A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA.

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.

Risk of arboviral transmission and insecticide resistance status of Aedes mosquitoes during a yellow fever outbreak in Ghana.

BACKGROUND: In late 2021, Ghana was hit by a Yellow Fever outbreak that started in two districts in the Savannah region and spread to several other Districts in three regions. Yellow fever is endemic in Ghana. However, there is currently no structured vector control programme for Aedes the arboviral vector in Ghana. Knowledge of Aedes bionomics and insecticide susceptibility status is important to control the vectors. This study therefore sought to determine Aedes vector bionomics and their insecticide resistance status during a yellow fever outbreak. METHODS: The study was performed in two yellow fever outbreak sites (Wenchi, Larabanga) and two non-outbreak sites (Kpalsogu, Pagaza) in Ghana. Immature Aedes mosquitoes were sampled from water-holding containers in and around human habitations. The risk of disease transmission was determined in each site using stegomyia indices. Adult Aedes mosquitoes were sampled using Biogents Sentinel (BG) traps, Human Landing Catch (HLC), and Prokopack (PPK) aspirators. Phenotypic resistance to permethrin, deltamethrin and pirimiphos-methyl was determined with WHO susceptibility tests using Aedes mosquitoes collected as larvae and reared into adults. Knockdown resistance (kdr) mutations were detected using allele-specific multiplex PCR. RESULTS: Among the 2,664 immature Aedes sampled, more than 60% were found in car tyres. Larabanga, an outbreak site, was classified as a high-risk zone for the Yellow Fever outbreak (BI: 84%, CI: 26.4%). Out of 1,507 adult Aedes mosquitoes collected, Aedes aegypti was the predominant vector species (92%). A significantly high abundance of Aedes mosquitoes was observed during the dry season (61.2%) and outdoors (60.6%) (P < 0.001). Moderate to high resistance to deltamethrin was observed in all sites (33.75% to 70%). Moderate resistance to pirimiphos-methyl (65%) was observed in Kpalsogu. Aedes mosquitoes from Larabanga were susceptible (98%) to permethrin. The F1534C kdr, V1016I kdr and V410 kdr alleles were present in all the sites with frequencies between (0.05-0.92). The outbreak sites had significantly higher allele frequencies of F1534C and V1016I respectively compared to non-outbreak sites (P < 0.001). CONCLUSION: This study indicates that Aedes mosquitoes in Ghana pose a significant risk to public health. Hence there is a need to continue monitoring these vectors to develop an effective control strategy.

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.

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.

Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 - 2018.

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species.

Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay.

Due to the concurrent prevalence and increasing risk of coinfection of the clinically important Arboviruses, timely and accurate differential diagnosis is important for clinical management and the epidemiological investigation. A two-tube multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection of Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was developed and optimized with high specificity and sensitivity. The detection limit for all the six viruses could reach as low as five genome equivalent copies and 2.8 × 10-3 tissue culture infectious doses (TCID50 ) for ZIKV, YFV, CHIKV and 2.8 × 10-2 TCID50  for JEV per reaction, with high accuracy and precision (R2 > 0.99). The coefficient of variation of intra-assay and inter-assay for our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was low, and the obtained positive rates ad Ct values of this assay were comparable with singleplex commercial kits. Moreover, the multiplex qRT-PCR assay was able to detect possible co-infections without competitive inhibition of target viral genomes. In conclusion, our rapid, sensitive, cost-effective multiplex qRT-PCR will be of great use for differential diagnosis in a clinical setting and epidemiological investigation during surveillance.

Fast surveillance response reveals the introduction of a new yellow fever virus sub-lineage in 2021, in Minas Gerais, Brazil.

BACKGROUND: In Brazil, the yellow fever virus (YFV) is maintained in a sylvatic cycle involving wild mosquitoes and non-human primates (NHPs). The virus is endemic to the Amazon region; however, waves of epidemic expansion reaching other Brazilian states sporadically occur, eventually causing spillovers to humans. OBJECTIVES: To report a surveillance effort that led to the first confirmation of YFV in NHPs in the state of Minas Gerais (MG), Southeast region, in 2021. METHODS: A surveillance network was created, encompassing the technology of smartphone applications and coordinated actions of several research institutions and health services to monitor and investigate NHP epizootics. FINDINGS: When alerts were spread through the network, samples from NHPs were collected and YFV infection confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and genome sequencing at an interval of only 10 days. Near-complete genomes were generated using the Nanopore MinION sequencer. Phylogenetic analysis indicated that viral genomes were related to the South American genotype I, clustering with a genome detected in the Amazon region (state of Pará) in 2017, named YFVPA/MG sub-lineage. Fast YFV confirmation potentialised vaccination campaigns. MAIN CONCLUSIONS: A new YFV introduction was detected in MG 6 years after the beginning of the major outbreak reported in the state (2015-2018). The YFV strain was not related to the sub-lineages previously reported in MG. No human cases have been reported, suggesting the importance of coordinated surveillance of NHPs using available technologies and supporting laboratories to ensure a quick response and implementation of contingency measures to avoid YFV spillover to humans.

Callitrichine gammaherpesvirus 3 and Human alphaherpesvirus 1 in New World Primate negative for yellow fever virus in Rio de Janeiro, Brazil.

BACKGROUND: Herpesvirus transmission between humans and non-human primate (NHP) can occur through contact scratches with lesions, infected saliva, and mainly through contaminated food. Therefore, cross-infection can lead to severe illness or even death for both the animal and human. In 2017, during the yellow fever (YF) outbreak in Brazil, species of the New World Primates (NWP) from Rio de Janeiro state, tested negative for yellow fever virus (YFV) detection. OBJECTIVES: To evaluate herpesvirus in the population NWP in Rio de Janeiro. METHODS: To investigate, liver samples of 283 NWP, from several regions of the state of Rio de Janeiro, were tested for the herpesvirus family using a Pan-polymerase chain reaction (Pan-PCR) and sequencing. FINDINGS: 34.6% (98/283) tested positive for at least one herpesvirus; 29.3% (83/283) tested positive to Human alphaherpesvirus 1 (HSV-1), this virus from humans can be lethal to New World monkey; 13% (37/283) were detected Callitrichine gammaherpesvirus 3 (CalHV-3), responsible for lymphoproliferative disease that can be fatal in NWP. In addition, CalHV-3 / HSV-1 co-infection was in 11.6% (33/283) of the samples. MAIN CONCLUSIONS: Pan-herpesvirus was useful to identify species-specific herpesviruses and virus from human that can infect animals. Furthermore, during an outbreak of YF other infections should be monitored.

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.

The host range restriction of bat-associated no-known-vector flaviviruses occurs post-entry.

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.

Circularization of flavivirus genomic RNA inhibits de novo translation initiation.

Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome.

Fatal Yellow Fever in a Kidney Transplant Patient.

A kidney-transplanted patient, unvaccinated against yellow fever (YF), developed high fever, progressed rapidly to hepatic insufficiency and coma, and died 8 days later. Real-time polymarase chain reaction for YF virus collected on the seventh day of symptoms was positive. Autopsy showed disseminated infection and midzonal hepatitis with apoptotic hepatocytes and minimal inflammatory reaction.

Yellow Fever Virus Reemergence and Spread in Southeast Brazil, 2016-2019.

The recent reemergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the rapid dissemination of the virus in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil, we generated 18 complete and nearly complete genomes from the peak of the epidemic curve from nonhuman primates (NHPs) and human infected cases across the Espírito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the estimated timing, source, and likely routes of yellow fever virus transmission and dispersion during one of the largest outbreaks ever registered in Brazil. We showed that during the recent epidemic, YFV was reintroduced from Minas Gerais to the Espírito Santo and Rio de Janeiro states multiple times between 2016 and 2019. The analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce the idea that continued genomic surveillance strategies can provide information on virus genetic diversity and transmission dynamics that might assist in understanding arbovirus epidemics.IMPORTANCE Arbovirus infections in Brazil, including yellow fever, dengue, zika, and chikungunya, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak by analyzing genomic data from areas in southeastern Brazil not covered by other previous studies. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from nonhuman primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control.

Concurrent amplification of Zika, chikungunya, and yellow fever virus in a sylvatic focus of arboviruses in Southeastern Senegal, 2015.

BACKGROUND: Chikungunya (CHIKV), yellow fever (YFV) and Zika (ZIKV) viruses circulate in sylvatic transmission cycles in southeastern Senegal, where they share common hosts and vectors. All three viruses undergo periodic amplifications, during which they are detected in mosquitoes and sometimes in hosts. However, little is known about their spatio-temporal patterns in years in which they undergo concurrent amplification. The aim of this study was to describe the co-amplification of ZIKV, CHIKV, and YFV, and the daily dynamics of these arboviruses and theirs vectors within villages in southeastern Senegal. RESULTS: Mosquitoes were collected monthly from July to December 2015. Each evening, from 6 to 9 PM, landing collections were performed by teams of 3 persons working simultaneously in 70 sites situated in forest (canopy and ground), savannah, agriculture, barren, and village (indoor and outdoor) land covers. Collections within villages were continued until 6 AM. Mosquitoes were tested for virus infection by virus isolation and RT-PCR. Seventy-five mosquito pools comprising 10 mosquito species contained at least one virus. Ae. furcifer and Ae. luteocephalus were infected by all three viruses, Ae. taylori by YFV and ZIKV, and remaining seven species by only, only YFV or only ZIKV. No single mosquito pool contained more than one virus. CHIKV was the only virus detected in all land cover classes and was found in the greatest number of sampling sites (32.9%, n = 70). The proportion of sites in which more than one virus was detected was less than 6%. Ae. aegypti formosus, Ae. furcifer, Ae. luteocephalus, Ae. minutus, Ae. vittatus, and An. gambiae were found within villages. These vectors were mainly active around dusk but Ae. furcifer was collected until dawn. All viruses save ZIKV were detected indoors and outdoors, mainly around dusk. Virus positive pools were detected over 2, 3 and 4 months for YFV, CHIKV and ZIKV, respectively. CONCLUSION: Our data indicate that the distribution of different vector species and different arboviruses vary substantially between sites, suggesting that CHIKV, YFV, and ZIKV may have different transmission cycles in Southeastern Senegal.

Seasonal population dynamics of the primary yellow fever vector Haemagogus leucocelaenus (Dyar & Shannon) (Diptera: Culicidae) is mainly influenced by temperature in the Atlantic Forest, southeast Brazil.

BACKGROUND: Southeast Brazil has recently experienced a Yellow Fever virus (YFV) outbreak where the mosquito Haemagogus leucocelaenus was a primary vector. Climatic factors influence the abundance of mosquito vectors and arbovirus transmission. OBJECTIVES: We aimed at describing the population dynamics of Hg. leucocelaenus in a county touched by the recent YFV outbreak. METHODS: Fortnightly egg collections with ovitraps were performed from November 2012 to February 2017 in a forest in Nova Iguaçu, Rio de Janeiro, Brazil. The effects of mean temperature and rainfall on the Hg. leucocelaenus population dynamics were explored. FINDINGS: Hg. leucocelaenus eggs were continuously collected throughout the study, with a peak in the warmer months (December-March). The climatic variables had a time-lagged effect and four weeks before sampling was the best predictor for the positivity of ovitraps and total number of eggs collected. The probability of finding > 50% positive ovitraps increased when the mean temperature was above 24ºC. The number of Hg. leucocelaenus eggs expressively increase when the mean temperature and accumulated precipitation surpassed 27ºC and 100 mm, respectively, although the effect of rainfall was less pronounced. MAIN CONCLUSIONS: Monitoring population dynamics of Hg. leucocelaenus and climatic factors in YFV risk areas, especially mean temperature, may assist in developing climate-based surveillance procedures to timely strengthening prophylaxis and control.

Wild-Type Yellow Fever Virus RNA in Cerebrospinal Fluid of Child.

We report a 3-year-old child who was hospitalized because of severe manifestations of the central nervous system. The child died after 6 days of hospitalization. Analysis of postmortem cerebrospinal fluid showed the presence of yellow fever virus RNA. Nucleotide sequencing confirmed that the virus was wild-type yellow fever virus.

Yellow fever virus modulates cytokine mRNA expression and induces activation of caspase 3/7 in the human hepatocarcinoma cell line HepG2.

Yellow fever virus (YFV) penetrates the skin through the bite of a vector mosquito and spreads to various organs, mainly the liver, where it causes lesions and induces necrosis and apoptosis. We evaluated the mRNA expression of various cytokines and the activation of caspases in HepG2 cells infected with YFV. We observed that interferon-α (IFN-α) expression decreased and IFN-ß, transforming growth factor (TGF)-ß IIIR, interleukin (IL)-6, and IL-8 expression increased in cells infected with genotype 1. In contrast, TNF-α expression increased in cells infected with genotype 2 but not with genotype 1. This provides insights into the role of cytokine regulation in yellow fever.

Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

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.

Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons.

BACKGROUND: In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES: To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS: We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS: Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS: Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Genomic sequence of yellow fever virus from a Dutch traveller returning from the Gambia-Senegal region, the Netherlands, November 2018.

In November 2018, yellow fever was diagnosed in a Dutch traveller returning from a bicycle tour in the Gambia-Senegal region. A complete genome sequence of yellow fever virus (YFV) from the case was generated and clustered phylogenetically with YFV from the Gambia and Senegal, ruling out importation into the Netherlands from recent outbreaks in Brazil or Angola. We emphasise the need for increased public awareness of YFV vaccination before travelling to endemic countries.