Negevirus: a proposed new taxon of insect-specific viruses with wide geographic distribution.

Six novel insect-specific viruses, isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, the United States, Ivory Coast, Israel, and Indonesia, are described. Their genomes consist of single-stranded, positive-sense RNAs with poly(A) tails. By electron microscopy, the virions appear as spherical particles with diameters of ∼45 to 55 nm. Based on their genome organization and phylogenetic relationship, the six viruses, designated Negev, Ngewotan, Piura, Loreto, Dezidougou, and Santana, appear to form a new taxon, tentatively designated Negevirus. Their closest but still distant relatives are citrus leposis virus C (CiLV-C) and viruses in the genus Cilevirus, which are mite-transmitted plant viruses. The negeviruses replicate rapidly and to high titer (up to 10(10) PFU/ml) in mosquito cells, producing extensive cytopathic effect and plaques, but they do not appear to replicate in mammalian cells or mice. A discussion follows on their possible biological significance and effect on mosquito vector competence for arboviruses.

Quaranfil, Johnston Atoll, and Lake Chad viruses are novel members of the family Orthomyxoviridae.

Arboviral infections are an important cause of emerging infections due to the movements of humans, animals, and hematophagous arthropods. Quaranfil virus (QRFV) is an unclassified arbovirus originally isolated from children with mild febrile illness in Quaranfil, Egypt, in 1953. It has subsequently been isolated in multiple geographic areas from ticks and birds. We used high-throughput sequencing to classify QRFV as a novel orthomyxovirus. The genome of this virus is comprised of multiple RNA segments; five were completely sequenced. Proteins with limited amino acid similarity to conserved domains in polymerase (PA, PB1, and PB2) and hemagglutinin (HA) genes from known orthomyxoviruses were predicted to be present in four of the segments. The fifth sequenced segment shared no detectable similarity to any protein and is of uncertain function. The end-terminal sequences of QRFV are conserved between segments and are different from those of the known orthomyxovirus genera. QRFV is known to cross-react serologically with two other unclassified viruses, Johnston Atoll virus (JAV) and Lake Chad virus (LKCV). The complete open reading frames of PB1 and HA were sequenced for JAV, while a fragment of PB1 of LKCV was identified by mass sequencing. QRFV and JAV PB1 and HA shared 80% and 70% amino acid identity to each other, respectively; the LKCV PB1 fragment shared 83% amino acid identity with the corresponding region of QRFV PB1. Based on phylogenetic analyses, virion ultrastructural features, and the unique end-terminal sequences identified, we propose that QRFV, JAV, and LKCV comprise a novel genus of the family Orthomyxoviridae.

Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales.

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.

Antigenic relationships between sylvatic and endemic dengue viruses.

Sylvatic dengue viruses (DENVs) are transmitted between non-human primates and arboreal Aedes spp. mosquitoes in Southeast Asia and west Africa. Recent evidence suggests that the risk for re-emergence of sylvatic DENV into the urban endemic/epidemic cycle may be high, which could limit the potential for eradicating the human transmission cycle with vaccines now under development. We assessed the likelihood of sylvatic DENV re-emergence in the face of immunity to current endemic strains or vaccines by evaluating the neutralization capacity of sera from DENV vaccinees and convalescent patients after primary infection with DENV-2 and DENV-3 serotypes. Our data indicate robust homotypic cross-immunity between human sera and sylvatic DENV strains, but limited heterotypic neutralization. Should a licensed vaccine lead to the eradication of the urban transmission cycle in the future, re-emergence of sylvatic strains into the urban cycle would be limited by homotypic immunity mediated by virus-neutralizing antibodies.

An animal model for studying the pathogenesis of chikungunya virus infection.

Newborn and 14-day-old mice inoculated subcutaneously with chikungunya virus (CHIKV) developed lethargy, difficulty walking, dragging of hind limbs, and reduced weight gain within 7-10 days after infection (PI). During the initial 6-7 days PI, the animals had viremia; high levels (10(6)-10(8) PFU) of CHIKV were also present in leg muscle. The virus persisted in muscle for several days after viremia disappeared. The major histopathologic changes were in skeletal muscle, which were focal necrosis and inflammation, followed by fibrosis and dystrophic calcification. Some mice also showed dystrophic calcification in the joint cartilage, but there were few deaths, and most of the animals eventually recovered. CHIKV antigen was shown by immunohistochemistry in the muscle for several weeks after infection. Based on the clinical and pathologic similarities with CHIKV infection in humans, young ICR and CD-1 mice offer a useful and realistic model for further study of the pathogenesis and treatment of CHIKV infection.

Venezuelan equine encephalitis virus infection of cotton rats.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogen of equids and humans, but infection of its rodent reservoir hosts has received little study. To determine whether responses to infection vary among geographic populations, we inoculated 3 populations of cotton rats with 2 enzootic VEEV strains (Co97-0054 [enzootic ID subtype] and 68U201 [enzootic IE subtype]). The 3 populations were offspring from wild-caught cotton rats collected in a VEE-enzootic area of south Florida, USA; wild-caught cotton rats from a non-VEE-enzootic area of Texas, USA; and commercially available (Harlan) colony-reared cotton rats from a non-VEE-enzootic region. Although each population had similar early viremia titers, no detectable disease developed in the VEE-sympatric Florida animals, but severe disease and death affected the Texas and Harlan animals. Our findings suggest that the geographic origins of cotton rats are important determinants of the outcome of VEE infection and reservoir potential of these rodents.

Antigenic and genetic relationships among Rift Valley fever virus and other selected members of the genus Phlebovirus (Bunyaviridae).

Preliminary serologic data indicated that two South American phleboviruses (Belterra virus [BELTV] and Icoaraci virus [ICOV]) may be related to Rift Valley fever virus (RVFV), an African phlebovirus that causes severe hepatitis and hemorrhagic fever in humans. To further define this relationship and to investigate the underlying genetic basis, comparative serologic and genetic sequence analyses were performed with RVFV and five other New World phleboviruses (ICOV, BELTV, Salobo virus, Joa virus, and Frijoles virus). Serologically, a one-way cross reaction was confirmed between antibodies against these New World viruses and RVFV antigen. In contrast, phylogenetic analysis demonstrated clear separation of these viruses from RVFV, into distinct phylogenies, based on sequences of the small, medium, and large RNA segments.

Efficacy of the antipoxvirus compound ST-246 for treatment of severe orthopoxvirus infection.

Efficacy of the new antipoxvirus compound ST-246 was evaluated as treatment of monkeypox (MPX) virus infection in a ground squirrel model of the disease. Ground squirrels were given a lethal dose of MPX virus and were then treated orally at various times post-inoculation (pi) with 100 mg/kg/day of ST-246. Morbidity and mortality, clinical laboratory results, viral load, and pathology of placebo and treatment groups were compared. All animals that started treatment with ST-246 on days 0, 1, 2, and 3 pi survived lethal challenge with MPX virus; 67% of animals treated on day 4 pi also survived. In contrast, 100% of the placebo group died. Most of the ST-246-treated animals showed no evidence of clinical disease or alteration of baseline clinical laboratory values and had minimal histopathologic changes. These results suggest that ST-246 is a promising candidate for early treatment of severe orthopoxvirus infection.

Dhori virus (Orthomyxoviridae: Thogotovirus) infection in mice: a model of the pathogenesis of severe orthomyxovirus infection.

After intranasal, subcutaneous, or intraperitoneal infection with Dhori virus (DHOV), adult mice developed a fulminant and uniformly fatal illness with many of the clinical and pathologic findings seen in mice infected with H5N1 highly pathogenic avian influenza A virus. Histopathologic findings in lungs of DHOV-infected mice consisted of hemorrhage, inflammation, and thickening of the interstitium and the alveolar septa and alveolar edema. Extra-pulmonary findings included hepatocellular necrosis and steatosis, widespread severe fibrinoid necrosis in lymphoid organs, marked lymphocyte loss and karyorrhexis, and neuronal degeneration in brain. Similar systemic histopathologic findings have been reported in the few fatal human H5N1 cases examined at autopsy. Because of the relationship of DHOV to the influenza viruses, its biosafety level 2 status, and its similar pathology in mice, the DHOV-mouse model may offer a low-cost, relatively safe, and realistic animal model for studies on the pathogenesis and management of H5N1 virus infection.

Chronic St. Louis encephalitis virus infection in the golden hamster (Mesocricetus auratus).

To further study the phenomenon of flavivirus persistent infection, golden hamsters (Mesocricetus auratus) were inoculated intraperitoneally with a low pathogenicity strain of St. Louis encephalitis virus (SLEV). After inoculation, the animals remained asymptomatic and developed high levels of specific neutralizing antibodies in their sera. However, about one half of the hamsters continued to shed infectious SLEV in their urine for prolonged periods of time. By co-cultivation, SLEV was recovered from selected tissues (kidney, lung, and brain) of some of the animals for up to 185 days after initial infection. Although no specific histopathologic changes were observed in these tissues, SLEV antigen was shown by immunohistochemistry in the interstitium and tubular epithelium of the renal cortex and in a few large neurons of the cerebral cortex. Seventeen SLEV isolates from urine and tissues of the chronically infected hamsters were sequenced. In comparison with the infecting parent SLEV strain, two common mutations and amino acid substitutions were observed in all of the hamster isolates. The findings of this study were very similar to previous descriptions of chronic West Nile, Modoc, and tick-borne encephalitis virus infections in mammals, and they re-emphasize the potential importance of persistent flavivirus infection in vertebrates.

West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection.

Primary cultures of embryonic murine neurons and newborn mouse astrocytes were inoculated with West Nile virus (WNV) strain NY385-99 to compare the pathogenesis of WNV infection in these types of CNS cells. Two different outcomes were observed. WNV infection in the neurons was rapidly progressive and destructive; within 5 days, all of the neurons were destroyed through apoptosis. WNV infection in the astrocytes evolved more slowly and did not seem to be highly lethal to the cells. The infected astrocytes continued to produce infectious virus (10(4.6)-10(6.5) PFU/mL) for 114 days, in a permissive, persistent infection. During this period, WNV antigen could be shown in the cytoplasm of the infected astrocytes by immunocytochemical assay, transmission electron microscopy of ultrathin sections, and in the cell culture medium by complement fixation test. Our results with this in vitro experimental murine cell model indicate that astrocytes can develop chronic or persistent infection with WNV, suggesting that these cells may play a role in the maintenance of WNV in the CNS.

Effects of immunosuppression on West Nile virus infection in hamsters.

A research study, comparing the pathogenesis of experimental West Nile virus (WNV) infection in immunocompetent and immunosuppressed golden hamsters, is described. Cyclophosphamide was used to immunosuppress the animals. The immunosuppressed hamsters had a prolonged period of viremia, depressed humoral immune response, more extensive and severe pathology, and higher fatality rate than the untreated immunocompetent animals. Histopathological and immunohistochemical studies of tissues from the two groups showed that pathologic changes in the untreated infected animals were confined to the brain and spinal cord, whereas the histopathological changes and WNV antigen distribution in the immunosuppressed animals were much more extensive and diffuse, involving the adrenal, kidney, heart and lung, and brain and spinal cord. Results of this study in the hamster model provide insight into the increased severity of WNV infection observed in immunosuppressed people.

Araguari virus, a new member of the family Orthomyxoviridae: serologic, ultrastructural, and molecular characterization.

This paper reports the results of serologic, structural, biochemical, and genetic studies indicating that Araguari virus, a previously unassigned viral agent, is a member of the family Orthomyxoviridae and genus Thogotovirus. Araguari virus has six RNA fragments; biologically, it shares several properties with other viruses in the family Orthomyxoviridae. Nucleotide sequencing of the RNA segments 4 (glycoprotein) and 5 (nucleoprotein) of Araguari virus aligned with the orthomyxoviruses, showing the closest relationship with Thogoto virus (sequence similarity = 61.9% and 69.1%, respectively, for glycoprotein and nucleoprotein), but also sharing a more distant similarity with Dhori and Influenza C viruses, especially for the glycoprotein gene. Based on these results, we propose that Araguari virus should be assigned as a new member of the family Orthomyxoviridae and genus Thogotovirus.

Oropouche virus isolation, southeast Brazil.

An Oropouche virus strain was isolated from a novel host (Callithrix sp.) in Arinos, Minas Gerais State, southeastern Brazil. The virus was identified by complement fixation test and confirmed by reverse transcription-polymerase chain reaction. Phylogenetic analysis identified this strain as a genotype III isolate previously recognized only in Panama.

Short report: serological evidence of West Nile virus activity in El Salvador.

Epizootics of encephalitis in El Salvador killed 203 equines between November 2001 and April 2003. During an investigation of the outbreaks, 18 (25%) of 73 serum samples collected from stablemates of deceased animals in 2003 had antibodies to West Nile virus. Ten of these infections were confirmed by plaque reduction neutralization tests, suggesting West Nile virus has extended its range and spread to Central America.

Experimental infection of prairie dogs with monkeypox virus.

Studies of experimental infection of prairie dogs (Cynomys ludovicianus) with monkeypox virus are described. After intraperitoneal infection, all of the animals died within 11 days. Virus was cultured from their blood and oropharynx several days before death; at necropsy, most of the organs tested contained monkeypox virus. Marked hepatic and splenic necrosis were observed, along with mild inflammatory changes in the lungs. After intranasal (IN) infection, the primary pathologic changes were in the lungs and pleural cavity. Some of the IN infected animals (40%) survived, and monkeypox virus could be cultured from their nasal discharge and oropharynx for <22 days. Ulcerative lesions also developed on the lips, tongue, and buccal mucosa of the surviving animals. Our findings support an earlier report, which suggested that infected prairie dogs can transmit monkeypox virus by respiratory and mucocutaneous contact with susceptible animals and persons.

Persistent shedding of West Nile virus in urine of experimentally infected hamsters.

Adult hamsters that survived experimental West Nile virus (WNV) infection developed persistent viruria. Infectious WNV could be cultured from their urine for up to 52 days. Immunohistochemical examination of kidneys of viruric animals showed foci of WNV antigen in renal tubular epithelial and vascular endothelial cells. These findings are compatible with virus replication and persistent infection of renal epithelial cells. The potential clinical and virologic significance of these findings as well as their possible epidemiologic importance are discussed.

Oral transmission of West Nile virus in a hamster model.

The results of experiments comparing the pathogenesis of West Nile virus (WNV) following infection by mosquito bite, needle inoculation, and ingestion are reported. Adult hamsters were readily infected by all three routes. The level and duration of viremia, clinical manifestations, pathology, and antibody response in the hamsters following mosquito infection and needle inoculation were similar; after oral infection, the onset of viremia was delayed and the mortality was lower, but the level and duration of viremia, histopathology, and antibody response were similar to the other routes. The results from this and previously published studies indicate that a wide variety of animal species are susceptible to oral infection with WNV and that orally infected animals develop a viremia and illness similar to that following the bite of infected mosquitoes. Oral infection appears to be an alternative transmission mechanism used by a number of different flaviviruses; its potential role in the natural history of WNV is discussed.

Alteration of clinical outcome and histopathology of yellow fever virus infection in a hamster model by previous infection with heterologous flaviviruses.

Using a recently described hamster model of severe yellow fever (YF), we examined the hypothesis that prior infection with heterologous flaviviruses protects against severe or fatal YF. Hamsters were singly or sequentially infected with Japanese encephalitis, St. Louis encephalitis, West Nile, and/or dengue-1 viruses, and then challenged with a virulent strain of yellow fever virus (YFV). In contrast to control (naive) hamsters, many of which appeared clinically ill or died after YFV infection, the flavivirus-immune animals remained asymptomatic. The flavivirus-immune hamsters also had a reduced viremia and lower serum levels of alanine aminotransferase and total bilirubin, compared with naive hamsters, following YFV infection. Histologically, livers of animals in the flavivirus-immune and control groups showed comparable levels of multifocal necrapoptosis. However, steatosis was not observed in the flavivirus-immune animals, whereas naive hamsters developed extensive microvesicular steatosis in the liver following YFV infection. These findings suggest that hepatocytic steatosis is an adverse microscopic feature associated with severe disease in YFV infection. Our experimental results support earlier anecdotal reports that prior exposure of humans to heterologous flaviviruses reduces subsequent risk of fatal YFV infection.

West Nile virus infection in nonhuman primate breeding colony, concurrent with human epidemic, southern Louisiana.

During the summer of 2002, an epidemic of West Nile meningoencephalitis occurred in southern Louisiana. Following the outbreak, blood samples were collected from 1,692 captive rhesus monkeys (Macaca mulatta), pigtail macaques (M. nemestrina), and baboons (Papio spp.) that were permanently housed outdoors at a nonhuman primate breeding facility in St. Tammany Parish, Louisiana. The serum samples were examined for antibodies to West Nile virus (WNV). Overall, 36% of the captive nonhuman primates had WNV antibodies; comparison of these samples with banked serum samples from previous blood collections indicated that the animals were infected subclinically from February to August 2002. WNV activity was demonstrated in surveillance at the nonhuman primate-breeding colony and in the neighboring community during this same period. The high infection rate in this captive nonhuman primate population illustrates the intensity of WNV transmission that can occur silently in nature among other susceptible vertebrates during epidemic periods.