Nocodazole delays viral entry into the brain following footpad inoculation with West Nile virus in mice.

West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log(10) greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), did not occur until day 7. At these later time points (7 and 9 days PI), nocodazole-treated WNV-infected animals attained viral titers in these tissues similar to titers in the untreated WNV-infected control animals. These results demonstrate that a single dose of nocodazole delays, but does not block, WNV infection of the brain.

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Nipah virus: a recently emergent deadly paramyxovirus.

A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.

The emergence of West Nile virus in North America: ecology, epidemiology, and surveillance.

In late summer 1999, the first domestically acquired human cases of WN encephalitis were documented in the USA. Aggressive vector-control and public education efforts by state and local public health officials limited the extent of human involvement. The discovery of virus-infected, overwintering mosquitoes during the winter of 1999-2000, predicted renewed virus activity for the following spring, and prompted early season vector-control activities and disease surveillance efforts in NYC and the surrounding areas. These surveillance efforts were focused on identifying WN virus infections in birds and mosquitoes as predictors of the potential risk of transmission to humans. By the end of the 2000 mosquito-borne disease transmission season, WN virus activity had been documented as far north as the states of Vermont and New Hampshire, and as far south as the state of North Carolina. The ongoing impacts that WN virus will have on wildlife, domestic animal and human populations of the western hemisphere are not yet known. Plans are in place for public health officials and scientists to monitor the further expansion of WN virus with the establishment or enhancement of vector-borne disease surveillance and control programs throughout the eastern seaboard. The valuable lessons learned from the detection and response to the introduction of WN virus into NYC should prove useful if and when subsequent intrusions of new disease agents occur.

West Nile virus in the United States: guidelines for detection, prevention, and control.

The epidemic/epizootic of West Nile (WN) encephalitis in the northeastern United States in the summer and fall of 1999 was an unprecedented event, underscoring the ease with which emerging infectious pathogens can be introduced into new geographic areas in today's era of rapid transportation and increased movement of people, animals, and commodities. This epidemic/epizootic and the increased frequency of other exotic pathogens being imported into the United States raises the issue of whether local, state, and national public health agencies are prepared to deal with epidemics/epizootics of vector-borne infectious diseases. The overwintering of WN virus and the epizootic transmission in the summer of 2000 reinforces the need to rebuild the public health infrastructure to deal with vector-borne diseases in this country. This article summarizes guidelines for surveillance, prevention, and control of WN virus that were drafted in December 1999 to help prepare state and local health departments for monitoring WN virus activity in the spring and summer of 2000 and also summarizes the data collected from those surveillance systems through September 2000.

Antibody prophylaxis and therapy for flavivirus encephalitis infections.

The outbreak of West Nile (WN) encephalitis in the United States has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. Although equine WN vaccines are currently being developed, a WN vaccine for humans is years away. There is also no specific therapeutic agent for flaviviral infections. The incidence of human WN virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the vaccine's economic feasibility. It has been shown, however, that prophylactic application of antiflaviviral antibody can protect mice from subsequent virus challenge. This model of antibody prophylaxis using murine monoclonal antibodies (MAbs) has been used to determine the timing of antibody application and specificity of applied antibody necessary for successful prophylaxis. The major flaviviral antigen is the envelope (E) glycoprotein that binds cellular receptors, mediates cell membrane fusion, and contains an array of epitopes that elicit virus-neutralizing and nonneutralizing antibodies. The protective efficacy of an E-glycoprotein-specific MAb is directly related to its ability to neutralize virus infectivity. The window for successful application of prophylactic antibody to prevent flaviviral encephalitis closes at about 4 to 6 days postinfection concomitant with viral invasion of the brain. Using murine MAbs to modify human disease results in a human antimouse antibody (HAMA) response that eventually limits the effectiveness of subsequent murine antibody applications. To reduce the HAMA response and make these MAbs more generally useful for humans, murine MAbs can be "humanized" or human MAbs with analogous reactivities can be developed. Antiflaviviral human or humanized MAbs might be practical and cost-effective reagents for preventing or modifying flaviviral diseases.

Monoclonal antibodies define antigenic variation in the ID variety of Venezuelan equine encephalitis virus.

Three monoclonal antibodies were generated that are specific for the E2 glycoprotein of Venezuelan equine encephalitis (VEE) virus and have useful reactivities in an enzyme-linked immunosorbent assay (ELISA). Antibody 1A1B-9 distinguished between the IC (epizootic) and ID (enzootic) varieties of VEE virus by ELISA. Clone 7A1A-1 antibody distinguished the Panamanian prototype virus (3880) from Colombian ID isolates by a 500-fold difference in titer by endpoint ELISA, and it detected antigenic variation in ID isolates from southern Colombia and Ecuador. Antibody 7A3A-4 defined a cryptic antigenic site on the latter two isolates. These monoclonal antibodies complement others in identifying VEE isolates by a simple ELISA.

Genetic variation of Venezuelan equine encephalitis virus strains of the ID variety in Colombia.

To determine the degree of genetic variation within one serologic group of Venezuelan equine encephalitis virus and the relatedness of viruses with different epidemiologic backgrounds isolated within the same country, virion RNA from 16 isolates belonging to subtype I were compared by RNase T1 oligonucleotide fingerprinting. RNA fingerprints of 12 enzootic isolates showed a large degree of heterogeneity, even though they were serologically indistinguishable. A reference enzootic strain from Colombia showed more genetic relatedness to three epizootic strains isolated in the same country, than to its own serogroup prototype strain isolated in Panama. Thus, genetic relatedness within Venezuelan equine encephalitis strains in Colombia seems to be a function of geography rather than epidemiology.

Identification of Highlands J virus from a Florida horse.

A virus, strain 64A-1519, isolated from the brain of a horse dying of encephalitis in Florida in 1964, was identified as western equine encephalomyelitis (WEE) virus. Recently, we used polyclonal and monoclonal immune reagents to identify this isolate by comparing it to 2 strains of WEE virus and to Highlands J (HJ) virus in hemagglutination-inhibition, immunofluorescent antibody, and plaque-reduction neutralization tests. These tests demonstrate that strain 64A-1519 is a strain of HJ virus distinct from WEE virus.

Detection of eastern equine encephalitis virus in infected mosquitoes using a monoclonal antibody-based antigen-capture enzyme-linked immunosorbent assay.

Surveillance of mosquito populations for virus activity is not often performed by small, vector-control districts because they do not have the financial resources to use virus isolation, or newer methods such as the polymerase chain reaction. Consequently, development and refinements of rapid, sensitive, and simple enzyme-linked immunosorbent assays (ELISAs) applicable to a wide variety of public health settings are justified. We have developed an antigen-capture ELISA for the detection of eastern equine encephalitis (EEE) virus in mosquitoes that uses both monoclonal capture and detector antibodies. The sensitivity of this assay is 4.0-5.0 log10 plaque-forming units/ml, which is comparable to previously published EEE antigen-capture assays developed with polyclonal antibody reagents. This test identifies only North American strains of EEE virus and does not react with either western equine encephalitis or Highlands J viruses. Test sensitivity was enhanced by sonicating mosquito pools, treating them with Triton X-100, and increasing the time and temperature of antigen incubation. The conversion of this ELISA to a monoclonal antibody-based format should result in a readily standardizable and transferable assay that will permit laboratories lacking virus isolation facilities to conduct EEE virus surveillance.

Nucleotide sequences of the 26S mRNAs of the viruses defining the Venezuelan equine encephalitis antigenic complex.

Genetic relationships among viruses defining the Venezuelan equine encephalitis (VEE) virus antigenic complex were determined by analyzing the 3'-terminal 561 nucleotides of the nonstructural protein 4 gene and the entire 26S RNA region of the genome. New sequence information is reported for VEE 78V-3531 (VEE subtype-variety IF), Mucambo (IIIA), Tonate (IIIB), 71D-1252 (IIIC), Pixuna (IV), Cabassou (V), and AG80-663 (VI) viruses. The results reported here and by previous investigators largely support the current classification scheme of these viruses, while clearly identifying Everglades (II) as a subtype I virus. A genetic relationship between 78V-3531 (IF) and AG80-663 (VI) viruses contradicted previous serologic results. Mutations near the amino terminus of the E2 envelope proteins of Pixuna and AG80-663 viruses probably account for the previously reported low reactivity of the protective monoclonal antibody 1A2B-10 with these two viruses. Variations in the distribution of potential glycosylation sites in the E2 glycoprotein are discussed.

Identification of monoclonal antibodies capable of differentiating antigenic varieties of eastern equine encephalitis viruses.

We have isolated and characterized 3 monoclonal antibody (Mab) reagents useful in the serological identification of varieties of eastern equine encephalitis (EEE) viruses. These antibodies were specific for the E1 glycoprotein of their homologous viruses. One Mab, 1B5C-3, reacted specifically with all North American (NA) EEE viruses isolated over a 50 year period. This antigenic stability of NA isolates was genetically confirmed by oligonucleotide fingerprinting. Evolutionary stability is a unique feature among alphaviruses. The Mab, 1C1J-4 reacted specifically with 1 South American isolate of EEE virus. A third Mab, 1B1C-4, was EEE virus complex reactive. While none of these antibodies had virus neutralizing activity, the identified reactivities could be demonstrated in the more rapid serological tests of enzyme-linked immunosorbent assay and indirect immunofluorescence.

Venezuelan equine encephalitis febrile cases among humans in the Peruvian Amazon River region.

A survey was conducted from October 1, 1993 to June 30, 1995 to determine the arboviral etiologies of febrile illnesses in the city of Iquitos in the Amazon River Basin of Peru. The study subjects were patients who were enrolled at medical care clinics or in their homes by Peruvian Ministry of Health (MOH) workers as part of the passive and active disease surveillance program of the MOH. The clinical criterion for enrollment was the diagnosis of a suspected viral-associated, acute, undifferentiated febrile illness of < or = 5 days duration. A total of 598 patients were enrolled in the study. Demographic information, medical history, clinical data, and blood samples were obtained from each patient. The more common clinical features were fever, headache, myalgia, arthralgia, retro-ocular pain, and chills. Sera were tested for virus by the newborn mouse and cell culture assays. Viral isolates were identified initially by immunofluorescence using polyclonal antibody. An ELISA using viral-specific monoclonal antibodies and nucleotide sequence analysis were used to determine the specific variety of the viruses. In addition, thin and thick blood smears were observed for malaria parasites. Venezuelan equine encephalitis (VEE) virus subtype I, variety ID virus was isolated from 10 cases, including three cases in October, November, and December 1993, five cases in January and February 1994, and two cases in June 1995. The ELISA for IgM and IgG antibody indicated that VEE virus was the cause of an additional four confirmed and four presumptive cases, including five from January through March 1994 and three in August 1994. Sixteen cases were positive for malaria. The 18 cases of VEE occurred among military recruits (n = 7), agriculture workers (n = 3), students (n = 3), and general laborers (n = 5). These data indicated that an enzootic strain of VEE virus was the cause of at least 3% (18 of 598) of the cases of febrile illnesses studied in the city of Iquitos in the Amazon Basin region of Peru.

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

Immunogens of encephalitis viruses.

The equine encephalitis viruses are members of the genus Alphavirus, in the family Togaviridae. Three main virus serogroups represented by western (WEE), eastern (EEE) and Venezuelan equine encephalitis (VEE) viruses cause epizootic and enzootic infection of horses throughout the western hemisphere. All equine encephalitis viruses are transmitted through the bite of an infected mosquito. The first equine encephalitis virus vaccines were produced by virus inactivation. Problems with inadequate inactivation, which may have caused a major epidemic/epizootic of VEE in central America and Texas in the 1970s, led to the development of a live attenuated VEE virus vaccine (TC-83) derived by cell culture passage. Inactivated vaccines are still used to prevent equine infections with WEE and EEE viruses. Alphaviruses are small single stranded, positive sense RNA viruses. The 12000 nucleotide genome is enclosed in an icosahedral nucleocapsid composed of multiple copies of the capsid (C) protein. The virion is enveloped. The membrane is modified by the insertion of heterodimers of two glycoproteins: E1 and E2. Monoclonal antibody analysis of the surface glycoproteins have provided a detailed understanding of important protective antigens. Recent studies comparing gene sequences from virulent and avirulent VEE viruses have begun to delineate mechanisms of alphavirus attenuation.

Differentiation of parapoxviruses by application of orf virus-specific monoclonal antibodies against cell surface proteins.

Monoclonal antibodies were produced against orf virus-specified cell surface proteins in an attempt to develop reagents capable of differentiating between members of the Parapoxviridae. Two immunization protocols were used to induce an anti-orf response in BALB/c mice, one of which resulted in virus replication in the recipient. The monoclonal antibodies produced were tested for crossreactivity with bovine papular stomatitis virus (BPS) and milker's node virus (MNV) by indirect immunofluorescence assay (IFA) and immunoblotting. The results indicate that significant antigenic overlap exists between isolates of orf, MNV and BPS, even at the level of specificity provided by monoclonal antibodies. One monoclonal antibody reacted strongly in IFA with orf virus isolates, very weakly with MNV, and not at all with BPS. On immunoblots this same antibody recognized a 40-43 kDa protein in orf virus-infected cells, and also a 45-48 kDa protein in cells infected with MNV or BPS virus. The data suggest that it may be possible to define parapoxvirus strains on the basis of small variations in specific virus-directed cell surface proteins.

Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus.

We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.

Review on flavivirus vaccine development. Proceedings of a meeting jointly organised by the World Health Organization and the Thai Ministry of Public Health, 26-27 April 2004, Bangkok, Thailand.

In light of the continuous spread of human pathogenic flaviviruses, in particular the mosquito-transmitted species, vaccine development remains a high priority on the public health agenda. On 26-27 April 2004, a conference was held in Bangkok, Thailand, to review current status of flavivirus vaccine development and related issues, focussing on dengue (DEN) and Japanese encephalitis (JE). This event, co-sponsored by the World Health Organization (WHO) and the Thai Ministry of Public Health, reviewed the progress made with vaccine development, sero-epidemiological studies and other accompanying activities critical for vaccine development and vaccination. The considerable interest in and awareness of the flavivirus diseases and their prevention by public health decision makers, as well as the establishment of two dedicated programmes for dengue and Japanese encephalitis vaccine development raise hopes that new or improved vaccines will become available in the coming years.

Development of an enzyme-linked immunosorbent assay for the identification of arthropod-borne togavirus antibodies.

The applicability of the standard enzyme-linked immunosorbent assay (ELISA) for the identification of togavirus infections was investigated. Optimal concentration of gradient-purified antigen was 2.5 micrograms/well for alphaviruses or flaviviruses when coating polystyrene microtitre plates. A procedure for producing antigen in suckling mouse brain was developed. Results obtained with ELISA could be correlated with standard serology, but in general the ELISA was more sensitive. The ELISA was specific in differentiating alphavirus antigens. Flavivirus cross-reactivity was magnified by ELISA. ELISA should be useful as a rapid screening assay for non-related antigens, avoiding the extensive techniques currently used in standard serodiagnosis.