Host range specificity of flaviviruses: correlation with in vitro replication.

Vector-borne flaviviruses have been traditionally grouped into either mosquito-borne or tick-borne group. However, this vector range specificity has sometimes been questioned because of the puzzling records of occasional isolation of mosquito-borne viruses from ticks and of tick-borne viruses from mosquitoes. In this study, host range of the flaviviruses representing not only the two vector-borne groups but also insect flaviviruses and vertebrate viruses that are not arboviruses was comprehensively reexamined by a serial passage experiment in vitro by using cell cultures derived from mosquitoes, ticks, and vertebrates. The results showed that the host range specificity in the four groups of viruses, based on replication for five consecutive passages as a criterion to evaluate the ability of viruses to replicate in three different cell cultures, agreed with the conventional grouping as well as phylogenetic clustering. Thus, this assay provides useful, supplementary information regarding host range for those flaviviruses when their natural host range is unknown, ambiguous, or questionable.

Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.

Many members of the genus Flavivirus are the agents of important diseases of humans, livestock, and wildlife. Currently, no complete genome sequence is available for the three African viruses, Bagaza, Zika, and Kedougou viruses, each representing a distinct virus subgroup according to the latest virus classification. In this study, we obtained a complete genome sequence of each of those three viruses and characterized the open reading frames (ORFs) with respect to gene sizes, cleavage sites, potential glycosylation sites, distribution of cysteine residues, and unique motifs. The sequences of the three viruses were then scanned across the entire length of the ORF against available sequences of other African flaviviruses and selected reference viruses for genetic relatedness. The data collectively indicated that Kedougou virus was close to dengue viruses but nonetheless distinct, while Bagaza virus shared genetic relatedness with West Nile virus in several genomic regions. In the non-coding regions, it was found that a particular organizational pattern of conserved sequences in the 3' terminal region generally correlated with the current virus grouping.

A survey of the relationships among the viruses not considered arboviruses, vertebrates, and arthropods.

No single group of organisms demonstrates more extensive and diverse associations with animal viruses than the phylum Arthropoda. Compared with the well-recognized relationship found in arboviruses, however, most of the atypical arthropod-vertebrate relationships of the viruses normally not considered arboviruses have received much less attention, as they remain in the marginal areas of interest for most researchers in animal virology, veterinary medicine, medical entomology, and invertebrate pathology. However, this comprehensive review of the information gathered from several branches of virology by profession reveals highly valuable information potentially useful in the fields of research ranging from investigations of the mode of transmission of poorly understood or emerging viral diseases to studies of the evolution of biological transmission of animal viruses by arthropod vectors. The observations and data obtained for the animal virus relationships with arthropods and vertebrates outside the boundaries of arboviruses, in turn, can be used to reexamine more closely the definition of arboviruses. With increasing number of reports challenging one of the basic tenets of the definition of arbovirus (requirement of viremia in vertebrate host) and others describing virus-host relationships that complicate the definition of arbovirus, the accumulated information clearly demonstrates the difficulty of defining the boundaries of arboviruses.

Phylogeny of Thogoto virus.

Thogoto virus is a tick-borne member of the family Orthomyxoviridae. Previously, based on the similarity in antigenic relationship by cross-neutralization test, all virus strains were concluded to have derived from the same origin. In this study, we obtained partial gene sequences of 4 genes (PB1-like protein, PA-like protein, glycoprotein, and nucleoprotein) of 8 Thogoto virus strains isolated in Africa, Asia, and Europe and studied the genetic variation and phylogeny. Unrooted phylogenetic trees created by both neighbor-joining and maximum likelihood methods based on nucleotide and amino acid sequences for 4 genes were mostly similar and revealed two lineages, Euro-Asian and African. Intra-lineage nucleotide sequence variation was greater in the Euro-Asian lineage than in the African lineage for all 4 genes. Furthermore, for the strains of Euro-Asian lineage, variations for two genes associated with RNA-dependent RNA polymerase activities were greater than those for glycoprotein or nucleoprotein gene, based on both nucleotide and amino acid sequence differences as well as on synonymous and nonsynonymous differences, indicating greater mutation rates for the polymerase activity genes in these strains.

Genomic sequencing of deer tick virus and phylogeny of powassan-related viruses of North America.

Powassan (POW) virus is responsible for central nervous system infection in humans in North America and the eastern parts of Russia. Recently, a new flavivirus, deer tick (DT) virus, related to POW virus was isolated in the United States, but neither its pathogenic potential in human nor the taxonomic relationship with POW virus has been elucidated. In this study, we obtained the near-full-length genomic sequence of the DT virus and complete sequences of 3 genomic regions of 15 strains of POW-related virus strains. The phylogeny revealed 2 lineages, one of which had the prototype POW virus and the other DT virus. Both lineages can cause central nervous system infection in humans. By use of the combination of molecular definition of virus species within the genus Flavivirus and serological distinction in a 2-way cross-neutralization test, the lineage of DT virus is classified as a distinct genotype of POW virus.

Persistence of arboviruses and antiviral antibodies in vertebrate hosts: its occurrence and impacts.

The recent isolation of West Nile virus from a bird in mid-winter in New York immediately raised, as one of a few explanations, the possibility of long-term persistence of arboviruses in vertebrate hosts. Although it was a highly popular topic for research many years ago, generally it has since been neglected and its meaning under appreciated. This comprehensive survey of literature worldwide uncovered, contrary to the general perception that it is a rather infrequent phenomenon, a large number of important observations involving all groups of arboviruses that have been accumulating over the years without drawing much attention. In this review, the data and observations were analysed in terms of the occurrence, role in natural transmission, mechanisms and genesis of persistence, source of problems in research and impact. The outcome of the analyses clearly demonstrates that asymptomatic, long-term infection in the absence of viraemia with or without the induction of neutralising antibody, the most frequent characteristics of arboviral persistence, presents a serious question about the validity of some of the past animal experiments that were conducted without the consideration of such a possibility. Likewise, significant impacts are felt on diverse fields ranging from epidemiology to diagnostic virology and from veterinary medicine to agricultural commerce. Published in 2001 by John Wiley & Sons, Ltd.

Flavivirus DNA vaccines: current status and potential.

The use of DNA-based vaccines is a novel and promising immunization approach for the development of flavivirus vaccines. This approach has been attempted in vaccine development for various virus species, including St. Louis encephalitis, Russian spring-summer encephalitis, Central European encephalitis, dengue serotypes 1 and 2, Murray Valley encephalitis, Japanese encephalitis, and West Nile viruses. However, very little is known about the factors affecting its efficacy. Recently, we demonstrated that a single intramuscular immunization of DNA vaccine of Japanese encephalitis and West Nile viruses protected mice and horses from virus challenge. Administration of these recombinant plasmid vectors resulted in endogenous expression and secretion of extracellular virus-like particles that correlated well with the induction of protective immunity. These results provided evidence that the virus-like particles composed of premembrane/membrane and envelope proteins are essential for eliciting immune responses similar to those induced by live, attenuated virus vaccines. The biosynthesis and protein processing of premembrane/membrane and envelope proteins that preserve the native conformation and glycosylation profiles identical to virion proteins could be determined by the effectiveness of the transmembrane signal sequence located at the amino-terminus of premembrane protein. The use of DNA vaccines in multivalent and/or combination vaccines designed to immunize against multiple flaviviruses is also a promising area of development.

Transmission of arboviruses without involvement of arthropod vectors.

Transmission of arboviruses (arthropod-borne viruses belonging to various virus families) without involvement of arthropod vectors has been documented for years, but the reports have not been reviewed systematically. The recent report of West Nile (WN) virus isolation from a hawk in mid-winter in New York (Garmendia et al., J. Clin. Microbiol. 38, 3110-3111, 2000) generated a considerable interest in this mode of arbovirus transmission. In this article, the data available worldwide are analyzed according to the factors involved in such a transmission under natural conditions, mode of infection, virus entry mechanism, administration and efficacy evaluation of vaccines, and significance in agricultural trade and public health. Analysis of numerous reports compiled for this review revealed that peroral and intranasal/aerosol transmissions are very common among arboviruses. The mechanism of virus infections in animals was most extensively studied for intranasal/aerosol infection, confirming two routes of virus spread to central nervous system (CNS), olfactory and hematogenous. To rule out the possibility of asymptomatic, cryptic infection the efficacy evaluation of candidates for vaccines against neurotropic arboviruses should include virus isolation from tissues of not only symptomatic but also of asymptomatic animals that survive intranasal virus challenge. Human activities, such as feeding livestock animals with food containing virus-contaminated meat and assembling a large number of livestock from many geographically-separated locations, have been identified as a cause of spread of some arboviral diseases. Despite numerous laboratory reports, the significance of this mode of transmission of arboviruses under natural conditions was rarely investigated, except for a few viruses important for veterinary medicine.

Evaluation of an IgM immunoblot kit for dengue diagnosis.

A commercial IgM immunoblot kit was evaluated for dengue diagnosis with a panel of serum specimens collected from patients in a dengue endemic area. The kit is not recommended for use in its present form because of its undesirable rate of false-positive results. However, by substituting internal controls with the reference positive and negative controls that are more representative of those seen in endemic areas and by modifying the positive and negative scoring criteria, sensitivity and specificity of 80.3% and 94.5%, respectively, were obtained. These results are comparable with those obtained with the IgM ELISA on specimens, most of which were obtained from outpatient health care facilities. With further technical modifications, inclusion of a visual guide to ensure scoring standardization, and a more complete elaboration of the limitations of the test, wide application of the kit in diagnostic laboratories should be possible.

Universal diagnostic RT-PCR protocol for arboviruses.

A selected number of PCR protocols were evaluated to determine if they could serve as a universal protocol for detecting and identifying all arboviruses. In this study, four parameters that affect the efficacy of RT-PCR (RNA extraction method, choice of reverse transcriptase, choice of DNA polymerase and thermocycling program) were evaluated in combination. The most optimal combination of those parameters employed use of silica gel membrane spin column, RAV-2 reverse transcriptase, Tth DNA polymerase, and a simple modification of a published thermocycling program. By this modified protocol, viral RNA could be amplified satisfactorily with more than 50 pairs of primers designed for diagnosis of arboviruses representing five families. The sensitivity and specificity obtained by this universal protocol were comparable to those obtained by the original protocol for each primer pair tested; and for some primers, improved sensitivity was observed. It was also found that a simple modification of a suggested protocol of a commercial RT-PCR kit could produce nearly identical results and serve as another universal protocol. With the use of a universal diagnostic reverse transcriptase-polymerase chain reaction (RT-PCR) protocol, simultaneous screening of clinical or biological specimens against a large number of RNA viruses belonging to many families can be performed more efficiently for etiologic determination in the situations complicated by the difficulty of differential diagnosis. Furthermore, such a universal protocol facilitates reducing the cost of PCR-based diagnostic operation and standardizing the qualities of PCR-based diagnosis within an institution or among collaborating institutions. A logical strategy is to conduct diagnosis in two stages by using broadly group-reactive primers in the first stage to narrow the range of possible etiologic agents and using virus-specific primers in the second stage for identification. Before such a strategy is employed, however, more group-reactive primers for a large number of arboviruses, for which no such primers currently exist, must be made available. Furthermore, the best pair or pairs of primers need to be selected for each virus for the second stage of the strategy.

Phylogeny of the genus Flavivirus.

We undertook a comprehensive phylogenetic study to establish the genetic relationship among the viruses of the genus Flavivirus and to compare the classification based on molecular phylogeny with the existing serologic method. By using a combination of quantitative definitions (bootstrap support level and the pairwise nucleotide sequence identity), the viruses could be classified into clusters, clades, and species. Our phylogenetic study revealed for the first time that from the putative ancestor two branches, non-vector and vector-borne virus clusters, evolved and from the latter cluster emerged tick-borne and mosquito-borne virus clusters. Provided that the theory of arthropod association being an acquired trait was correct, pairwise nucleotide sequence identity among these three clusters provided supporting data for a possibility that the non-vector cluster evolved first, followed by the separation of tick-borne and mosquito-borne virus clusters in that order. Clades established in our study correlated significantly with existing antigenic complexes. We also resolved many of the past taxonomic problems by establishing phylogenetic relationships of the antigenically unclassified viruses with the well-established viruses and by identifying synonymous viruses.

The 1986 dengue and dengue hemorrhagic fever epidemic in Puerto Rico: epidemiologic and clinical observations.

In 1986 Puerto Rico experienced its eleventh dengue outbreak of this century, but the first with simultaneous transmission of three dengue virus serotypes, and the first with significant numbers of severe and fatal hemorrhagic disease. Overall, 10,659 cases were reported; 1,257 cases were laboratory confirmed as having current or recent dengue infection. Dengue 4 (DEN-4) was the predominant serotype (160/363 isolates, 44%) followed by dengue 1 (DEN-1) with 134 isolates (37%) and dengue 2 (DEN-2), 69 isolates (19%). Transmission peaked during September, but large numbers of cases occurred through November. Seventy-one (91%) of Puerto Rico's 78 municipalities had laboratory-confirmed cases. Fifty-one percent of all confirmed cases occurred in metropolitan San Juan. Most cases presented clinically as classical dengue fever, but 37% of all confirmed cases were reported to have developed some type of hemorrhagic manifestation, and 6% reported hematemesis. In addition, 29 laboratory confirmed cases met the WHO case definition for dengue hemorrhagic fever, 3 of which were fatal. Among the 29 laboratory-confirmed cases of dengue hemorrhagic fever/ dengue shook syndrome, virus was isolated from 12; one DEN-1, three DEN-2, and eight DEN-4. Among laboratory confirmed cases, infants less than one year of age were at greater risk of developing dengue hemorrhagic fever/ dengue shook syndrome, hematemesis and any reported hemorrhage than were the other age groups evaluated.

The 1986 dengue and dengue hemorrhagic fever epidemic in Puerto Rico: epidemiologic and clinical observations

In 1986 Puerto Rico experienced its eleventh dengue outbreak of this century, but the first with simultaneous transmission of three dengue virus serotypes, and the first with significant numbers of severe and fatal hemorrhagic disease. Overall, 10,659 cases were reported; 1,257 cases were laboratory confirmed as having current or recent dengue infection. Dengue 4 (DEN-4) was the predominant serotype (160/363 isolates, 44 percent) followed by dengue 1 (DEN-1) with 134 isolates (37 percent) and dengue 2 (DEN-2), 69 isolates (19 percent). Transmission peaked during September, but large numbers of cases occurred through November. Seventy-one (91 percent) of Puerto Rico's 78 municipalities had laboratory-confirmed cases. Fifty-one percent of all confirmed cases occurred in metropolitan San Juan. Most cases presented clinically as classical dengue fever, but 37 percent of all confirmed cases were reported to have developed some type of hemorrhagic manifestation, and 6 percent reported hematemesis. In addition, 29 laboratory confirmed cases met the WHO case definition for dengue hemorrhagic fever, 3 of which were fatal. Among the 29 laboratory-confirmed cases of dengue hemorrhagic fever/ dengue shook syndrome, virus was isolated from 12; one DEN-1, three DEN-2, and eight DEN-4. Among laboratory confirmed cases, infants less than one year of age were at greater risk of developing dengue hemorrhagic fever/ dengue shook syndrome, hematemesis and any reported hemorrhage than were the other age groups evaluated

Detecting bunyaviruses of the Bunyamwera and California serogroups by a PCR technique.

Many bunyaviruses of the Bunyamwera and California serogroups are medically important human pathogens. The development of an effective technique to detect the viruses by using molecular biologic tools, such as PCR, improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, we evaluated eight pairs of primers for reactivity with 44 viruses of the genus Bunyavirus, using a reverse transcriptase PCR technique. With a pair of serogroup-specific primers we designed, all viruses of the serogroups tested could be detected. Further, virus-specific primer pairs were identified for California encephalitis virus, Jamestown Canyon virus, La Crosse virus, and snowshoe hare virus for use in North America. Using this technique, we could detect one La Crosse virus-infected mosquito in a pool of 100 mosquitoes with undetectable plaque titers.

Symptoms of dengue fever in relation to host immunologic response and virus serotype, Puerto Rico, 1990-1991.

The authors investigated the role of secondary immunologic response, virus serotype, age, and sex on the clinical manifestations of dengue fever in Puerto Rico. From surveillance data for 1990 and 1991, this study identified 3,926 laboratory-positive cases, including 889 for whom dengue immunologic status and symptoms could be ascertained. Of those, 622 cases were virologically confirmed, and 267 cases were serologically confirmed. More than 50% of all positive patients reported fever, chills, headache, eye pain, body pains, joint pains, nausea, vomiting, or skin rash. The frequency of reporting signs, symptoms, and hospitalization was significantly higher among persons with secondary infections diagnosed by serological methods. Only rash was more common among those with primary infections. Symptom reporting increased with age; body pains, joint pains, and rash were significantly more frequently reported by female patients. No significant difference in symptom frequency was found among the virologically confirmed cases, comparing primary and secondary cases or infections due to different serotypes. The data for serologically confirmed cases suggest that in Puerto Rico the manifestations of dengue fever are, as with dengue hemorrhagic fever in Asia, more prominent among those who are experiencing secondary infections, and this effect may be more marked in the younger age groups.

Dengue severity throughout seasonal changes in incidence in Puerto Rico, 1989-1992. The Puerto Rico Association of Epidemiologists.

To determine whether the proportion of severe dengue cases increased with the yearly seasonal increase in dengue incidence, we examined reports of disease symptoms in case surveillance data and laboratory testing results in Puerto Rico from January 1989 to July 1992. A computer algorithm was designed to identify severe cases, i.e., those that fulfilled three or all four of the World Health Organization criteria for dengue hemorrhagic fever (DHF). A monthly severity index (SI) was defined as the ratio of severe cases to laboratory-positive and indeterminate (all non-negative) cases for each month, while a more restrictive severity rate (SR) was defined as the ratio of severe laboratory-positive cases to the total number of laboratory-positive cases for each month. Monthly SI and SR were compared in two ways: within an epidemic cycle, and month-by-month. Linear regression analysis was performed over the monthly averages of the SI and SR. For a month-by-month examination of SI and SR, we examined the 43-month sequence by means of a linear model with autocorrelated disturbances. We found no statistically significant or cyclical change in the proportion of severe cases from month to month in this period. Our conclusions differ from the observations during the Cuban DHF epidemic of 1981, in which case severity was shown to increase markedly as the epidemic progressed; they agree with the conclusions of most previous studies in that dengue severity does not change significantly throughout a period of increased incidence.

A PCR-restriction enzyme technique for determining dengue virus subgroups within serotypes.

The polymerase chain reaction (PCR) and restriction enzyme analysis were used to develop a rapid and simple procedure for identifying geographic subgroups of dengue virus within serotypes for epidemiologic investigations. The entire structural protein region of dengue viruses was amplified and the products were digested with the endonucleases AluI or DdeI. By comparing the restriction fragment length polymorphisms (RFLPs), we recognized dengue-2 and dengue-3 subgroups that corresponded to those previously determined by oligonucleotide fingerprinting or genomic sequencing. This procedure can be performed in 2 days without the use of radioisotopes, and results can be interpreted without computer analysis. For those analyses which require only subgroup affiliations, this is a useful tool for rapidly screening multiple virus isolates.

Cytokine responses to dengue infection among Puerto Rican patients.

Recently, a strong correlation between high concentration of tumor necrosis factor (TNF alpha) in blood and severity of dengue hemorrhagic fever/dengue shock syndrome has been reported from Asia and the Pacific. We wished to determine if a similar relationship could be found in dengue patients in the Americas where adult patients with severe syndromes have been observed more frequently than in Asia where severe cases have been observed mostly among children. The concentrations of interleukin-1 (IL-1 beta) in hospitalized adult groups were significantly lower than that in outpatient adults. In contrast, the levels of interleukin 6 (IL-6) were significantly higher in hospitalized adults and children than in the corresponding outpatients. Levels of TNF alpha were higher in hospitalized children than in outpatient children or hospitalized adults. There was no significant difference in the levels of these three cytokines among hospitalized patients with or without hemorrhagic manifestations. Thus, an elevated IL-6 level was positively associated with severity of dengue infection in both children and adults, but IL-1 beta level was negatively associated with severity in adults.