Vaccination reshapes the virus-specific T cell repertoire in unexposed adults.

We examined how baseline CD4+ T cell repertoire and precursor states impact responses to pathogen infection in humans using primary immunization with yellow fever virus (YFV) vaccine. YFV-specific T cells in unexposed individuals were identified by peptide-MHC tetramer staining and tracked pre- and post-vaccination by tetramers and TCR sequencing. A substantial number of YFV-reactive T cells expressed memory phenotype markers and contained expanded clones in the absence of exposure to YFV. After vaccination, pre-existing YFV-specific T cell populations with low clonal diversity underwent limited expansion, but rare populations with a reservoir of unexpanded TCRs generated robust responses. These altered dynamics reorganized the immunodominance hierarchy and resulted in an overall increase in higher avidity T cells. Thus, instead of further increasing the representation of dominant clones, YFV vaccination recruits rare and more responsive T cells. Our findings illustrate the impact of vaccines in prioritizing T cell responses and reveal repertoire reorganization as a key component of effective vaccination.

Molecular Determinants of Flavivirus Virion Assembly.

Virion assembly is an important step in the life cycle of all viruses. For viruses of the Flavivirus genus, a group of enveloped positive-sense RNA viruses, the assembly step represents one of the least understood processes in the viral life cycle. While assembly is primarily driven by the viral structural proteins, recent studies suggest that several nonstructural proteins also play key roles in coordinating the assembly and packaging of the viral genome. This review focuses on describing recent advances in our understanding of flavivirus virion assembly, including the intermolecular interactions between the viral structural (capsid) and nonstructural proteins (NS2A and NS2B-NS3), host factors, as well as features of the viral genomic RNA required for efficient flavivirus virion assembly.

Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

BACKGROUND: Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. METHODS: Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. RESULTS: Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. CONCLUSIONS: LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Zafirlukast, as a viral inactivator, potently inhibits infection of several flaviviruses, including Zika virus, dengue virus, and yellow fever virus.

Zika virus (ZIKV) is one of the mosquito-borne flaviviruses that exhibits a unique tropism to nervous systems and is associated with Guillain-Barre syndrome and congenital Zika syndrome (CZS). Dengue virus (DENV) and yellow fever virus (YFV), the other two mosquito-borne flaviviruses, have also been circulating for a long time and cause severe diseases, such as dengue hemorrhagic fever and yellow fever, respectively. However, there are no safe and effective antiviral drugs approved for the treatment of infections or coinfections of these flaviviruses. Here, we found that zafirlukast, a pregnancy-safe leukotriene receptor antagonist, exhibited potent antiviral activity against infections of ZIKV strains from different lineages in different cell lines, as well as against infections of DENV-2 and YFV 17D. Mechanistic studies demonstrated that zafirlukast directly and irreversibly inactivated these flaviviruses by disrupting the integrity of the virions, leading to the loss of viral infectivity, hence inhibiting the entry step of virus infection. Considering its efficacy against flaviviruses, its safety for pregnant women, and its neuroprotective effect, zafirlukast is a promising candidate for prophylaxis and treatment of infections or coinfections of ZIKV, DENV, and YFV, even in pregnant women.

Specialized cis-Acting RNA Elements Balance Genome Cyclization to Ensure Efficient Replication of Yellow Fever Virus.

Genome cyclization is essential for viral RNA (vRNA) replication of the vertebrate-infecting flaviviruses, and yet its regulatory mechanisms are not fully understood. Yellow fever virus (YFV) is a notorious pathogenic flavivirus. Here, we demonstrated that a group of cis-acting RNA elements in YFV balance genome cyclization to govern efficient vRNA replication. It was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) is conserved in the YFV clade and is important for efficient YFV propagation. By using two different replicon systems, we found that the function of the DCS-HP is determined primarily by its secondary structure and, to a lesser extent, by its base-pair composition. By combining in vitro RNA binding and chemical probing assays, we found that the DCS-HP orchestrates the balance of genome cyclization through two different mechanisms, as follows: the DCS-HP assists the correct folding of the 5' end in a linear vRNA to promote genome cyclization, and it also limits the overstabilization of the circular form through a potential crowding effect, which is influenced by the size and shape of the DCS-HP structure. We also provided evidence that an A-rich sequence downstream of the DCS-HP enhances vRNA replication and contributes to the regulation of genome cyclization. Interestingly, diversified regulatory mechanisms of genome cyclization, involving both the downstream of the 5'-cyclization sequence (CS) and the upstream of the 3'-CS elements, were identified among different subgroups of the mosquito-borne flaviviruses. In summary, our work highlighted how YFV precisely controls the balance of genome cyclization to ensure viral replication. IMPORTANCE Yellow fever virus (YFV), the prototype of the Flavivirus genus, can cause devastating yellow fever disease. Although it is preventable by vaccination, there are still tens of thousands of yellow fever cases per year, and no approved antiviral medicine is available. However, the understandings about the regulatory mechanisms of YFV replication are obscure. In this study, by a combination of bioinformatics, reverse genetics, and biochemical approaches, it was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) promotes efficient YFV replication by modulating the conformational balance of viral RNA. Interestingly, we found specialized combinations for the downstream of the 5'-cyclization sequence (CS) and upstream of the 3'-CS elements in different groups of the mosquito-borne flaviviruses. Moreover, possible evolutionary relationships among the various downstream of the 5'-CS elements were implied. This work highlighted the complexity of RNA-based regulatory mechanisms in the flaviviruses and will facilitate the design of RNA structure-targeted antiviral therapies.

Elongation of N6-benzyladenosine scaffold via Pd-catalyzed C-C bond formation leads to derivatives with antiflaviviral activity.

Decoration of nucleoside analogues with lipophilic groups often leads to compounds with improved antiviral activity. For example, N6-benzyladenosine derivatives containing elongated lipophilic substituents in the benzyl core efficiently inhibit reproduction of tick-borne encephalitis virus (TBEV), while N6-benzyladenosine itself potently inhibits reproduction of human enterovirus A71 (EV-A71). We have extended a series of N6-benzyladenosine analogues using effective synthetic methods of CC bond formation based on Pd-catalyzed cross-coupling reactions (Sonogashira and Suzuki) in order to study the influence of bulky lipophilic substituents in the N6 position of adenosine on the antiviral activity against flaviviruses, such as TBEV, yellow fever virus (YFV) and West Nile virus (WNV), as well as a panel of enteroviruses including EV-A71, Echovirus 30 (E30), and poliovirus type 2 (PV2). Reproduction of tested flaviviruses appeared to be inhibited by the micromolar concentrations of the compounds, while cytotoxicity in most cases was beyond the detection limit. Time-of-addition studies demonstrated that the hit compounds inhibited the stage of viral RNA synthesis, but not the stages of the viral entry or protein translation. As a result, several new promising antiflaviviral leads have been identified. On the other hand, none of the synthesized compounds inhibited enterovirus reproduction, indicating a possibility of involvement of flavivirus-specific pathways in their mechanism of action.

Circulation of yellow fever virus in Benin city, Edo state Nigeria.

Yellow fever (YF) is one of the most acute viral hemorrhagic diseases of the 18th and 19th centuries, which continues to cause severe morbidity and mortality in Africa. After 21 years of no reported cases of yellow fever in Nigeria, till 2017 where a case was confirmed in Kwara State, also in November 2018,WHO was informed of a cluster of suspected yellow fever cases and deaths in Edo state, Nigeria. The study was among all age group attending health centres in Benin City, Edo state. A total of 280 blood samples were collected from consented febrile patients and were screened for antibodies to Zika virus using rapid diagnostic test (RDT) kits. Blood samples positive to Zika virus (IgM/IgG RDT), were subjected to molecular characterization. Using the flavividae family primers, six (6) samples where confirmed positive by Hemi-nested reverse transcription PCR (hnRT-PCR) sequencing. Nucleotide sequence blast revealed the sequenceswere similar to Yellow fever virus strains. Phylogenetic analysis revealed that the yellow fever virus sequences are closely related to the African strains. Despite the safe and effective yellow fever vaccine, yellow fever virus is seen to be in circulation, hence the need for continues mass vaccination.

Enhanced production of yellow fever virus through tailored culture media optimization.

In the present study, an initial screening was conducted using 12 types of cell culture media, and four media with the best performance were selected for further study. The optimization of four media blend for YFV production was evaluated using an Augmented simplex centroid mixture design. Among all the different models that were investigated, the quadratic model was found to be the most appropriate model for exploring mixture design. It was found that M10 exhibited the greatest impact on YFV production, followed by M9, M4, and M1. The utilization of M1 and M4 media individually yielded higher compared to their blends with other media. The YFV titers were reduced when M1 media was combined with other media. The utilization of M9 and M10 media in combination resulted a higher viral yield compared to their respective concentrations. The optimal ratio for achieving a higher titer of YFV from primary CEFs was found to be approximately 38:62, with M9 and M10 being the most favorable media blend. The use of a media mixture led to a significant increase of virus titer up to 2.6 × 108 PFU/ml or 2 log titer yield, which is equivalent to 1.92 × 105 doses, without any changes to growth conditions or other process factors. This study concluded that the utilization of a mixture design could be efficiently employed to choose the optimal combination of media blends for enhanced viral production from cell culture.

Molecular Characterization of Circulating Yellow Fever Viruses from Outbreak in Ghana, 2021-2022.

Yellow fever virus, transmitted by infected Aedes spp. mosquitoes, causes an acute viral hemorrhagic disease. During October 2021-February 2022, a yellow fever outbreak in some communities in Ghana resulted in 70 confirmed cases with 35 deaths (case-fatality rate 50%). The outbreak started in a predominantly unvaccinated nomadic community in the Savannah region, from which 65% of the cases came. The molecular amplification methods we used for diagnosis produced full-length DNA sequences from 3 confirmed cases. Phylogenetic analysis characterized the 3 sequences within West Africa genotype II; strains shared a close homology with sequences from Cote d'Ivoire and Senegal. We deployed more sensitive advanced molecular diagnostic techniques, which enabled earlier detection, helped control spread, and improved case management. We urge increased efforts from health authorities to vaccinate vulnerable groups in difficult-to-access areas and to educate the population about potential risks for yellow fever infections.

Cell Strain-Derived Induced Pluripotent Stem Cells as an Isogenic Approach To Investigate Age-Related Host Response to Flaviviral Infection.

The expansion of the geographical footprint of dengue viruses (DENVs) and their mosquito vectors have affected more than half of the global population, including older adults who appear to show elevated risk of severe dengue. Despite this epidemiological trend, how aging contributes to increased dengue pathogenesis is poorly understood. A limitation has been the lack of useful in vitro experimental approaches; cell lines commonly used for infection studies are immortal and hence do not age. Cell strains such as WI-38 and MRC-5 with diploid genomes do age with in vitro passaging, but these cell strains were isolated decades ago and are now mostly highly passaged. Here, we show that reprogramming of cell strains with finite life span into induced pluripotent stem cells (iPSCs), followed by conversion back into terminally differentiated cells, can be an approach to derive genetically identical cells at different stages of aging. The iPSC-derived differentiated cells were susceptible to wild-type DENV infection and produced greater levels of type I interferon expression with increased passaging, despite similar levels of infection. In contrast, infection with the attenuated DENV-2 PDK53 and YF17D-204 strains showed reduced and increased levels of infection with increasing passages, respectively; the latter could be clinically pertinent, as YF17D-204 vaccination in older adults is associated with increased risk of severe adverse outcome. The differences in infection susceptibility and host response collectively suggest the potential of iPSC-derived cell strains as a genetically controlled approach to understanding how aging impacts viral pathogenesis. IMPORTANCE Aging has been a risk factor for poor clinical outcome in several infectious diseases, including dengue. However, age-dependent responses to dengue and other flaviviral infection or vaccination have remained incompletely understood due partly to lack of suitable laboratory tools. We thus developed an in vitro approach to examine age-related changes in host response to flaviviral infection. Notably, this approach uses cell strains with diploid rather than aneuploidic genomes, which are unstable. Conversion of these cells into iPSCs ensures sustainability of this resource, and reprogramming back into terminally differentiated cells would, even with a limited number of passages, produce cells at different stages of aging for infection studies. Our findings suggest that this in vitro system has the potential to serve as a genetically controlled approach to define the age-related response to flavivirus infection.

A Brief History of Human Challenge Studies (1900-2021) Emphasising the Virology, Regulatory and Ethical Requirements, Raison D'etre, Ethnography, Selection of Volunteers and Unit Design.

Venetian quarantine 400 years ago was an important public health measure. Since 1900 this has been refined to include "challenge" or deliberate infection with pathogens be they viruses, bacteria, or parasites. Our focus is virology and ranges from the early experiments in Cuba with Yellow Fever Virus to the most widespread pathogen of our current times, COVID-19. The latter has so far caused over four million deaths worldwide and 190 million cases of the disease. Quarantine and challenge were also used to investigate the Spanish Influenza of 1918 which caused over 100 million deaths. We consider here the merits of the approach, that is the speeding up of knowledge in a practical sense leading to the more rapid licensing of vaccines and antimicrobials. At the core of quarantine and challenge initiatives is the design of the unit to allow safe confinement of the pathogen and protection of the staff. Most important though is the safety of volunteers. We can see now, as in 1900, that members of our society are prepared and willing to engage in these experiments for the public good. Our ethnology study, where the investigator observed the experiment from within the quarantine, gave us the first indication of changing attitudes amongst volunteers whilst in quarantine. These quarantine experiments, referred to as challenge studies, human infection studies, or "controlled human infection models" involve thousands of clinical samples taken over two to three weeks and can provide a wealth of immunological and molecular data on the infection itself and could allow the discovery of new targets for vaccines and therapeutics. The Yellow Fever studies from 121 years ago gave the impetus for development of a successful vaccine still used today whilst also uncovering the nature of the Yellow Fever agent, namely that it was a virus. We outline how carefully these experiments are approached and the necessity to have high quality units with self-contained air-flow along with extensive personal protective equipment for nursing and medical staff. Most important is the employment of highly trained scientific, medical and nursing staff. We face a future of emerging pathogens driven by the increasing global population, deforestation, climate change, antibiotic resistance and increased global travel. These emerging pathogens may be pathogens we currently are not aware of or have not caused outbreaks historically but could also be mutated forms of known pathogens including viruses such as influenza (H7N9, H5N1 etc.) and coronaviruses. This calls for challenge studies to be part of future pandemic preparedness as an additional tool to assist with the rapid development of broad-spectrum antimicrobials, immunomodulators and new vaccines.

Molecular surveillance of arboviruses in Nigeria.

Arboviral infections are fast becoming a global public health concern as a result of its high fatality rate and sporadic spread. From the outbreak of Zika virus in the Americas, the endemicity of Yellow fever in West Africa and South America, outbreaks of West Nile virus in South Africa to the year-round and national risk of Dengue fever in Mainland China and India. The war against emerging and re-emerging viral infection could probably lead to the next pandemic. To be above the pending possible arboviral pandemic, consistent surveillance of these pathogens is necessary in every society. This study was aimed at conducting a surveillance for Yellow fever virus, Zika virus, Chikungunya virus, Dengue virus and Rift Valley fever virus in four states in Nigeria using molecular techniques. A cross-sectional study involving 1600 blood samples collected from febrile patients in Lagos, Kwara, Ondo and Delta States between 2018 and 2021 was conducted using Real time polymerase chain reaction for detection of the pathogens. Extraction and purification of viral RNA were done using Qiagen Viral RNA Mini Kit. Samples were analyzed using One Step PrimeScript III RT-PCR mix (Takara Bio) alongside optimized primers and probes designed in-house. Positive samples were sequenced on MinION platform (Nanopore technologies). Bioinformatic and phylogenetic analysis were performed with DNASTAR Lasergene 17.3. All the RNA extracted from samples collected from the four states were negative for ZIKV RNA, RVFV RNA, CHIKV RNA and DENV RNA. However, twelve of the samples (2%) tested positive for YFV RNA. Three full genomes of sizes 10,751 bp, 10,500 bp and 10,715 bp were generated and deposited in GenBank with accession numbers: ON323052, ON323053 and ON323054 respectively. Phylogenetic analysis shows clustering within lineage 3 of West African genotype. This result shows an active spread of Yellow fever in Delta State, Nigeria. However, there is no emergence of a new genotype There is a need for an intense surveillance of Yellow fever virus in Nigeria to avert a major outbreak.

Consumptive coagulopathy of severe yellow fever occurs independently of hepatocellular tropism and massive hepatic injury.

Yellow fever (YF) is a mosquito-transmitted viral disease that causes tens of thousands of deaths each year despite the long-standing deployment of an effective vaccine. In its most severe form, YF manifests as a hemorrhagic fever that causes severe damage to visceral organs. Although coagulopathy is a defining feature of severe YF in humans, the mechanism by which it develops remains uncertain. Hepatocytes are a major target of yellow fever virus (YFV) infection, and the coagulopathy in severe YF has long been attributed to massive hepatocyte infection and destruction that results in a defect in clotting factor synthesis. However, when we analyzed blood from Brazilian patients with severe YF, we found high concentrations of plasma D-dimer, a fibrin split product, suggestive of a concurrent consumptive process. To define the relationship between coagulopathy and hepatocellular tropism, we compared infection and disease in Fah-/-, Rag2-/-, and Il2rɣ-/- mice engrafted with human hepatocytes (hFRG mice) and rhesus macaques using a highly pathogenic African YFV strain. YFV infection of macaques and hFRG mice caused substantial hepatocyte infection, liver damage, and coagulopathy as defined by virological, clinical, and pathological criteria. However, only macaques developed a consumptive coagulopathy whereas YFV-infected hFRG mice did not. Thus, infection of cell types other than hepatocytes likely contributes to the consumptive coagulopathy associated with severe YF in primates and humans. These findings expand our understanding of viral hemorrhagic disease and associated coagulopathy and suggest directions for clinical management of severe YF cases.

Longitudinal dynamics of the human B cell response to the yellow fever 17D vaccine.

A comprehensive understanding of the development and evolution of human B cell responses induced by pathogen exposure will facilitate the design of next-generation vaccines. Here, we utilized a high-throughput single B cell cloning technology to longitudinally track the human B cell response to the yellow fever virus 17D (YFV-17D) vaccine. The early memory B cell (MBC) response was mediated by both classical immunoglobulin M (IgM) (IgM+CD27+) and switched immunoglobulin (swIg+) MBC populations; however, classical IgM MBCs waned rapidly, whereas swIg+ and atypical IgM+ and IgD+ MBCs were stable over time. Affinity maturation continued for 6 to 9 mo following vaccination, providing evidence for the persistence of germinal center activity long after the period of active viral replication in peripheral blood. Finally, a substantial fraction of the neutralizing antibody response was mediated by public clones that recognize a fusion loop-proximal antigenic site within domain II of the viral envelope glycoprotein. Overall, our findings provide a framework for understanding the dynamics and complexity of human B cell responses elicited by infection and vaccination.

Tetrahydroquinazoline N-oxide derivatives inhibit reproduction of tick-borne and mosquito-borne flaviviruses.

Tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and West Nile virus (WNV) are flaviviruses causing emerging arthropod-borne infections of a great public health concern. Clinically approved drugs are not available to complement or replace the existing vaccines, which do not provide sufficient coverage. Thus, the discovery and characterization of new antiflaviviral chemotypes would advance studies in this field. In this study, a series of tetrahydroquinazoline N-oxides was synthesized, and the antiviral activity of the compounds was assessed against TBEV, YFV, and WNV using the plaque reduction assay along with the cytotoxicity to the corresponding cell lines (porcine embryo kidney and Vero). Most of the studied compounds were active against TBEV (EC50 2 to 33 µM) and WNV (EC50 0.15 to 34 µM) and a few also demonstrated inhibitory activity against YFV (EC50 0.18 to 41 µM). To investigate the potential mechanism of action of the synthesized compounds, time-of-addition (TOA) experiments and virus yield reduction assays were performed for TBEV. The TOA studies suggested that the antiviral activity of the compounds should affect the early stages of the viral replication cycle after cell entry. Compounds with tetrahydroquinazoline N-oxide scaffold show a broad spectrum of activity against flaviviruses and represent a promising chemotype for antiviral drug discovery.

3-[N,N-Bis(sulfonyl)amino]isoxazolines with Spiro-Annulated or 1,2-Annulated Cyclooctane Rings Inhibit Reproduction of Tick-Borne Encephalitis, Yellow Fever, and West Nile Viruses.

Spirocyclic compounds containing heterocyclic moieties represent promising 3D scaffolds for modern drug design. In the search for novel anti-flaviviral agents, we have obtained a series of 3-[N,N-bis(sulfonyl)amino]isoxazolines containing spiro-annulated cyclooctane rings and assessed their antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses. The structural analogs of spirocyclic compounds with a single sulfonyl group or 1,2-annulated cyclooctane ring were also investigated. Almost all the studied 3-[N,N-bis(sulfonyl)amino]isoxazolines revealed antiviral activity against TBEV and WNV. The most active against TBEV was spiro-isoxazoline derivative containing p-nitrophenyl groups in the sulfonyl part (EC50 2.0 ± 0.5 µM), while the highest potency against WNV was found for the compounds with lipophilic substituents in sulfonyl moiety, naphtyl being the most favorable one (EC50 1.3 ± 0.5 µM). In summary, two novel scaffolds of anti-flaviviral agents based on N,N-bis(sulfonyl)amino]isoxazoline were proposed, and the compounds of this type demonstrated activity against TBEV and WNV.

Yellow Fever Molecular Diagnosis Using Urine Specimens during Acute and Convalescent Phases of the Disease.

Prior studies have demonstrated prolonged presence of yellow fever virus (YFV) RNA in saliva and urine as an alternative to serum. To investigate the presence of YFV RNA in urine, we used RT-PCR for YFV screening in 60 urine samples collected from a large cohort of naturally infected yellow fever (YF) patients during acute and convalescent phases of YF infection from recent YF outbreaks in Brazil (2017 to 2018). Fifteen urine samples from acute phase infection (up to 15 days post-symptom onset) and four urine samples from convalescent phase infection (up to 69 days post-symptom onset), were YFV PCR-positive. We genotyped YFV detected in seven urine samples (five collected during the acute phase and two collected during the YF convalescent phase). Genotyping indicated the presence of YFV South American I genotype in these samples. To our knowledge, this is the first report of wild-type YFV RNA detection in the urine this far out from symptom onset (up to 69 DPS), including YFV RNA detection during the convalescent phase of YF infection. The detection of YFV RNA in urine is an indicative of YFV infection; however, the results of RT-PCR using urine as sample should be interpreted with care, since a negative result does not exclude the possibility of YFV infection. With a possible prolonged period of detection beyond the viremic phase, the use of urine samples coupled with serological tests, epidemiologic inquiry, and clinical assessment could provide a longer diagnostic window for laboratory YF diagnosis.

Bis(Benzofuran-1,3-N,N-heterocycle)s as Symmetric and Synthetic Drug Leads against Yellow Fever Virus.

The yellow fever virus (YFV) is an emerging RNA virus and has caused large outbreaks in Africa and Central and South America. The virus is often transmitted through infected mosquitoes and spreads from area to area because of international travel. Being an acute viral hemorrhagic disease, yellow fever can be prevented by an effective, safe, and reliable vaccine, but not be eliminated. Currently, there is no antiviral drug available for its cure. Thus, two series of novel bis(benzofuran−1,3-imidazolidin-4-one)s and bis(benzofuran−1,3-benzimidazole)s were designed and synthesized for the development of anti-YFV lead candidates. Among 23 new bis-conjugated compounds, 4 of them inhibited YFV strain 17D (Stamaril) on Huh-7 cells in the cytopathic effect reduction assays. These conjugates exhibited the most compelling efficacy and selectivity with an EC50 of <3.54 µM and SI of >15.3. The results are valuable for the development of novel antiviral drug leads against emerging diseases.

Brazilian Aedes aegypti as a Competent Vector for Multiple Complex Arboviral Coinfections.

BACKGROUND: Aedes aegypti is a highly competent vector in the transmission of arboviruses, such as chikungunya, dengue, Zika, and yellow fever viruses, and causes single and coinfections in the populations of tropical countries. METHODS: The infection rate, viral abundance (VA), vector competence (VC), disseminated infection, and survival rate were recorded after single and multiple infections of the vector with 15 combinations of chikungunya, dengue, Zika, and yellow fever arboviruses. RESULTS: Infection rates were 100% in all single and multiple infection experiments, except in 1 triple coinfection that presented a rate of 50%. The VC and disseminated infection rate varied from 100% (in single and quadruple infections) to 40% (in dual and triple infections). The dual and triple coinfections altered the VC and/or VA of ≥1 arbovirus. The highest viral VAs were detected for a single infection with chikungunya. The VAs in quadruple infections were similar when compared with each respective single infection. A decrease in survival rates was observed in a few combinations. CONCLUSIONS: A. aegypti was able to host all single and multiple arboviral coinfections. The interference of the chikungunya virus suggests that distinct arbovirus families may have a significant role in complex coinfections.

Differential Yellow Fever Susceptibility in New World Nonhuman Primates, Comparison with Humans, and Implications for Surveillance.

A major outbreak of yellow fever (YF) occurred in Brazil during 2016-2018. Epizootics in New World nonhuman primates are sentinel events for YF virus circulation. However, genus-specific susceptibilities and suitability for YF surveillance remain poorly understood. We obtained and compared epidemiologic, histopathologic, immunohistochemical, and molecular results from 93 human and 1,752 primate cases submitted during the recent YF outbreak in Brazil (2017), with the support of the Brazilian National YF Surveillance Program. We detected heterogeneous YF-associated profiles among the various genera of primates we analyzed. Alouatta primates were the most reliable sentinel; Sapajus and Callicebus primates had higher viral loads but lower proportional mortality rates. Callithrix primates were the least sensitive, showing lower viral loads, lower proportional mortality rates, and no demonstrable YF virus antigen or extensive lesions in liver, despite detectable viral RNA. These differences in susceptibility, viral load, and mortality rates should be considered in strategic surveillance of epizootics and control measures for YF.