Mechanisms of Yellow Fever Transmission: Gleaning the Overlooked Records of Importance and Identifying Problems, Puzzles, Serious Issues, Surprises and Research Questions.

In viral disease research, few diseases can compete with yellow fever for the volume of literature, historical significance, richness of the topics and the amount of strong interest among both scientists and laypersons. While the major foci of viral disease research shifted to other more pressing new diseases in recent decades, many critically important basic tasks still remain unfinished for yellow fever. Some of the examples include the mechanisms of transmission, the process leading to outbreak occurrence, environmental factors, dispersal, and viral persistence in nature. In this review, these subjects are analyzed in depth, based on information not only in old but in modern literatures, to fill in blanks and to update the current understanding on these topics. As a result, many valuable facts, ideas, and other types of information that complement the present knowledge were discovered. Very serious questions about the validity of the arbovirus concept and some research practices were also identified. The characteristics of YFV and its pattern of transmission that make this virus unique among viruses transmitted by Ae. aegypti were also explored. Another emphasis was identification of research questions. The discovery of a few historical surprises was an unexpected benefit.

Contrasting the Practices of Virus Isolation and Characterization between the Early Period in History and Modern Times: The Case of Japanese Encephalitis Virus.

Japanese encephalitis is a serious disease transmitted by mosquitoes. With its recent spread beyond the traditional territory of endemicity in Asia, the magnitude of global threat has increased sharply. While much of the current research are largely focused on changing epidemiology, molecular genetics of virus, and vaccination, little attention has been paid to the early history of virus isolation and phenotypic characterization of this virus. In this review, using this piece of history as an example, I review the transition of the concept and practice of virus isolation and characterization from the early period of history to modern times. The spectacular development of molecular techniques in modern times has brought many changes in practices as well as enormous amount of new knowledge. However, many aspects of virus characterization, in particular, transmission mechanism and host relationship, remain unsolved. As molecular techniques are not perfect in all respects, beneficial accommodation of molecular and biologic data is critically important in many branches of research. Accordingly, I emphasize exercising caution in applying only these modern techniques, point out unrecognized communication problems, and stress that JE research history is a rich source of interesting works still valuable even today and waiting to be discovered.

Widespread interspecific phylogenetic tree incongruence between mosquito-borne and insect-specific flaviviruses at hotspots originally identified in Zika virus.

Intraspecies (homologous) phylogenetic incongruence, or 'tree conflict' between different loci within the same genome of mosquito-borne flaviviruses (MBFV), was first identified in dengue virus (DENV) and subsequently in Japanese encephalitis virus (JEV), St Louis encephalitis virus, and Zika virus (ZIKV). Recently, the first evidence of phylogenetic incongruence between interspecific members of the MBFV was reported in ZIKV and its close relative, Spondweni virus. Uniquely, these hybrid proteomes were derived from four incongruent trees involving an Aedes-associated DENV node (1 tree) and three different Culex-associated flavivirus nodes (3 trees). This analysis has now been extended across a wider spectrum of viruses within the MBFV lineage targeting the breakpoints between phylogenetic incongruent loci originally identified in ZIKV. Interspecies phylogenetic incongruence at these breakpoints was identified in 10 of 50 viruses within the MBFV lineage, representing emergent Aedes and Culex-associated viruses including JEV, West Nile virus, yellow fever virus, and insect-specific viruses. Thus, interspecies phylogenetic incongruence is widespread amongst the flaviviruses and is robustly associated with the specific breakpoints that coincide with the interspecific phylogenetic incongruence previously identified, inferring they are 'hotspots'. The incongruence amongst the emergent MBFV group was restricted to viruses within their respective associated epidemiological boundaries. This MBFV group was RY-coded at the third codon position ('wobble codon') to remove transition saturation. The resulting 'wobble codon' trees presented a single topology for the entire genome that lacked any robust evidence of phylogenetic incongruence between loci. Phylogenetic interspecific incongruence was therefore observed for exactly the same loci between amino acid and the RY-coded 'wobble codon' alignments and this incongruence represented either a major part, or the entire genomes. Maximum likelihood codon analysis revealed positive selection for the incongruent lineages. Positive selection could result in the same locus producing two opposing trees. These analyses for the clinically important MBFV suggest that robust interspecific phylogenetic incongruence resulted from amino acid selection. Convergent or parallel evolutions are evolutionary processes that would explain the observation, whilst interspecific recombination is unlikely.

The Absence of Yellow Fever in Asia: History, Hypotheses, Vector Dispersal, Possibility of YF in Asia, and Other Enigmas.

Since the recent epidemics of yellow fever in Angola and Brazil as well as the importation of cases to China in 2016, there has been an increased interest in the century-old enigma, absence of yellow fever in Asia. Although this topic has been repeatedly reviewed before, the history of human intervention has never been considered a critical factor. A two-stage literature search online for this review, however, yielded a rich history indispensable for the debate over this medical enigma. As we combat the pandemic of COVID-19 coronavirus worldwide today, we can learn invaluable lessons from the historical events in Asia. In this review, I explore the history first and then critically examine in depth major hypotheses proposed in light of accumulated data, global dispersal of the principal vector, patterns of YF transmission, persistence of urban transmission, and the possibility of YF in Asia. Through this process of re-examination of the current knowledge, the subjects for research that should be conducted are identified. This review also reveals the importance of holistic approach incorporating ecological and human factors for many unresolved subjects, such as the enigma of YF absence in Asia, vector competence, vector dispersal, spillback, viral persistence and transmission mechanisms.

Recombination of B- and T-cell epitope-rich loci from Aedes- and Culex-borne flaviviruses shapes Zika virus epidemiology.

Sporadic human Zika virus (ZIKV) infections have been recorded in Africa and Asia since the 1950s. Major epidemics occurred only after ZIKV emerged in the Pacific islands and spread to the Americas. Specific biological determinants of the explosive epidemic nature of ZIKV have not been identified. Phylogenetic studies revealed incongruence in ZIKV placement in relation to Aedes-borne dengue viruses (DENV) and Culex-borne flaviviruses. We hypothesized that this incongruence reflects interspecies recombination resulting in ZIKV evasion of cross-protective T-cell immunity. We investigated ZIKV phylogenetic incongruence in relation to: DENV T-cell epitope maps experimentally identified ex vivo, published B-cell epitope loci, and CD8+ T-cell epitopes predicted in silico for mosquito-borne flaviviruses. Our findings demonstrate that the ZIKV proteome is a hybrid of Aedes-borne DENV proteins interspersed amongst Culex-borne flavivirus proteins derived through independent interspecies recombination events. These analyses infer that DENV-associated proteins in the ZIKV hybrid proteome generated immunodominant human B-cell responses, whereas ZIKV recombinant derived Culex-borne flavivirus-associated proteins generated immunodominant CD8+ and/or CD4+ T-cell responses. In silico CD8+ T-cell epitope ZIKV cross-reactive prediction analyses verified this observation. We propose that by acquiring cytotoxic T-cell epitope-rich regions from Culex-borne flaviviruses, ZIKV evaded DENV-generated T-cell immune cross-protection. Thus, Culex-borne flaviviruses, including West Nile virus and Japanese encephalitis virus, might induce cross-protective T-cell responses against ZIKV. This would explain why explosive ZIKV epidemics occurred in DENV-endemic regions of Micronesia, Polynesia and the Americas where Culex-borne flavivirus outbreaks are infrequent and why ZIKV did not cause major epidemics in Asia where Culex-borne flaviviruses are widespread.

Vertebrate Reservoirs of Arboviruses: Myth, Synonym of Amplifier, or Reality?

The rapid succession of the pandemic of arbovirus diseases, such as dengue, West Nile fever, chikungunya, and Zika fever, has intensified research on these and other arbovirus diseases worldwide. Investigating the unique mode of vector-borne transmission requires a clear understanding of the roles of vertebrates. One major obstacle to this understanding is the ambiguity of the arbovirus definition originally established by the World Health Organization. The paucity of pertinent information on arbovirus transmission at the time contributed to the notion that vertebrates played the role of reservoir in the arbovirus transmission cycle. Because this notion is a salient feature of the arbovirus definition, it is important to reexamine its validity. This review addresses controversial issues concerning vertebrate reservoirs and their role in arbovirus persistence in nature, examines the genesis of the problem from a historical perspective, discusses various unresolved issues from multiple points of view, assesses the present status of the notion in light of current knowledge, and provides options for a solution to resolve the issue.

A Re-Examination of the History of Etiologic Confusion between Dengue and Chikungunya.

Contrary to the perception of many researchers that the recent invasion of chikungunya (CHIK) in the Western Hemisphere marked the first episode in history, a recent publication reminded them that CHIK had prevailed in the West Indies and southern regions of the United States from 1827-1828 under the guise of "dengue" (DEN), and that many old outbreaks of so-called "dengue" actually represented the CHIK cases erroneously identified as "dengue." In hindsight, this confusion was unavoidable, given that the syndromes of the two diseases-transmitted by the same mosquito vector in urban areas-are very similar, and that specific laboratory-based diagnostic techniques for these diseases did not exist prior to 1940. While past reviewers reclassified problematic "dengue" outbreaks as CHIK, primarily based on manifestation of arthralgia as a marker of CHIK, they neither identified the root cause of the alleged misdiagnosis nor did they elaborate on the negative consequences derived from it. This article presents a reconstructed history of the genesis of the clinical definition of dengue by emphasizing problems with the definition, subsequent confusion with CHIK, and the ways in which physicians dealt with the variation in dengue-like ("dengue") syndromes. Then, the article identifies in those records several factors complicating reclassification, based on current practice and standards. These factors include terms used for characterizing joint problems, style of documenting outbreak data, frequency of manifestation of arthralgia, possible involvement of more than one agent, and occurrence of the principal vector. The analysis of those factors reveals that while some of the old "dengue" outbreaks, including the 1827-1828 outbreaks in the Americas, are compatible with CHIK, similar reclassification of other "dengue" outbreaks to CHIK is difficult because of a combination of the absence of pathognomonic syndrome in these diseases and conflicting background information.

Genetic divergence among members of the Kokobera group of flaviviruses supports their separation into distinct species.

The Kokobera virus group comprises mosquito-borne flaviviruses that cluster together phylogenetically. These viruses are unique to Australia and Papua New Guinea, and have been associated with a mild polyarticular disease in humans. Recent isolation of genetically diverse viruses within this group has prompted analysis of their genetic and phenotypic relationships. Phylogenetic analysis based on complete ORF, the envelope gene or the NS5/3' untranslated region supported the separation of the group into distinct species: Kokobera virus (KOKV), Stratford virus, New Mapoon virus, MK7979 and TS5273. Virulence studies in 3-week-old mice also provided the first evidence that a member of the KOKV group (MK7979) was neuroinvasive after intraperitoneal inoculation. In this context, our recent detection of KOKV group-specific antibodies in horses in the field suggests that these viruses should be considered in the epidemiology of flavivirus encephalitis in Australia.

Revisiting Houston and Memphis: the background histories behind the discovery of the infestation by Aedes albopictus (Diptera: Culicidae) in the United States and their significance in the contemporary research.

The discovery of Aedes albopictus Skuse infestation in Texas in 1985 and subsequent global spread of this mosquito have spawned a large number of publications worldwide. The unique discovery in this episode was used tire trade as mechanism by which this exotic mosquito was introduced to the United States. While most researchers have focused their attention primarily on recent events and scientific progress, far fewer examined in depth early contributions made in the United States still very much relevant for contemporary discussion and other important subjects associated with its infestation. The first part of this review covers early background histories of research on Ae. albopictus in the institutions in the United States and of introduction of Ae. albopictus and Ae. aegypti to the U.S. territories in the eastern Pacific as well as dengue outbreaks therein. The second part covers evolution of recognition of the importance of tire as breeding site of domesticated or peridomestic dengue vectors and of its transport in the dispersal of these mosquitoes. In the third part, the significance of vector-borne viral disease outbreak potential of Ae. albopictus and Ae. aegypti in temperate regions and a variety of problems concerning vector spread and commercial practices learned in the past are reassessed in the context of contemporary research. I further identified as byproducts of this review two potentially important epidemiological mechanisms in the transmission of dengue in temperate regions, increasing tendency of indoor activity of Ae. albopictus and massive human movement associated with unpredictable political development.

Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains.

The yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culicidae), is well recognized for its extensive adaptation to diverse ecological conditions and for genetic variation. Recognizing the importance of strain variation of this mosquito, researchers have established a large number of laboratory strains. Some of the popular strains have been used for research for years in many laboratories around the world. However, the exact origins of many of these strains are unknown. In this review, publications and archival records were examined to report the early laboratory mosquito rearing practices around the world and to identify the origins of selected strains. The records showed that inter-laboratory sharing of strains was already underway in the early part of the 20th century because of the ease of breeding Ae. aegypti and of sending eggs by mail. It also was found that the four strains established in major U.S. institutions by the mid-1930s, including the "ROCK" (short for Rockefeller) strain, had been derived from Cuba, Nigeria, Philippines, or Puerto Rico, all known for a long history of transmission of yellow fever virus or dengue virus rather than from North America. The strains used for research in Europe were primarily derived from West Africa, but strains of Asian, Caribbean, and South American origins also were used for comparative experiments among geographic strains. Neglected issues related to strain designation and original source identification in scientific publications were found and their relevance to current research is discussed.

Emergence of the severe syndrome and mortality associated with dengue and dengue-like illness: historical records (1890 to 1950) and their compatibility with current hypotheses on the shift of disease manifestation.

Outbreaks of the severe dengue syndrome, dengue hemorrhagic fever (DHF), emerged beginning in the 1950s, marking a dramatic change in the dengue syndrome. While intense investigations in multiple directions have been conducted for many years to elucidate the intrinsic mechanisms conducive to the development of DHF, no consensus has yet emerged. Meanwhile, relatively little attention has been paid to the occurrence of severe dengue and death prior to the 1950s. This comprehensive review was designed to evaluate outbreak records in the early dengue history to better understand the epidemiologic background and other factors that existed before the emergence of DHF outbreaks. By applying a set of stringent criteria to remove unreliable data as much as possible and by interpreting the results conservatively, a short list of etiologically more reliable outbreaks with high mortality was obtained. The results show that severe dengue syndrome, clinically very much compatible with DHF, occurred far more frequently in multiple locations than it had been assumed before; that the magnitudes of mortality in several outbreaks were not negligible; and that the epidemiologic background features shared among these outbreaks in the early period were, with the exceptions of more limited demographic changes, generally similar to the post-1950 conditions.

Complete genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in Sao Paulo state.

The flaviviruses of major medical importance in South American countries are yellow fever, dengue, Saint Louis encephalitis, West Nile and Rocio viruses. Rocio virus (ROCV) has been responsible for epidemics of severe encephalitis in Brazil with a case-fatality rate of 10 % and development of sequelae in 20 % of the survivors. We have sequenced and characterized the entire genome of ROCV for the first time, by determining the general traits of the open reading frame and the characteristics of viral genes including the potential cleavage sites, conserved or unique motifs, cysteine residues and potential glycosylation sites. The conserved sequences in the 3'-non-coding region were identified, and the predicted secondary structures during cyclization between 5'- and 3'-non-coding regions were studied. Multiple protein and phylogenetic analyses based on antigenically important and phylogenetically informative genes confirmed a close relationship between ROCV and Ilheus virus (ILHV), together constituting a unique and distinct phylogenetic subgroup as well as the genetic relationship of ROCV with several members of the Japanese encephalitis group. Although ROCV is phylogenetically related to ILHV, our study shows that it is still a virus distinct from the latter virus. This is the first flavivirus uniquely indigenous to Brazil that has been sequenced completely and the genome characterized. The data should be useful for further studies at the molecular level, including construction of infectious clone, identification of gene function, improved disease surveillance based on molecular diagnostic tools and vaccine development.

Research on dengue and dengue-like illness in East Asia and the Western Pacific during the First Half of the 20th century.

Dengue has become an enormous medical problem worldwide since the end of the World War II (WWII). Despite a voluminous amount of research conducted worldwide for many years to elucidate the mechanism of the development of the two severe forms of dengue (dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS)), to develop effective vaccines and to design reliable disease control and prevention strategies, the goals of achieving these objectives are not in sight yet. For such a medical research that requires a multi-directional approach to resolve a variety of research questions, sometimes it is necessary to step back and re-examine historically how the current status of the expanding global problems and sudden emergence of severe forms of dengue have evolved in the first place. To examine the history of dengue epidemiology, it is first necessary to put in perspective all relevant documents, including a large number of nearly forgotten, old Japanese publications regarding dengue outbreaks and allied background information in East Asia and the Western Pacific that were documented before the emergence of the haemorrhagic disease outbreaks in the 1950s there. The compiled data in this review, thus, fill in for the first time many blanks in the early epidemiologic history of dengue in this part of the world. Besides the data for epidemiologists, virologic data, clinical studies of haemorrhagic manifestation, significance in military medicine and entomologic investigations covered in this review should be useful for the current researchers investigating any of those subjects.

Characterization of Sepik and Entebbe bat viruses closely related to yellow fever virus.

Yellow fever virus has a special place in medical history as the first animal virus isolated and as the prototype virus in the genus Flavivirus, which contains many serious human pathogens. Only recently, its closely related viruses within the group were identified phylogenetically. In this study, we obtained complete or near complete genome sequences of two viruses most closely related to yellow fever virus: Sepik virus of Papua New Guinea and Entebbe bat virus of Africa. Based on full-genomic characterization and genomic traits among related viruses, we identified Sepik virus to be most closely related to yellow fever virus and analyzed the pattern of repeat and conserved sequence motifs in the 3'-noncoding region among the members of yellow fever virus cluster. We also discuss the geographic dispersal as a part of ecological traits of this lineage of flaviviruses.

Biological transmission of arboviruses: reexamination of and new insights into components, mechanisms, and unique traits as well as their evolutionary trends.

Among animal viruses, arboviruses are unique in that they depend on arthropod vectors for transmission. Field research and laboratory investigations related to the three components of this unique mode of transmission, virus, vector, and vertebrate host, have produced an enormous amount of valuable information that may be found in numerous publications. However, despite many reviews on specific viruses, diseases, or interests, a systematic approach to organizing the available information on all facets of biological transmission and then to interpret it in the context of the evolutionary process has not been attempted before. Such an attempt in this review clearly demonstrates tremendous progress made worldwide to characterize the viruses, to comprehend disease transmission and pathogenesis, and to understand the biology of vectors and their role in transmission. The rapid progress in molecular biologic techniques also helped resolve many virologic puzzles and yielded highly valuable data hitherto unavailable, such as characterization of virus receptors, the genetic basis of vertebrate resistance to viral infection, and phylogenetic evidence of the history of host range shifts in arboviruses. However, glaring gaps in knowledge of many critical subjects, such as the mechanism of viral persistence and the existence of vertebrate reservoirs, are still evident. Furthermore, with the accumulated data, new questions were raised, such as evolutionary directions of virus virulence and of host range. Although many fundamental questions on the evolution of this unique mode of transmission remained unresolved in the absence of a fossil record, available observations for arboviruses and the information derived from studies in other fields of the biological sciences suggested convergent evolution as a plausible process. Overall, discussion of the diverse range of theories proposed and observations made by many investigators was found to be highly valuable for sorting out the possible mechanism(s) of the emergence of arboviral diseases.

Identification of new flaviviruses in the Kokobera virus complex.

Novel flavivirus isolates from mosquitoes collected in northern Australia were analysed by partial genomic sequencing, monoclonal antibody-binding assays and polyclonal cross-neutralization tests. Two isolates were found to be antigenically distinct from, but related to, viruses of the Kokobera virus complex, which currently contains Kokobera (KOKV) and Stratford (STRV) viruses. Nucleotide sequence comparison of two separate regions of the genome revealed that an isolate from Saibai Island in the Torres Strait in 2000 (TS5273) was related closely to KOKV and STRV, with 74-80 and 75-76 % nucleotide similarity, respectively. An isolate from mainland Cape York in 1998 (CY1014) was found to be more divergent from KOKV and STRV, with <70 % nucleotide sequence similarity to either virus. It is proposed that isolate TS5273 represents a new subtype of KOKV and that CY1014 be classified as a novel species within the Kokobera virus complex of flaviviruses, named New Mapoon virus.

Recent advancement in flavivirus vaccine development.

Lately, the magnitude of cumulative diseases burden caused by flaviviruses, such as dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus and yellow fever virus, has reached an unprecedented level with the sizes of human and animal populations at risk increasing sharply. These diseases present highly complex medical, economic and ecologic problems, some effecting primarily human and others affecting human, livestock and wildlife. The large body of recent publications on the development of vaccines taking advantage of new generations of bio-engineering techniques clearly reflects the profound interests and deep sense of urgency in the scientific and medical communities in combating those diseases. This review reveals a collection of remarkable progresses thus far made in flaviviral vaccine research not only employing a diverse range of new strategies but also re-tooling old techniques to improve the existing vaccines. The efficacy and safety of some of the new vaccine candidates have been evaluated and proven in human clinical trials. Besides the technical advancement in vaccine development, in this review, the importance of somewhat neglected and yet critical subjects, such as adequacy of animal model, vaccine safety, vaccine formulation and delivery, complication in serodiagostics and economic factor, was examined in-depth.