Sensitivity of host-seeking nymphal lone star ticks (Acari: Ixodidae) to immersion in heated water.

Host-seeking nymphal Amblyomma americanum (L.) (Acari: Ixodidae) were placed into heated water, and their survival or their torpidity was recorded as a function of exposure time. Exposures were determined that either kill the nymphs or affect their mobility. All nymphs died when exposed for a minute or more to a temperature > 51 degrees C. Nearly all nymphs remained motionless for a period of time when exposed for 3 min to a temperature > 44 degrees C.

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.

Dengue virus: molecular basis of cell entry and pathogenesis, 25-27 June 2003, Vienna, Austria.

Multivalent dengue vaccines now in late stage development pose unique vaccine safety challenges in that primary or secondary vaccine failures might place vaccines at risk to antibody-dependent enhanced (ADE) wild-type dengue infections. This conference was organized to address this unique vaccine safety issue. New data were presented on the structure of dengue and other flaviviruses, the cellular receptors of dengue virus for biologically relevant cells, dengue viral cell entry mechanisms and mechanisms underlying in vivo protection, neutralization and enhancement of dengue virus infection. It was concluded that a targeted research program should aim to develop an in vitro test to characterize persons immunized with dengue vaccines as completely or partially protected. Achievement of this aim will require a better understanding of the basic mechanisms by which dengue viruses recognize, attach, enter and infect relevant human cells and how antibodies protect against dengue infections.

Molecular determinants of virulence of West Nile virus in North America.

West Nile virus (WNV) is a mosquito-borne flavivirus that until very recently had not been found in the Americas. In 1999, there was an outbreak of West Nile encephalitis in New York and surrounding areas, involving 62 human cases, including 7 fatalities. The virus has subsequently become established in the United States of America (U.S.) with 4156 human cases, including 284 deaths, in 2002. The WNV strains found in the U.S. are members of "lineage I", a genetic grouping that includes viruses from Europe, Asia and Africa. Molecular epidemiologic studies indicate that two genetic variants of WNV emerged in 2002. The major genetic variant is found in most parts of the U.S., while the minor genetic variant has been identified only on the southeast coast of Texas. Investigation of WNV in mouse and hamster models demonstrated that strains from the U.S. are highly neurovirulent and neuroinvasive in these laboratory rodents. Other strains, such as Ethiopia 76a from lineage I, are not neuroinvasive and represent important viruses which can be used to elucidate the molecular basis of virulence and attenuation of WNV. To identify putative molecular determinants of virulence and attenuation, we have undertaken comparative nucleotide sequencing of Ethiopia 76a and strains from the U.S. The results show that the two viruses differ by 5 amino acids in the envelope (E) protein, including loss of the glycosylation site. Comparison of our panel of 27 WNV strains suggests that E protein glycosylation is a major determinant of the mouse neuroinvasive phenotype.

Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis.

Two approaches for presentation of a part of the rickettsial outer membrane protein A (OmpA) of Rickettsia rickettsii, namely (1) recombinant Mycobacterium vaccae (rMV) or (2) recombinant DNA vaccine, stimulated protective immunity against a lethal challenge with the closely related bacterium, R. conorii, in mice. After primary immunization with rMV and booster immunization with homologous recombinant protein, 67 and 55% of mice were protected against challenge in two experiments. DNA vaccination with booster recombinant protein immunization protected six out of eight animals from a lethal challenge. Production of IFN-gamma by antigen-exposed T-lymphocytes of DNA vaccine recipients indicated that cellular immunity had been stimulated.

Elevated stress hormone levels relate to Epstein-Barr virus reactivation in astronauts.

OBJECTIVE: The objective of this study was to determine the effects of stress and spaceflight on levels of neuroendocrine hormones and Epstein-Barr virus (EBV)-specific antibodies in astronauts. METHODS: Antiviral antibody titers and stress hormones were measured in plasma samples collected from 28 astronauts at their annual medical exam (baseline), 10 days before launch (L-10), landing day (R+0), and 3 days after landing (R+3). Urinary stress hormones were also measured at L-10 and R+0. RESULTS: Significant increases (p <.01) in EBV virus capsid antigen antibodies were found at all three time points (L-10, R+0, and R+3) as compared with baseline samples. Anti-EBV nuclear antigen antibodies were significantly decreased at L-10 (p <.05) and continued to decrease after spaceflight (R+0 and R+3, p <.01). No changes were found in antibodies to the nonlatent measles virus. The 11 astronauts who showed evidence of EBV reactivation had significant increases in urinary epinephrine and norepinephrine as compared with astronauts without EBV reactivation. CONCLUSION: These findings indicate that physical and psychological stresses associated with spaceflight resulted in decreased virus-specific T-cell immunity and reactivation of EBV.

Nucleotide sequencing and serological evidence that the recently recognized deer tick virus is a genotype of Powassan virus.

Deer tick virus (DTV) is a recently recognized North American virus isolated from Ixodes dammini ticks. Nucleotide sequencing of fragments of structural and non-structural protein genes suggested that this virus was most closely related to the tick-borne flavivirus Powassan (POW), which causes potentially fatal encephalitis in humans. To determine whether DTV represents a new and distinct member of the Flavivirus genus of the family Flaviviridae, we sequenced the structural protein genes and 5' and 3' non-coding regions of this virus. In addition, we compared the reactivity of DTV and POW in hemagglutination inhibition tests with a panel of polyclonal and monoclonal antisera, and performed cross-neutralization experiments using anti-DTV antisera. Nucleotide sequencing revealed a high degree of homology between DTV and POW at both nucleotide (>80% homology) and amino acid (>90% homology) levels, and the two viruses were indistinguishable in serological assays and mouse neuroinvasiveness. On the basis of these results, we suggest that DTV should be classified as a genotype of POW virus.

Phylogenetic and evolutionary relationships among yellow fever virus isolates in Africa.

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.

Amino acid substitution(s) in the stem-anchor region of langat virus envelope protein attenuates mouse neurovirulence.

The identification of variants that are unable to bind membrane receptor preparations (MRPs) has previously been shown to select attenuated yellow fever and Japanese encephalitis viruses. In this study, this methodology has been extended to the tick-borne serocomplex of flaviviruses. Langat (LGT) virus strain TP21 was bound to mouse or human brain MRPs and viruses that escaped binding were isolated and characterized. In addition, variant viruses escaping neutralization by the monoclonal antibody (MAb) 9F9 were also isolated. All of the variant viruses were attenuated for mouse neurovirulence (> or =13-fold). Sequence analysis of the prM/E region of the variant viruses identified mutations within the stem-anchor region of the E protein in variants isolated following incubation with mouse or human brain MRPs at a pH > or = 7.0. The MAb 9F9 variants and MRP variants isolated at pH 5.0, which should induce a conformational shift in the viral E protein, had nearly identical mutations in the prM/M protein immediately N-terminal to the prM/E cleavage site. MAb 9F9 neutralized none of the variant viruses and hemagglutination inhibition assays suggest that the variant virus surface proteins have slightly different conformations compared to the parental virus. These data support previous work indicating that the stem-anchor region of the E protein is important to the surface architecture of the tick-borne flaviviruses. In addition, this study demonstrates that the M protein is at least partially solvent accessible on the virion surface and that the M protein plays a role in maintaining the conformation of the M/E surface complex.

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.

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.

Langat virus M protein is structurally homologous to prM.

Langat (LGT) virus M protein has been generated in a recombinant system. Antiserum raised against the LGT virus M protein neutralizes tick-borne encephalitis serocomplex flaviviruses but not mosquito-borne flaviviruses, indicating that the M protein is exposed on the surface of virions. The antiserum recognizes intracellular LGT virus prM/M and binds to prM and M in Western blots of whole-cell lysates and purified virus, respectively. These data suggest that the prM and M proteins are structurally similar under native conditions and support the hypothesis that the "pr" portion of prM facilitates proper folding of the M protein for expression on the virion surface.

Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein.

The molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain III of the envelope protein has been implicated in these functions. We examined the solution properties and antagonist activity of Langat virus domain III. Our results suggest that domain III adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. Three clusters of phylogenetically conserved residues are identified that may be responsible for the vector-specific antagonist activity of domain III.

Nucleotide sequence and deduced amino acid sequence of the medium RNA segment of Oropouche, a Simbu serogroup virus: comparison with the middle RNA of Bunyamwera and California serogroup viruses.

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50-52%) and amino acid (30-32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743-748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.

Genetic variation in the 3' non-coding region of dengue viruses.

The 3' non-coding region (3'NCR) of strains of dengue 1 (DEN 1), DEN 2, DEN 3, and DEN 4 viruses, isolated in different geographical regions, was sequenced and compared to published sequences of the four dengue viruses. A total of 50 DEN 2 strains was compared: 7 West African strains, 3 Indonesian mosquito strains, 1 Indonesian macaque isolate, and 39 human isolates from Southeast Asia, the South Pacific, and the Caribbean and Americas. Nucleotide sequence alignment revealed few deletions and no repeat sequences in the 3' NCR of DEN 2 viruses and showed that much of the 3' NCR was well conserved. The strains could be divided into two groups, sylvatic and human/mosquito/macaque, based on nucleotide sequence homology. A hypervariable region was identified immediately following the NS5 stop codon, which involved a 2-10 nucleotide deletion in human, mosquito, and macaque isolates compared with the sylvatic strains. The DEN 2 3'NCR was also compared with 3'NCR sequences from strains of DEN 1, DEN 3, and DEN 4 viruses. DEN 1 was found to have four copies of an eight nucleotide imperfect repeat following the NS5 stop codon, while DEN 4 virus had a deletion of 75 nucleotides in the 3'NCR. We propose that the variation in nucleotide sequence in the 3'NCR may have evolved as a function of DEN virus transmission and replication in different mosquito and non-human primate/human host cycles. The results from this study are consistent with the hypothesis that DEN viruses arose from sylvatic progenitors and evolved into human epidemic strains. However, the data do not support the hypothesis that variation in the 3'NCR correlates with DEN virus pathogenesis.

Current status of flavivirus vaccines.

Although there are approximately 68 flaviviruses recognized, vaccines have been developed to control very few human flavivirus diseases. Licensed live attenuated vaccines have been developed for yellow fever (strain 17D) and Japanese encephalitis (strain SA14-14-2) viruses, and inactivated vaccines have been developed for Japanese encephalitis and tick-borne encephalitis viruses. The yellow fever live attenuated 17D vaccine is one of the most efficacious and safe vaccines developed to date and has been used to immunize more than 300 million people. A number of experimental vaccines are being developed, most notably for dengue. Candidate tetravalent live attenuated dengue vaccines are undergoing clinical trials. Other vaccines are being developed using reverse genetics, DNA vaccines, and recombinant immunogens. In addition, the yellow fever 17D vaccine has been used as a backbone to generate chimeric viruses containing the premembrane and envelope protein genes from other flaviviruses. The "Chimerivax" platform has been used to construct chimeric Japanese encephalitis and dengue viruses that are in different phases of development. Similar strategies are being used by other laboratories.

The French neurotropic vaccine strain of yellow fever virus accumulates mutations slowly during passage in cell culture.

This study of the yellow fever French neurotropic vaccine strain from the Institut Pasteur (FNV-IP) demonstrates that this viral genome is not as stable as that of the 17D-204 vaccine virus. FNV-IP was plaque-purified three times and then passaged eight times in Vero cells. Viral populations from the second and eighth passage post purification were sequenced and compared to the published sequences of FNV-IP. The passage-2 viral population had 31 nucleotide and nine amino acid changes compared to the parental virus while the passage-8 virus had six additional nucleotide changes encoding a single amino acid substitution. The plaque-purified virus also had two sequence deletions in the 3'-noncoding region. The plaque purification resulted in selection of a passage-2 virus that had a mouse LD(50) of 20 pfu/ml, 67-fold greater than parental FNV-IP which had an LD(50) of 0.3 pfu/ml. Subsequent passage in Vero cells resulted in a passage-8 virus which had increased neurovirulence with an LD(50) of 3.2 pfu/ml. The only amino acid difference between the passage-2 and passage-8 viruses was at amino acid 638 of NS5 which lies within domain V of the RNA-dependent-RNA polymerase. Overall, these data indicate that FNV-IP virus has an inherently less stable genome than 17D vaccine virus and a variable viral population.