Seroepidemiology of selected alphaviruses and flaviviruses in bats in Trinidad.

A serosurvey of antibodies against selected flaviviruses and alphaviruses in 384 bats (representing 10 genera and 14 species) was conducted in the Caribbean island of Trinidad. Sera were analysed using epitope-blocking enzyme-linked immunosorbent assays (ELISAs) specific for antibodies against West Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), all of which are zoonotic viruses of public health significance in the region. Overall, the ELISAs resulted in the detection of VEEV-specific antibodies in 11 (2.9%) of 384 bats. Antibodies to WNV and EEEV were not detected in any sera. Of the 384 sera, 308 were also screened using hemagglutination inhibition assay (HIA) for antibodies to the aforementioned viruses as well as St. Louis encephalitis virus (SLEV; which also causes epidemic disease in humans), Rio Bravo virus (RBV), Tamana bat virus (TABV) and western equine encephalitis virus (WEEV). Using this approach, antibodies to TABV and RBV were detected in 47 (15.3%) and 3 (1.0%) bats, respectively. HIA results also suggest the presence of antibodies to an undetermined flavivirus(es) in 8 (2.6%) bats. Seropositivity for TABV was significantly (P<0.05; χ2) associated with bat species, location and feeding preference, and for VEEV with roost type and location. Differences in prevalence rates between urban and rural locations were statistically significant (P<0.05; χ2) for TABV only. None of the aforementioned factors was significantly associated with RBV seropositivity rates.

Genetic and phenotypic characterization of sylvatic dengue virus type 4 strains.

Four serotypes of dengue virus (DENV 1-4) currently circulate between humans and domestic/peridomestic Aedes mosquitoes, resulting in 100 million infections per year. All four serotypes emerged, independently, from sylvatic progenitors transmitted among non-human primates by arboreal Aedes mosquitoes. This study investigated the genetic and phenotypic changes associated with emergence of human DENV-4 from its sylvatic ancestors. Analysis of complete genomes of 3 sylvatic and 4 human strains revealed high conservation of both the 5'- and 3'-untranslated regions but considerable divergence within the open reading frame. Additionally, the two ecotypes did not differ significantly in replication dynamics in cultured human liver (Huh-7), monkey kidney (Vero) or mosquito (C6/36) cells, although significant inter-strain variation within ecotypes was detected. These findings are in partial agreement with previous studies of DENV-2, where human strains produced a larger number of progeny than sylvatic strains in human liver cells but not in monkey or mosquito cells.

Genome-scale phylogeny of the alphavirus genus suggests a marine origin.

The genus Alphavirus comprises a diverse group of viruses, including some that cause severe disease. Using full-length sequences of all known alphaviruses, we produced a robust and comprehensive phylogeny of the Alphavirus genus, presenting a more complete evolutionary history of these viruses compared to previous studies based on partial sequences. Our phylogeny suggests the origin of the alphaviruses occurred in the southern oceans and spread equally through the Old and New World. Since lice appear to be involved in aquatic alphavirus transmission, it is possible that we are missing a louse-borne branch of the alphaviruses. Complete genome sequencing of all members of the genus also revealed conserved residues forming the structural basis of the E1 and E2 protein dimers.

Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein.

The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic (SH) protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase protein indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses.

Silent circulation of arboviruses in Cameroon.

OBJECTIVES: To investigate the silent circulation and transmission of arthropod-borne viruses (arboviruses) in the Fako Division of Cameroon. DESIGN: This survey was conducted based on clinical observations and laboratory diagnosis; field collections of mosquitoes. SETTING: This study was conducted in the Fako Division of South West Cameroon. SUBJECTS: One hundred and two sera were obtained from febrile patients (with negative laboratory findings for malaria and typhoid fever) at clinics in the Fako Division, and diurnal anthropophilic mosquitoes (4,764) collected. INTERVENTIONS: Virus isolation was attempted from these, and sera were screened for antibodies against 18 African arboviruses by haemagglutination inhibition (HI) and complement fixation (CF) tests. RESULTS: No virus was isolated. Fifty three of 79 (67.1%) sera reacted with one or more viral antigens. Twenty nine sera (36.7%) reacted with members of the genus Alphavirus, with Chikungunya (CHIKV) and O'nyong-nyong (ONNV) viruses as the most frequent (34.2%). Forty six sera (58.2%) reacted with members of the genus Flavivirus: 24 (30.4%) were cross-reactive, but 11.4% reacted monotypically with Zika, 5.1% with yellow fever virus (YFV), 5.1% with dengue virus-2 (DENV-2), 2.5% with DENV-1 and 1.3% with Wesselsbron virus, respectively. The plaque reduction neutralisation test used to specify the agent that elicited the response could not resolve 33.3% of the cross reactions between CHIKV and ONNV. Neutralising antibody titres against ONNV and CHIKV were very high indicating probable re-infection. CONCLUSION: Our results indicate previously undetected circulation of arboviruses in Cameroon, and suggest that they are important, overlooked public health problems.

Development of multiplex PCR-ligase detection reaction assay for detection of West Nile virus.

We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

Risk factors for encephalitis and death from West Nile virus infection.

We conducted a nested case-control study to determine potential risk factors for developing encephalitis from West Nile virus (WNV) infection. Retrospective medical chart reviews were completed for 172 confirmed WNV cases hospitalized in Houston between 2002 and 2004. Of these cases, 113 had encephalitis, including 17 deaths, 47 had meningitis, and 12 were fever cases; 67% were male. Homeless patients were more likely to be hospitalized from WNV compared to the general population. A multiple logistic regression model identified age [odds ratio (OR) 1.1, P<0.001], history of hypertension, including those cases taking hypertension-inducing drugs (OR 2.9, P=0.012), and history of cardiovascular disease (OR 3.5, P=0.061) as independent risk factors for developing encephalitis from WNV infection. After adjusting for age, race/ethnicity (being black) (OR 12.0, P<0.001), chronic renal disease (OR 10.6, P<0.001), hepatitis C virus (OR 23.1, P=0.0013), and immunosuppression (OR 3.9, P=0.033) were identified as risk factors for death from WNV infection.

Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

West Nile virus infection in the golden hamster (Mesocricetus auratus): a model for West Nile encephalitis.

This report describes a new hamster model for West Nile (WN) virus encephalitis. Following intraperitoneal inoculation of a New York isolate of WN virus, hamsters had moderate viremia of 5 to 6 days in duration, followed by the development of humoral antibodies. Encephalitic symptoms began 6 days after infection; about half the animals died between the seventh and 14th days. The appearance of viral antigen in the brain and neuronal degeneration also began on the sixth day. WN virus was cultured from the brains of convalescent hamsters up to 53 days after initial infection, suggesting that persistent virus infection occurs. Hamsters offer an inexpensive model for studying the pathogenesis and treatment of WN virus encephalitis.

Pirital virus (Arenaviridae) infection in the syrian golden hamster, Mesocricetus auratus: a new animal model for arenaviral hemorrhagic fever.

Adult Syrian golden hamsters inoculated intraperitoneally with Pirital virus, a recently discovered member of the Tacaribe complex of New World arenaviruses, developed a progressively severe, fatal illness with many of the pathologic features observed in fatal human cases of Lassa fever and other arenaviral hemorrhagic fevers. Most of the animals became moribund by Day 5 and were dead by Day 7 after inoculation. The most consistent histopathologic changes included interstitial pneumonitis, splenic lymphoid depletion and necrosis, and multifocal hepatic necrosis without significant inflammatory cell infiltration. The liver changes ranged from single cell death by apoptosis to coagulative necrosis of clusters of hepatocytes. Immunohistochemical studies of the liver demonstrated the presence and accumulation ot Pirital virus antigen within hepatocytes as well as Kupffer cells. An in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay showed progressively increasing apoptotic activity in the liver of infected hamsters. A human hepatoblastoma cell line (Hep G2/C3A) inoculated with Pirital virus also developed progressive cell destruction and accumulation of viral antigen, as demonstrated by immunofluorescence. Results of this pilot study suggest that the Pirital virus-hamster model is a very promising new small animal model for studying the pathogenesis of arenavirus infections, particularly, the mechanism of direct virus-induced hepatic injury. It may also be useful for testingantiviral agents for treatment of arenaviral hemorrhagic fevers.

Jatobal virus is a reassortant containing the small RNA of Oropouche virus.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucuruí, Pará state, Brazil and was classified as a distinct member of the Simbu serogroup of the Bunyavirus genus, family Bunyaviridae on the basis of neutralization tests. On the basis of nucleotide sequencing, we have found that the small (S) RNA of JAT virus is very similar (>95% identity) to that of Oropouche (ORO) virus, in particular, the Peruvian genotype of ORO virus. In comparison, limited nucleotide sequencing of the G2 protein gene, encoded by the middle (M) RNA, of JAT and ORO viruses, revealed relatively little identity (<66%) between these two viruses. Neutralization tests confirmed the lack of cross-reactivity between the viruses. These results suggest that JAT virus is a reassortant containing the S RNA of ORO virus. JAT virus was attenuated in hamsters compared to ORO virus suggesting that the S RNA of ORO virus is not directly involved in hamster virulence.

Geographical distribution of rodent-associated hantaviruses in Texas.

The purpose of this study was to increase our knowledge of the geographic distribution and natural host range of hantaviruses in Texas, southeastern New Mexico, and Mexico. Blood samples from 3,225 wild rodents, representing 34 species, were tested for hantavirus antibody (IgG), using an enzyme-linked immunosorbent assay. Hantavirus antibody was found in one or more rodents from each of 13 counties in Texas, Otero County in southeastern New Mexico, and Mexico State (central Mexico). The 133 antibody-positive rodents included seven Peromyscus species (P. attwateri, P. boylii, P. hylocetes, P. leucopus, P. maniculatis, P. melanotis, and P. pectoralis), Sigmodon hispidus, Oryzomys palustris, two Reithrodontomys species (R. fulvescens and R. megalotis), Neotoma albigula, and Perognathus merriami. This study provides further evidence that rodent-associated hantaviruses are geographically widely distributed in Texas. The discovery of antibody in P. hylocetes and P. melanotis is evidence that peromyscine rodents in Mexico are naturally associated with viruses belonging to the genus Hantavirus.

Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin.

This report describes Trocara virus, a newly recognized member of the genus Alphavirus, that has been isolated from Aedes serratus mosquitoes collected at two widely separated sites in the Amazon Basin. Biological, antigenic and genetic characteristics of the new virus are given. Results of these studies indicate that Trocara virus is the first member of a newly discovered antigenic complex within the family Togaviridae genus Alphavirus. The public health and veterinary importance of Trocara virus is still unknown.

Diagnosis of Oropouche virus infection using a recombinant nucleocapsid protein-based enzyme immunoassay.

Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.

Allpahuayo virus: a newly recognized arenavirus (arenaviridae) from arboreal rice rats (oecomys bicolor and oecomys paricola) in northeastern peru.

Allpahuayo virus was initially isolated from arboreal rice rats (Oecomys bicolor and Oecomys paricola) collected during 1997 at the Allpahuayo Biological Station in northeastern Peru. Serological and genetic studies identified the virus as a new member of the Tacaribe complex of the genus Arenavirus. The small (S) segment of the Allpahuayo virus prototype strain CLHP-2098 (Accession No. AY012686) was sequenced, as well as that of sympatric isolate CLHP-2472 (Accession No. AY012687), from the same rodent species. The S segment was 3382 bases in length and phylogenetic analysis indicated that Allpahuayo is a sister virus to Pichinde in clade A. Two ambisense, nonoverlapping reading frames were identified, which result in two predicted gene products, a glycoprotein precursor (GPC) and a nucleocapsid protein (NP). A predicted stable single hairpin secondary structure was identified in the intergenic region between GPC and NP. Details of the genetic organization of Allpahuayo virus are discussed.

Geographic distribution and genetic diversity of Whitewater Arroyo virus in the southwestern United States.

The purpose of this study was to extend our knowledge of the geographic distribution and genetic diversity of the arenavirus(es) associated with Neotoma species (woodrats) in the southwestern United States. Infectious arenavirus was recovered from 14 (3.3%) of 425 woodrats. The virus-positive species included N. albigula in New Mexico and Oklahoma, N. cinerea in Utah, N. mexicana in New Mexico and Utah, and N. micropus in Texas. Analyses of viral nucleocapsid protein gene sequence data indicated that all the isolates were strains of the Whitewater Arroyo virus, an arenavirus previously known only from northwestern New Mexico. Analyses of the sequence data also indicated that there can be substantial genetic diversity among strains of Whitewater Arroyo virus from conspecific woodrats collected from different localities and substantial genetic diversity among strains from different woodrat species collected from the same locality.

Extreme genetic diversity among Pirital virus (Arenaviridae) isolates from western Venezuela.

Pirital-like virus isolates from rodents collected in a variety of habitats within a six-state area of central Venezuela were analyzed genetically by amplifying a portion of the nucleocapsid protein gene using RT-PCR. Comparisons of the sequences from 30 selected Pirital-like virus isolates demonstrated up to 26% divergence in nucleotide sequences and up to 16% divergence in deduced amino acid sequences. Within the Pirital monophyletic group, 14 distinct lineages or genotypes, differing by at least 6% in nucleotide sequences, were identified. Although sample sizes were small for some lineages, many of the different genotypes were sampled in only one region or locality, suggesting allopatric divergence. Complement fixation tests with representatives of the most divergent Pirital virus lineages failed to delineate multiple species or subtypes within the Pirital clade. These results indicate that the previously proposed 12% nucleocapsid protein amino acid sequence divergence cutoff value for delineating arenavirus species is not appropriate for the entire family. When individual clones were examined from PCR amplicons, a mean of 0.17% sequence diversity vs the consensus sequences was detected, suggesting diverse quasispecies populations within infected rodent hosts. Possible explanations for the extreme genetic diversity within and among Pirital virus populations in infected rodents are discussed.

Experimental yellow fever virus infection in the Golden Hamster (Mesocricetus auratus). I. Virologic, biochemical, and immunologic studies.

This report describes the clinical laboratory findings in golden hamsters experimentally infected with yellow fever (YF) virus. An accompanying paper describes the pathologic findings. Following intraperitoneal inoculation of a virulent strain of YF virus, hamsters developed a high-titered viremia (up to 109/mL) lasting 5--6 days and abnormal liver function tests. YF hemagglutination-inhibiting antibodies appeared 4 or 5 days after infection, often while viremia was still present. The mortality rate in YF-infected hamsters was variable, depending on the virus strain and the age of the animals. Clinical and pathologic changes in the infected hamsters were very similar to those described in experimentally infected macaques and in fatal human cases of YF, which indicates that the golden hamster may be an excellent alternative animal model, in place of nonhuman primates, for research on the pathogenesis and treatment of YF and other viscerotropic flavivirus diseases.

Experimental yellow fever virus infection in the Golden hamster (Mesocricetus auratus). II. Pathology.

Subadult and adult hamsters were inoculated intraperitoneally with 10(6) TCID(50) of yellow fever (YF) virus (Jimenez strain). Four animals from each group were subjected daily to histologic examination for 9 days. The liver showed spotty necrosis on day 3 after infection, which was followed by steatosis and focally confluent necrosis. In surviving hamsters, hepatocyte regeneration began on day 8, which was accompanied by decreasing steatosis. The spleen initially exhibited lymphoid hyperplasia, which was followed by lymphoid depletion and increased phagocytosis by splenic macrophages. Focal pancreatic acinar necrosis and spotty adrenal cortical necrosis were seen transiently between days 5 and 7. Viral antigen was detected immunohistochemically in the liver and the spleen. TUNEL analysis showed a dynamic change of hepatocyte necrapoptosis, with activity corresponding to the severity of disease. The histopathologic changes were more severe in younger (subadult) animals. The YF-hamster model appears to be an accurate and inexpensive experimental system for studying the pathophysiology and treatment of YF.