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Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti , which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features., (© 2023 Cold Spring Harbor Laboratory Press.)

Genetic diversity and population structuring for the species Haemogogus leucocelaenus , a sylvatic vector of yellow fever virus, were found to vary with the degree of agricultural land use and isolation of fragments of Atlantic Forest in municipalities in the state of São Paulo where specimens were collected. Genotyping of 115 mitochondrial SNPs showed that the populations with the highest indices of genetic diversity (polymorphic loci and mean pairwise differences between the sequences) are found in areas with high levels of agricultural land use (northeast of the State). Most populations exhibited statistically significant negative values for the Tajima D and Fu FS neutrality tests, suggesting recent expansion. The results show an association between genetic diversity in this species and the degree of agricultural land use in the sampled sites, as well as signs of population expansion of this species in most areas, particularly those with the highest forest edge densities. A clear association between population structuring and the distance between the sampled fragments (isolation by distance) was observed: samples from a large fragment of Atlantic Forest extending along the coast of the state of São Paulo exhibited greater similarity with each other than with populations in the northwest of the state.

Background: The behaviors and ontogeny of Aedes aegypti are closely related to the spread of diseases caused by dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV) viruses. During the life cycle, Ae. aegypti undergoes drastic morphological, metabolic, and functional changes triggered by gene regulation and other molecular mechanisms. Some essential regulatory factors that regulate insect ontogeny have been revealed in other species, but their roles are still poorly investigated in the mosquito., Results: Our study identified 6 gene modules and their intramodular hub genes that were highly associated with the ontogeny of Ae. aegypti in the constructed network. Those modules were found to be enriched in functional roles related to cuticle development, ATP generation, digestion, immunity, pupation control, lectins, and spermatogenesis. Additionally, digestion-related pathways were activated in the larvae and adult females but suppressed in the pupae. The integrated protein‒protein network also identified cilium-related genes. In addition, we verified that the 6 intramodular hub genes encoding proteins such as EcKinase regulating larval molt were only expressed in the larval stage. Quantitative RT‒PCR of the intramodular hub genes gave similar results as the RNA-Seq expression profile, and most hub genes were ontogeny-specifically expressed., Conclusions: The constructed gene coexpression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. Ultimately, these findings will be key in identifying potential molecular targets for disease control., (© 2023. The Author(s).)

Vector-borne diseases are serious public health concern. Mosquito is one of the major vectors responsible for the transmission of a number of diseases like malaria, Zika, chikungunya, dengue, West Nile fever, Japanese encephalitis, St. Louis encephalitis, and yellow fever. Various strategies have been used for mosquito control, but the breeding potential of mosquitoes is such tremendous that most of the strategies failed to control the mosquito population. In 2020, outbreaks of dengue, yellow fever, and Japanese encephalitis have occurred worldwide. Continuous insecticide use resulted in strong resistance and disturbed the ecosystem. RNA interference is one of the strategies opted for mosquito control. There are a number of mosquito genes whose inhibition affected mosquito survival and reproduction. Such kind of genes could be used as bioinsecticides for vector control without disturbing the natural ecosystem. Several studies have targeted mosquito genes at different developmental stages by the RNAi mechanism and result in vector control. In the present review, we included RNAi studies conducted for vector control by targeting mosquito genes at different developmental stages using different delivery methods. The review could help the researcher to find out novel genes of mosquitoes for vector control., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 ( Dcr-2 ) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.

Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti , the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 ( Kr-h1 ), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1 , which suppresses the expression of E93 , resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.

Aedes aegypti is the vector of important human diseases, and genomic resources are crucial in facilitating the study of A. aegypti and its ecosystem interactions. Several laboratory-acclimated strains of this mosquito have been established, but the most used strain in toxicology studies is "Rockefeller," which was originally collected and established in Cuba 130 years ago. A full-length genome assembly of another reference strain, "Liverpool," was published in 2018 and is the reference genome for the species (AaegL5). However, genetic studies with the Rockefeller strain are complicated by the availability of only the Liverpool strain as the reference genome. Differences between Liverpool and Rockefeller have been known for decades, particularly in the expression of genes relevant to mosquito behavior and vector control (e.g. olfactory). These differences indicate that AaegL5 is likely not fully representative of the Rockefeller genome, presenting potential impediments to research. Here, we present a chromosomal-level assembly and annotation of the Rockefeller genome and a comparative characterization vs the Liverpool genome. Our results set the stage for a pan-genomic approach to understanding evolution and diversity within this important disease vector., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

The yellow fever mosquito Aedes aegypti is an obligatory blood feeder and a major arboviral disease vector, evoking severe public health concerns worldwide. In adult female mosquitoes, the gut is critical for blood digestion and pathogen entry. We aimed for a systematic exploration of microRNA expression dynamics in the gut during the gonadotrophic cycle. Small RNA libraries were constructed from female mosquito gut tissues at five time points. Unsupervised hierarchical clustering revealed three expression clusters (early, mid and late) peaking at sequential time points - 24, 48 and 72 h posteclosion. Differentially expressed miRNAs were identified at 24 h post-blood meal (PBM). Depletions of Methoprene-tolerant [Met; the juvenile hormone (JH) receptor] and Ecdysone receptor [EcR; the receptor to 20-hydroxyecdysone (20E)] were performed using dsRNA to these genes to investigate impacts on microRNA expressions. Our results suggest that Met-mediated signalling downregulates miRNA expression from the early cluster and upregulates that from the late cluster. EcR signalling either up- or downregulated miRNA levels at 24 h PBM, indicating a differential effect of this receptor in miRNA gene expression. Furthermore, miR-281, which is the most abundant miRNA in the gut tissue, is induced and repressed by Met- and EcR-mediated signalling, respectively. Systematic depletion using synthetic antagomir and phenotype examinations indicate that miR-281 is obligatory for the normal progression of blood digestion, ovarian development and reproduction. Collectively, this study unveils expression dynamics of microRNAs in the female gut tissue during the gonadotrophic cycle and demonstrates that they are affected by JH and 20E signalling.

The 2021 re-emergence of yellow fever in non-human primates in the state of Rio Grande do Sul (RS), southernmost Brazil, resulted in the death of many howler monkeys (genus Alouatta ) and led the state to declare a Public Health Emergency of State Importance, despite no human cases reported. In this study, near-complete genomes of yellow fever virus (YFV) recovered from the outbreak were sequenced and examined aiming at a better understanding of the phylogenetic relationships and the spatio-temporal dynamics of the virus distribution. Our results suggest that the most likely sequence of events involved the reintroduction of YFV from the state of São Paulo to RS through the states of Paraná and Santa Catarina, by the end of 2020. These findings reinforce the role of genomic surveillance in determining the pathways of distribution of the virus and in providing references for the implementation of preventive measures for populations in high risk areas.

Aedes aegypti is the primary vector of dengue, Zika, yellow fever and chikungunya viruses to humans. In Africa, two subspecies, Ae. aegypti aegypti (Aaa) and Ae. aegypti formosus (Aaf) have been described. Until very recently, it was considered that the two forms were sympatric in East Africa and that only Aaf was present in Central and West Africa. However, recent data suggests that Aaa was also common in Senegal without any clear evidence of genetic differences with Aaf. This study was carried out in different Ae. aegypti populations from Senegal to better clarify their taxonomic status. The larvae, pupae and eggs were collected between July and September 2018 and reared individually to adult stage. For each population, F1 progeny from eggs laid by a single female F0 were reared as sibling samples. The number of pale scales on the first abdominal tergite (T1) and the basal part of the second tergite (T2) were counted. Individuals with no pale scale on T1 were classified as Aaf while those with at least one pale scale on this tergite were classified as Aaa. The morphological variations within families of Aaf were studied across 4 generations. In total, 2400 individuals constituting 240 families were identified, of which 42.5% were heterogeneous (families with both forms). Multivariate statistical analysis of variance including T1 and T2 data together showed that populations were significantly different from each other. Statistical analysis of T1 alone showed a similarity between populations from the southeast while variations were observed within northwest population. The analysis of family composition across generations showed the presence of Aaa and Aaf forms in each generation. The classification of Ae. aegypti into two subspecies is invalid in Senegal. Populations exhibit morphological polymorphism at the intra-family level that could have biological and epidemiological impacts., Competing Interests: The authors have declared that no competing interests exist.

Among primates, susceptibility to yellow fever (YFV), a single-stranded (ss) RNA virus, ranges from complete resistance to high susceptibility. Howler monkeys (genus Alouatta) are the most susceptible to YFV. In order to identify Alouatta-specific genetic factors that may be responsible for their susceptibility, we collected skin samples from howler monkey museum specimens of the species A. caraya and A. guariba clamitans. We compared the rate of nonsynonymous to synonymous (dN/dS) changes of Toll-like receptor (TLR) 7 and TLR8, the two genes responsible for detecting all ssRNA viruses, across the Primate order. Overall, we found that the TLR7 gene is under stronger purifying selection in howler monkeys compared to other New World and Old World primates, but TLR8 is under the same selective pressure. When we evaluated dN/dS at each codon, we found six codons under positive selection in Alouatta TLR8 and two codons under positive selection in TLR7. The changes in TLR7 are unique to A. guariba clamitans and are found in functionally important regions likely to affect detection of ssRNA viruses by TLR7/TLR8, as well as downstream signaling. These amino acid differences in A. guariba clamitans may play a role in YFV susceptibility. These results have implications for identifying genetic factors affecting YFV susceptibility in primates., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aedes aegypti is among the best-studied mosquitoes due to its critical role as a vector of human pathogens and ease of laboratory rearing. Until now, this species was thought to have originated in continental Africa, and subsequently colonized much of the world following the establishment of global trade routes. However, populations of this mosquito on the islands in the southwestern Indian Ocean (SWIO), where the species occurs with its nearest relatives referred to as the Aegypti Group, have received little study. We re-evaluated the evolutionary history of Ae. aegypti and these relatives, using three data sets: nucleotide sequence data, 18,489 SNPs and 12 microsatellites. We found that: (a) the Aegypti Group diverged 16 MYA (95% HPD: 7-28 MYA) from its nearest African/Asian ancestor; (b) SWIO populations of Ae. aegypti are basal to continental African populations; (c) after diverging 7 MYA (95% HPD: 4-15 MYA) from its nearest formally described relative (Ae. mascarensis), Ae. aegypti moved to continental Africa less than 85,000 years ago, where it recently (<1,000 years ago) split into two recognized subspecies Ae. aegypti formosus and a human commensal, Ae. aegypti aegypti; (d) the Madagascar samples form a clade more distant from all other Ae. aegypti than the named species Ae. mascarensis, implying that Madagascar may harbour a new cryptic species; and (e) there is evidence of introgression between Ae. mascarensis and Ae. aegypti on Réunion, and between the two subspecies elsewhere in the SWIO, a likely consequence of recent introductions of domestic Ae. aegypti aegypti from Asia., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Introduction: The only currently available live vaccine against yellow fever (YF) based on chicken embryos infected with an attenuated 17D strain of the YF virus is one of the most effective vaccine preparations. However, the live vaccine is associated with "viscerotropic syndrome" (approximately 0.4 cases per 100 000 vaccinated). Therefore, the development and introduction of highly purified inactivated vaccine against YF is intended to ensure the maximum safety of vaccination against one of the most common human viral diseases.Goals and objectives. Development and evaluation of immunogenicity of the cultural inactivated vaccine against YF at the laboratory model level., Material and Methods: Adaptation of 17D strain of YF virus to Vero cell culture, cultivation, removal of cellular DNA, inactivation with β-propiolactone, concentration, chromatographic purification, determination of protein and antigen of YF virus, assessment of immunogenicity in mice in parallel with commercial live vaccine., Results and Discussion: Immunogenicity: the determination of specific antibodies of class G (IgG) and virus neutralizing antibodies in the sera of immunized mice showed high level of antibodies exceeding that of immunized with commercial live vaccine. The optimal dose of antigen in the vaccine (total protein) was 50 μg/ml (5 μg/0.1 ml -dose and volume per 1 vaccination of mice). Thus, the laboratory version of cultural inactivated vaccine against YF is as effective (and even superior) as the commercial live vaccine., Conclusion: Laboratory version of cultural inactivated vaccine against YF, which is not inferior in immunogenicity (in animal model) to commercial live vaccine, has been developed.

Background: In 2007-2009, a major yellow fever virus (YFV) outbreak in Northern Argentina decimated the local howler monkey (Alouatta) population., Aims: To evaluate whether the surviving howler monkeys possess advantageous genetic variants inherited from monkeys alive prior to the YFV outbreak, we explored the relationship between Toll-like receptor (TLR) 7 and TLR8 gene variation and YFV susceptibility., Methods: We used samples from Alouatta individuals in Misiones, Argentina alive before the YFV outbreak, individuals that died during the outbreak, and individuals that survived the outbreak and are alive today. We measured genetic divergence between Alouatta YFV exposure groups and evaluated Alouatta-specific substitutions for functional consequences., Results: We did not find different allele frequencies in the post-YFV exposure Alouatta group compared to the pre-exposure group. We identified three nonsynonymous variants in TLR7 in Alouatta guariba clamitans. Two of these substitutions are under positive selection in functionally important regions of the gene., Discussion and Conclusions: Our results did not indicate that surviving howler monkey spossess advantageous genetic variants at greater frequency than those alive before the YFV outbreak. However, the positively selected unique coding differences in A. guariba clamitans are in the region important in pathogen detection which may affect YFV resistance. Morework is necessary to fully explore this hypothesis., (© 2020 Wiley Periodicals, Inc.)

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-β and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8 + T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27 - CD28 - CD8 + T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27 - CD28 - CD8 + T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27 - CD28 - CD8 + T cells to acquire a broad-spectrum, innate-like killing activity.

Fifty patients with unexplained fever and poor outcomes presented at Irrua Specialist Teaching Hospital (ISTH) in Edo State, Nigeria, an area endemic for Lassa fever, between September 2018 - January 2019. After ruling out Lassa fever, plasma samples from these epidemiologically-linked cases were sent to the African Centre of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer's University, Ede, Osun State, Nigeria, where we carried out metagenomic sequencing which implicated yellow fever virus (YFV) as the etiology of this outbreak. Twenty-nine of the 50 samples were confirmed positive for YFV by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), 14 of which resulted in genome assembly. Maximum likelihood phylogenetic analysis revealed that these YFV sequences formed a tightly clustered clade more closely related to sequences from Senegal than sequences from earlier Nigerian isolates, suggesting that the YFV clade responsible for this outbreak in Edo State does not descend directly from the Nigerian YFV outbreaks of the last century, but instead reflects a broader diversity and dynamics of YFV in West Africa. Here we demonstrate the power of metagenomic sequencing for identifying ongoing outbreaks and their etiologies and informing real-time public health responses, resulting in accurate and prompt disease management and control.

Z ika virus (ZIKV) was first discovered in 1947 in Africa. Since then, sporadic ZIKV infections of humans have been reported in Africa and Asia. For a long time, this virus was mostly unnoticed due to its mild symptoms and low fatality rates. However, during the 2015-2016 epidemic in Central and South America, when millions of people were infected, it was discovered that ZIKV causes microcephaly in the babies of mothers infected during pregnancy. An examination of the M and C proteins of the ZIKV shell using the disorder predictor PONDR VLXT revealed that the M protein contains relatively high disorder levels comparable only to those of the yellow fever virus (YFV). On the other hand, the disorder levels in the C protein are relatively low, which can account for the low case fatality rate (CFR) of this virus in contrast to the more virulent YFV, which is characterized by high disorder in its C protein. A larger variation was found in the percentage of intrinsic disorder (PID) in the C protein of various ZIKV strains. Strains of African lineage are characterized by higher PIDs. Using both in vivo and in vitro experiments, laboratories have also previously shown that strains of African origin have a greater potential to inflict higher fetal morbidity than do strains of Asian lineage, with dengue-2 virus (DENV-2) having the least potential. Strong correlations were found between the potential to inflict fetal morbidity and shell disorder in ZIKV ( r 2 = 0.9) and DENV-2 (DENV-2 + ZIKV, r 2 = 0.8). A strong correlation between CFR and PID was also observed when ZIKV was included in an analysis of sets of shell proteins from a variety of flaviviruses ( r 2 = 0.8). These observations have potential implications for antiviral vaccine development and for the design of cancer therapeutics in terms of developing therapeutic viruses that penetrate hard-to-reach organs., Competing Interests: G.K.-M.G. is an independent researcher and the owner of Goh’s BioComputing, Singapore. G.K.-M.G. has also written a book (“Viral Shapeshifters: Strange Behaviors of HIV and Other Viruses”) on a related subject. The authors have no other potential conflict of interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Deep sequencing of live-attenuated viral vaccines has focused on vaccines in current use. Here we report characterization of a discontinued live yellow fever (YF) vaccine associated with severe adverse events. The French neurotropic vaccine (FNV) strain of YF virus was derived empirically in 1930 by 260 passages of wild-type French viscerotropic virus (FVV) in mouse brain. The vaccine was administered extensively in French-speaking Africa until discontinuation in 1982, due to high rates of post-vaccination encephalitis in children. Using rare archive strains of FNV, viral RNAs were sequenced and analyzed by massively parallel, in silico methods. Diversity and specific population structures were compared in reference to the wild-type parental strain FVV, and between the vaccine strains themselves. Lower abundance of polymorphism content was observed for FNV strains relative to FVV. Although the vaccines were of lower diversity than FVV, heterogeneity between the vaccines was observed. Reversion to wild-type identity was variably observed in the FNV strains. Specific population structures were recovered from vaccines with neurotropic properties; loss of neurotropism in mice was associated with abundance of wild-type RNA populations. The analysis provides novel sequence evidence that FNV is genetically unstable, and that adaptation of FNV contributed to the neurotropic adverse phenotype.

The repertoire of human αβ T-cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8 + T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8 + T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8 + T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for the A2/LLW epitope., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature., Competing Interests: The authors declare no conflict of interest.

Efficient and persisting immune memory is essential for long-term protection from infectious and malignant diseases. The yellow fever (YF) vaccine is a live attenuated virus that mediates lifelong protection, with recent studies showing that the CD8(+) T cell response is particularly robust. Yet, limited data exist regarding the long-term CD8(+) T cell response, with no studies beyond 5 years after vaccination. We investigated 41 vaccinees, spanning 0.27 to 35 years after vaccination. YF-specific CD8(+) T cells were readily detected in almost all donors (38 of 41), with frequencies decreasing with time. As previously described, effector cells dominated the response early after vaccination. We detected a population of naïve-like YF-specific CD8(+) T cells that was stably maintained for more than 25 years and was capable of self-renewal ex vivo. In-depth analyses of markers and genome-wide mRNA profiling showed that naïve-like YF-specific CD8(+) T cells in vaccinees (i) were distinct from genuine naïve cells in unvaccinated donors, (ii) resembled the recently described stem cell-like memory subset (Tscm), and (iii) among all differentiated subsets, had profiles closest to naïve cells. Our findings reveal that CD8(+) Tscm are efficiently induced by a vaccine in humans, persist for decades, and preserve a naïveness-like profile. These data support YF vaccination as an optimal mechanistic model for the study of long-lasting memory CD8(+) T cells in humans., (Copyright © 2015, American Association for the Advancement of Science.)

In vivo targeted gene disruption is a powerful tool to study gene function. Thus far, two tools for genome editing in Aedes aegypti have been applied, zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN). As a promising alternative to ZFN and TALEN, which are difficult to produce and validate using standard molecular biological techniques, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-it-yourself" genome editing tool. Here, we describe the use of CRISPR/Cas9 in the mosquito vector, Aedes aegypti. In a transgenic mosquito line expressing both Dsred and enhanced cyan fluorescent protein (ECFP) from the eye tissue-specific 3xP3 promoter in separated but tightly linked expression cassettes, we targeted the ECFP nucleotide sequence for disruption. When supplying the Cas9 enzyme and two sgRNAs targeting different regions of the ECFP gene as in vitro transcribed mRNAs for germline transformation, we recovered four different G1 pools (5.5% knockout efficiency) where individuals still expressed DsRed but no longer ECFP. PCR amplification, cloning, and sequencing of PCR amplicons revealed indels in the ECFP target gene ranging from 2-27 nucleotides. These results show for the first time that CRISPR/Cas9 mediated gene editing is achievable in Ae. aegypti, paving the way for further functional genomics related studies in this mosquito species.

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Exploiting the inhibition of host signaling pathways aiming for discovery of potential antiflaviviral compounds is clearly a beneficial strategy for the control of life-threatening diseases caused by flaviviruses. Here we describe the antiviral activity of the MEK1/2 inhibitor U0126 against Yellow fever virus 17D vaccine strain (YFV-17D). Infection of VERO cells with YFV-17D stimulates ERK1/2 phosphorylation early during infection. Pharmacological inhibition of MEK1/2 through U0126 treatment of VERO cells blockades not only the YFV-stimulated ERK1/2 phosphorylation, but also inhibits YFV replication by ∼99%. U0126 was also effective against dengue virus (DENV-2 and -3) and Saint-Louis encephalitis virus (SLEV). Levels of NS4AB, as detected by immunofluorescence, are diminished upon treatment with the inhibitor, as well as the characteristic endoplasmic reticulum membrane invagination stimulated during the infection. Though not protective, treatment of YFV-infected, adult BALB/c mice with U0126 resulted in significant reduction of virus titers in brains. Collectively, our data suggest the potential targeting of the MEK1/2 kinase as a therapeutic tool against diseases caused by flaviviruses such as yellow fever, adverse events associated with yellow fever vaccination and dengue., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: We calculated the incidence, mortality, and case fatality rates for Caucasians and non-Caucasians during 19th century yellow fever (YF) epidemics in the United States and determined statistical significance for differences in the rates in different populations. We evaluated nongenetic host factors, including socioeconomic, environmental, cultural, demographic, and acquired immunity status that could have influenced these differences. While differences in incidence rates were not significant between Caucasians and non-Caucasians, differences in mortality and case fatality rates were statistically significant for all epidemics tested (P < 0.01). Caucasians diagnosed with YF were 6.8 times more likely to succumb than non-Caucasians with the disease. No other major causes of death during the 19th century demonstrated a similar mortality skew toward Caucasians. Nongenetic host factors were examined and could not explain these large differences. We propose that the remarkably lower case mortality rates for individuals of non-Caucasian ancestry is the result of human genetic variation in loci encoding innate immune mediators., Importance: Different degrees of severity of yellow fever have been observed across diverse populations, but this study is the first to demonstrate a statistically significant association between ancestry and the outcome of yellow fever (YF). With the global burden of mosquito-borne flaviviral infections, such as YF and dengue, on the rise, identifying and characterizing host factors could prove pivotal in the prevention of epidemics and the development of effective treatments., (Copyright © 2014 Blake and Garcia-Blanco.)

The live-attenuated yellow fever 17D virus is one of the most outstanding human vaccines ever developed. It induces efficacious immune responses at a low production cost with a well-established manufacture process. These advantages make the YF17D virus attractive as a vector for the development of new vaccines. At the beginning of vector development studies, YF17D was genetically manipulated to express other flavivirus prM and E proteins, components of the viral envelope. While these 17D recombinants are based on the substitution of equivalent YF17D genes, other antigens from unrelated pathogens have also been successfully expressed and delivered by recombinant YF17D viruses employing alternative strategies for genetic manipulation of the YF17D genome. Herein, we discuss these strategies in terms of possibilities of single epitope or larger sequence expression and the main properties of these replication-competent viral platforms.

Gene expression profiling of peripheral blood mononuclear cells (PBMCs) has revealed a crucial role for type I interferon (IFN) in the pathogenesis of systemic lupus erythematosus (SLE). However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4(+)) and monocyte subsets (CD16(-) inflammatory and CD16(+) resident monocytes) isolated from patients with SLE, healthy donors (ND) immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4(+) T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.

Arboviruses such as yellow fever virus (YFV) are transmitted between arthropod vectors and vertebrate hosts. While barriers limiting arbovirus population diversity have been observed in mosquitoes, whether barriers exist in vertebrate hosts is unclear. To investigate whether arboviruses encounter bottlenecks during dissemination in the vertebrate host, we infected immunocompetent mice and immune-deficient mice lacking alpha/beta interferon (IFN-α/β) receptors (IFNAR⁻/⁻ mice) with a pool of genetically marked viruses to evaluate dissemination and host barriers. We used the live attenuated vaccine strain YFV-17D, which contains many mutations compared with virulent YFV. We found that intramuscularly injected immunocompetent mice did not develop disease and that viral dissemination was restricted. Conversely, 32% of intramuscularly injected IFNAR⁻/⁻ mice developed disease. By following the genetically marked viruses over time, we found broad dissemination in IFNAR⁻/⁻ mice followed by clearance. The patterns of viral dissemination were similar in mice that developed disease and mice that did not develop disease. Unlike our previous results with poliovirus, these results suggest that YFV-17D encounters no major barriers during dissemination within a vertebrate host in the absence of the type I IFN response.

The yellow fever virus (YFV), the first proven human-pathogenic virus, although isolated in 1927, is still a major public health problem, especially in West Africa where it causes outbreaks every year. Nevertheless, little is known about its genetic diversity and evolutionary dynamics, mainly due to a limited number of genomic sequences from wild virus isolates. In this study, we analyzed the phylogenetic relationships of 24 full-length genomes from YFV strains isolated between 1973 and 2005 in a sylvatic context of West Africa, including 14 isolates that had previously not been sequenced. By this, we confirmed genetic variability within one genotype by the identification of various YF lineages circulating in West Africa. Further analyses of the biological properties of these lineages revealed differential growth behavior in human liver and insect cells, correlating with the source of isolation and suggesting host adaptation. For one lineage, repeatedly isolated in a context of vertical transmission, specific characteristics in the growth behavior and unique mutations of the viral genome were observed and deserve further investigation to gain insight into mechanisms involved in YFV emergence and maintenance in nature.

Eukaryotic genomes contain numerous DNA transposons that move by a cut-and-paste mechanism. The majority of these elements are self-insufficient and dependent on their autonomous relatives to transpose. Miniature inverted repeat transposable elements (MITEs) are often the most numerous nonautonomous DNA elements in a higher eukaryotic genome. Little is known about the origin of these MITE families as few of them are accompanied by their direct ancestral elements in a genome. Analyses of MITEs in the yellow fever mosquito identified its youngest MITE family, designated as Gnome, that contains at least 116 identical copies. Genome-wide search for direct ancestral autonomous elements of Gnome revealed an elusive single copy Tc1/Mariner-like element, named as Ozma, that encodes a transposase with a DD37E triad motif. Strikingly, Ozma also gave rise to two additional MITE families, designated as Elf and Goblin. These three MITE families were derived at different times during evolution and bear internal sequences originated from different regions of Ozma. Upon close inspection of the sequence junctions, the internal deletions during the formation of these three MITE families always occurred between two microhomologous sites (6-8 bp). These results suggest that multiple MITE families may originate from a single ancestral autonomous element, and formation of MITEs can be mediated by sequence microhomology. Ozma and its related MITEs are exceptional candidates for the long sought-after endogenous active transposon tool in genetic control of mosquitoes.

Background: Mosquitoes are important pathogen vectors affecting human and other animals. Studies on genetic control of mosquito mediated disease transmission gained traction recently due to mosquito transgenesis technology. Active transposons are considered valuable tools to propagate pathogen resistance transgenes among mosquitoes, rendering the whole population recalcitrant to diseases. A major hurdle in this approach is the inefficient remobilization activity after the integration of heterologous transposon vectors bearing transgenes into chromosomes. Therefore, endogenous active transposons in mosquito genomes are highly desirable., Results: Starting with the transposable element database of the yellow fever mosquito Aedes aegypti genome, detailed analyses of the members of each TE family were performed to identify sequences with multiple identical copies, an indicator of their latest or current transposition activity. Among a dozen of potentially active TE families, two DNA elements (TF000728 and TF000742 in TEfam) are short and nonautonomous. Close inspection of the elements revealed that these two families were previously mis-categorized and, unlike other known TEs, insert specifically at dinucleotide "AT". These two families were therefore designated as ATon-I and ATon-II. ATon-I has a total copy number of 294, among which three elements have more than 10 identical copies (146, 61 and 17). ATon-II has a total copy number of 317, among which three elements have more than 10 identical copies (84, 15 and 12). Genome wide searches revealed additional 24 ATon families in A. aegypti genome with nearly 6500 copies in total. Transposon display analysis of ATon-1 family using different A. aegypti strains suggests that the elements are similarly abundant in the tested mosquito strains., Conclusion: ATons are novel mobile genetic elements bearing terminal inverted repeats and insert specifically at dinucleotide "AT". Five ATon families contain elements existing at more than 10 identical copies, suggesting very recent or current transposition activity. A total of 24 new TE families with nearly 6000 copies were identified in this study.

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes.

Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-alpha/beta) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-alpha/beta receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6-7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-alpha/beta response may be primate-specific such that infection of mice with a functional IFN-alpha/beta antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus.

Enantiomers differ only in the left or right handedness (chirality) of their orientations and exhibit identical chemical and physical properties. In chemical communication systems, enantiomers can be differentially active at the physiological and behavioral levels. Only recently were enantioselective odorant receptors demonstrated in mammals while their existence in insects has remained hypothetical. Using the two-microelectrode voltage clamp of Xenopus oocytes, we show that the yellow fever mosquito, Aedes aegypti, odorant receptor 8 (AaOR8) acts as a chiral selective receptor for the (R)-(-)-enantiomer of 1-octen-3-ol, which in the presence of other kairomones is an attractant used by blood-sucking insects to locate their hosts. In addition to steric constraints, chain length and degree of unsaturation play important roles in this recognition process. This is the first characterization of an enantioselective odorant receptor in insects and the results demonstrate that an OR alone, without helper proteins, can account for chiral specificity exhibited by olfactory sensory neurons (OSNs).

Candidate vaccine ChimeriVax viruses are attenuated, efficacious, safe, and highly unlikely to be transmitted by arthropod vectors. Nevertheless, concerns have been raised about the use of these vaccines because of the potential for recombination between vaccine and wild-type (WT) strains. To evaluate the vertebrate pathogenicity of such a worst-case recombinant, ChimeriVax-dengue (DEN) 4 virus was chimerized with the WT Asibi yellow fever virus. In this worst-case scenario, chimeric viruses remained fully attenuated in nonhuman primates. We therefore conclude that, even in the highly unlikely event of "virulent" backbone reversion, the safety of ChimeriVax-DEN vaccines would not be compromised.

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents.

Objective: Differential diagnosis of infections that cause similar diseases and may be active simultaneously in the same geographical areas is greatly needed. Dengue and yellow fever viruses (DENV and YFV) are transmitted by the same species of mosquito and both can cause haemorrhagic fever symptoms. These viruses are active mainly in regions where expensive and sophisticated technologies are not available. Our objective was to develop a simple, reliable and easy-to-perform method to detect and identify these viruses., Methods: We slightly modified a generic RT-PCR able to detect the mentioned viruses and other members of this genus: specific primers for each one of these viruses were designed and included in the nested reaction instead of one of the generic ones. The reaction was optimized and viruses are amplified giving rise to bands of different sizes distinguishable in agarose gels., Results: This test is able to detect and identify the four DENVs and YFV to a high level of sensitivity and specificity and can be used with clinical samples. This simple, reliable and easy-to-perform method able to detect and identify dengue 1-4 and YFV can be used in poor endemic countries.

Yellow fever (YF) virus is currently found in tropical Africa and South America, and is responsible for a febrile to severe illness characterized by organ failure and shock. The attenuated YF 17D strain, used in YF vaccine, was derived from the wild-type strain Asibi. Although studies have been done on genetic markers of YF virulence, differentiation of the two strains in terms of host-cell interaction during infection remains elusive. As YF wild-type strains are hepatotropic, we chose a hepatic cell line (HepG2) to study YF virus-host cell interaction. HepG2 cells rapidly produced high titres of infectious viral particles for 17D and Asibi YF strains. However, HepG2 cells were more susceptible to the attenuated 17D virus infection, and only this virus strain induced early apoptosis in these cells. Molecular markers specific for the 17D virus were identified by microarray analysis and confirmed by quantitative RT-PCR analysis. As early as 1h postinfection, three genes, (IEX-1, IRF-1, DEC-1) all implicated in apoptosis pathways, were upregulated. Later in infection (48 h) two other genes (HSP70-1A and 1B), expressed in cases of cellular stress, were highly upregulated in 17D-infected HepG2 cells. The early specific upregulation of these cellular genes in HepG2 cells may be considered markers of the 17D virus. This study on the YF attenuated strain gives a new approach to the analysis of the factors involved in virus attenuation.

Inherited resistance to flaviviruses in laboratory mice is a rare trait conferred by an autosomal dominant gene (Flvr). To provide information on genetic resistance to flaviviruses in wild mice, we analysed (i) wild M. m. domesticus trapped in Australia, and (ii) mice representing other species and subspecies in the genus Mus. Mice were screened for resistance relative to C3H/HeJ mice by intracerebral challenge with Murray Valley encephalitis virus or yellow fever virus, and breeding studies were undertaken to identify inherited resistance factors. Widespread flavivirus resistance was demonstrated in Australian M. m. domesticus. A single, autosomal dominant Flvr-like gene appeared to be primarily responsible, but there was some evidence for additional inherited resistance factors. Flavivirus resistance was also identified in other taxonomic groups, and a genetic basis for this resistance was demonstrated in M. m. musculus (Skive), M. spretus, and M. spicilegus. Interestingly, M. m. musculus (CZI-O) were more susceptible than C3H/HeJ mice. Our findings show that genetic resistance to flaviviruses is common in divergent taxonomic groups in the genus Mus, suggesting that the trait has an ancient evolutionary origin, but whether flavivirus resistance genes have an anti-viral role or serve some other function is unknown.

Acute- and convalescent-phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre-existing flavivirus antibodies were tested for cross-reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test. A fourfold or greater rise in N antibody titer in the convalescent-phase was considered significant. Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital, showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two cross-reactive JE sera revealed that IgM class antibody was specific for JE, while IgG class antibody was cross-reactive. Of three JE cases with pre-existing YF antibody obtained in Okinawa, Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross-reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test, in sequential flavivirus infection.

Studies of genetic resistance to flavivirus infection in laboratory mice have led to the development of a single model in which resistance is conferred by an autosomal dominant gene designated Flvr. Because of evidence suggesting that wild mice carry virus resistance genes which are not present in laboratory mice, we compared flavivirus resistance in the inbred strains CASA/Rk, CAST/Ei, and MOLD/Rk, which are derived directly from wild mice, and the congenic strains C3H/RV (Flvr/Flvr) and C3H/HeJ (Flvs/Flvs). Resistance to the Murray Valley encephalitis virus strain OR2 and the 17D vaccine strain of yellow fever virus was assessed by determining the lethality of intracerebral infection and by measuring virus replication in the brain. The resistance of the CASA/Rk and CAST/Ei strains resembled the resistance of C3H/RV mice, whereas the resistance of the MOLD/Rk strain was intermediate between those of C3H/RV and C3H/HeJ mice. Genetic analyses showed that resistance in both the CASA/Rk and MOLD/Rk strains is conferred by single autosomal dominant alleles at the Flv locus. Our data indicate that flavivirus resistance in the CASA/Rk strain is due to a gene which is similar or identical to Flvr, whereas resistance in the MOLD/Rk strain is due to a previously undescribed gene which we designate Flvmr to indicate minor resistance to flavivirus infection. Since genetic resistance to flaviviruses is rare in laboratory mice, the CASA/Rk and MOLD/Rk strains will be valuable for further investigation of this phenomenon.

Descendants of Dutch colonists, who emigrated to Surinam in the last century and survived epidemics of typhoid and yellow fever with a total mortality of about 60%, were tested for twenty-six polymorphisms. The gene frequencies were compared with those of a large Dutch control sample. An analysis of drift indicated that the variations in gene frequencies observed for C3, Gm, HLA-B, and GLO were unlikely to be due to drift. Therefore these data might indicate selection through genetic control of survival in these epidemics.

Arbovirus epidemics in a geographic region are believed to depend on the presence of susceptible or "competent" arthropod vectors. We demonstrate that an urban, Aedes aegypti-borne, epidemic of yellow fever occurred in 1987 although the mosquito vector was relatively resistant to infection and transmitted the virus inefficiently. Twenty-six percent of the experimental mosquitoes from the epidemic area that ingested yellow fever virus became infected and only 7% of these transmitted the virus. In contrast, 80% of an exotic susceptible strain of Ae. aegypti became infected and 43% were able to transmit. We also show that no other potential vectors were active during the epidemic and that the local Ae. aegypti were present in extremely large numbers. These results document, for the first time, that, in the presence of high population density an incompetent mosquito vector can initiate and maintain virus transmission resulting in an epidemic.