregon, Washington and Idaho), the other in the Sacramento River Basin (California). The first major IHNV outbreak in chinook salmon in 1973 in the Columbia River was genetically linked to importation of virus-infected fish eggs from the Sacramento River where outbreaks in chinook salmon are common. However, the introduced virus apparently did not persist, subsequent virus outbreaks in Columbia River chinook salmon being associated with Columbia River genetic lineages. In general, virus monoclonal antibody reactivity profiles and phylogenetic relationships correlated well.

The complete genome structure and phylogenetic relationship of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV), a member of the family Rhabdoviridae, causes a severe disease with high mortality in salmonid fish. The nucleotide sequence (11,131 bases) of the entire genome was determined for the pathogenic WRAC strain of IHNV from southern Idaho. This allowed detailed analysis of all 6 genes, the deduced amino acid sequences of their encoded proteins, and important control motifs including leader, trailer and gene junction regions. Sequence analysis revealed that the 6 virus genes are located along the genome in the 3' to 5' order: nucleocapsid (N), polymerase-associated phosphoprotein (P or M1), matrix protein (M or M2), surface glycoprotein (G), a unique non-virion protein (NV) and virus polymerase (L). The IHNV genome RNA was found to have highly complementary termini (15 of 16 nucleotides). The gene junction regions display the highly conserved sequence UCURUC(U)7RCCGUG(N)4CACR (in the vRNA sense), which includes the typical rhabdovirus transcription termination/polyadenylation signal and a novel putative transcription initiation signal. Phylogenetic analysis of M, G and L protein sequences allowed insights into the evolutionary and taxonomic relationship of rhabdoviruses of fish relative to those of insects or mammals, and a broader sense of the relationship of non-segmented negative-strand RNA viruses. Based on these data, a new genus, piscivirus, is proposed which will initially contain IHNV, viral hemorrhagic septicemia virus and Hirame rhabdovirus.

Genetic characterization of a human isolate of Puumala hantavirus from France.

PUU90-13 is a strain of Puumala (PUU) virus (family Bunyaviridae: genus Hantavirus) isolated from a human in northeastern France (Rollin et al., 1995). This report describes the full-length sequences of the small (S) and medium (M) genomic RNAs of PUU90-13. The terminal sequences of both the S and M genomic RNAs were found to be conserved and imperfectly complementary. The S RNA of PUU90-13 is 1847 nt in length and contains the nucleocapsid (N) protein gene and a potential overlapping open reading frame (ORF-2) previously described in other hantaviruses. Statistical analysis of the third base substitution frequency in the N ORFs of PUU90-13 and other PUU viruses suggests that the ORF-2 is functional. The M RNA is 3681 nt in length and encodes the glycoprotein precursor. Both genomic segments share the highest degree of nucleotide and amino acid sequence identity with PUUBerkel, a PUU virus from Germany. Phylogenetic analyses of sequences from both segments indicate that PUU90-13 occupies a distinct Western European PUU virus lineage that it shares with PUUBerkel. Both PUU90-13 and PUUBerkel lack a potential N-linked glycosylation site found on the G2 glycoprotein of other PUU viruses.

The genetic diversity and epizootiology of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes a serious disease in salmonid fish. The T1 ribonuclease fingerprinting method was used to compare the RNA genomes of 26 isolates of IHNV recovered from sockeye salmon (Oncorhynchus nerka), chinook salmon (O. tshawytscha), and steelhead trout (O. mykiss) throughout the enzootic portion of western North America. Most of the isolates analyzed in this study were from a single year (1987) to limit time of isolation as a source of genetic variation. In addition, isolates from different years collected at three sites were analyzed to investigate genetic drift or evolution of IHNV within specific locations. All of the isolates examined by T1 fingerprint analysis contained less than a 50% variation in spot location and were represented by a single fingerprint group. The observed variation was estimated to correspond to less than 5% variation in the nucleic acid sequence. However, sufficient variation was detected to separate the isolates into four subgroups which appeared to correlate to different geographic regions. Host species appeared not to be a significant source of variation. The evolutionary and epizootiologic significance of these findings and their relationship to other evidence of genetic variation in IHNV isolates are discussed.

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.

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.

Isolation, characterization and geographic distribution of Caño Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae).

Rodents collected from the Venezuelan llanos (plains) during field studies of viral hemorrhagic fever were tested for evidence of hantavirus infection. Hantavirus antibody was found in one (7.7%) of 13 Oryzomys bicolor, one (3.4%) of 29 Rattus rattus, 10 (6.0%) of 166 Sigmodon alstoni and one (2.2%) of 45 Zygodontomys brevicauda. Hantavirus-specific RNA was detected in lung tissues from four antibody-positive rodents: two S. alstoni from Portuguesa State and one S. alstoni each from Cojedes and Barinas States. A hantavirus isolate (herein identified as VHV-574) was recovered from lung tissue from a hantavirus RNA-positive S. alstoni collected from Portuguesa State. The results of serological tests and analyses of small and medium RNA segment nucleotide sequence data indicated that VHV-574 represents a novel hantavirus (proposed name 'Caño Delgadito') that is distinct from all previously characterized hantaviruses. The results of analyses of nucleotide sequence data from the four hantavirus RNA-positive S. alstoni suggested that Caño Delgadito virus is widely distributed in the Venezuelan llanos.

Short report: isolation and partial characterization of a Puumala virus from a human case of nephropathia epidemica in France.

Puumala (PUU) virus (Bunyaviridae: Hantavirus), the etiologic agent of nephropathia epidemica (NE), the mid form of hemorrhagic fever with renal syndrome, is enzootic in Europe and has been known to occur in France since 1983. We report the first isolation of PUU virus in France and western Europe from a case of NE acquired in France. The virus was isolated from a serum collected in the acute phase of the clinical course by successive blind passages in Vero E6 cells. Serologic typing using monoclonal antibodies confirmed the identity of the virus as PUU. The sequence of an 832-nucleotide fragment of the virus medium RNA segment obtained by the polymerase chain reaction (PCR) also classified it as a PUU virus. The sequence of this isolate from a human case in France is closely related to the sequence of a PUU virus obtained by the PCR from a German patient.

Occurrence of hantavirus within the rodent population of northeastern California and Nevada.

These studies were initiated to determine the prevalence and hosts of hantaviruses within the rodent population of Nevada and northeastern California. A total of 1,867 rodents were collected, sexed, weighed, identified, and tested by enzyme-linked immunosorbent assay for the presence of antibody against hantavirus nucleocapsid. The primary hosts for hantaviruses in this region were found within the family Muridae (Peromyscus maniculatus. Reithrodontomys megalotis. and Microtus montanus). Studies over time of animals within a defined geographic area indicated that animals with hantavirus antibody are not distributed uniformly over the rodent population in a specific area but were found in foci spanning a distance of only several hundred meters. The antibody prevalence in a given geographic area remained relatively constant with repeated sampling of between 0% and 30%. These data support the hypothesis that rodents within the family Muridae are the primary reservoir for hantaviruses, and the primary risk to biologists for exposure to hantavirus is by contact with members of this family.

Molecular investigation of a multisource outbreak of Crimean-Congo hemorrhagic fever in the United Arab Emirates.

During the investigation of an outbreak of Crimean-Congo hemorrhagic fever (CCHF) in the United Arab Emirates (UAE) between 1994 and 1995, blood samples from suspected CCHF cases and ticks collected from livestock were tested for CCHF virus by antigen-capture ELISA and by a reverse transcription-polymerase chain reaction. Phylogenetic analysis of partial small (S) segment nucleotide sequences from four ticks and five human samples showed that with one exception, all the human and tick viruses clustered along with samples from Pakistan and Madagascar in one distinct lineage. Within this lineage, sequences from the UAE patients were identical or closely related to those from three Hyalomma spp. ticks obtained from livestock recently imported from Somalia. Another sequence from a UAE patient was more closely related to a CCHF virus from Nigeria. These data indicate that the 1994-1995 CCHF epidemic in the UAE was a multisource outbreak possibly associated with importation of CCHF virus-infected livestock and ticks.

Isolation, genetic diversity, and geographic distribution of Bayou virus (Bunyaviridae: hantavirus).

Bayou hantavirus, previously implicated in human hantavirus pulmonary syndrome in Louisiana, was isolated from a rice rat (Oryzomys palustris) captured in Georgia. The presence of antibody among rice rats captured throughout the southeastern United States and the extent of diversity among the genetic variants of Bayou viruses suggest that the rice rat is the most likely natural reservoir of the virus and that both virus and host have probably co-evolved for some years.