Dynamics of emergence and genetic diversity of dengue virus in Reunion Island from 2012 to 2022.

BACKGROUND: Dengue is a major public health concern in Reunion Island, marked by recurrent epidemics, including successive outbreaks of dengue virus serotypes 1 and 2 (DENV1 and DENV2) with over 70,000 cases confirmed since 2017. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used Oxford Nanopore NGS technology for sequencing virologically-confirmed samples and clinical isolates collected between 2012 and 2022 to investigate the molecular epidemiology and evolution of DENV in Reunion Island. Here, we generated and analyzed a total of 499 DENV1, 360 DENV2, and 18 DENV3 sequences. By phylogenetic analysis, we show that different genotypes and variants of DENV have circulated in the past decade that likely originated from Seychelles, Mayotte and Southeast Asia and highly affected areas in Asia and Africa. CONCLUSIONS/SIGNIFICANCE: DENV sequences from Reunion Island exhibit a high genetic diversity which suggests regular introductions of new viral lineages from various Indian Ocean islands. The insights from our phylogenetic analysis may inform local health authorities about the endemicity of DENV variants circulating in Reunion Island and may improve dengue management and surveillance. This work emphasizes the importance of strong local coordination and collaboration to inform public health stakeholders in Reunion Island, neighboring areas, and mainland France.

Molecular epidemiology identifies the expansion of the DENV2 epidemic lineage from the French Caribbean Islands to French Guiana and mainland France, 2023 to 2024.

In 2023, dengue virus serotype 2 (DENV2) affected most French overseas territories. In the French Caribbean Islands, viral circulation continues with > 30,000 suspected infections by March 2024. Genome sequence analysis reveals that the epidemic lineage in the French Caribbean islands has also become established in French Guiana but not Réunion. It has moreover seeded autochthonous circulation events in mainland France. To guide prevention of further inter-territorial spread and DENV introduction in non-endemic settings, continued molecular surveillance and mosquito control are essential.

Generation and evaluation of protease inhibitor-resistant SARS-CoV-2 strains.

Since the start of the SARS-CoV-2 pandemic, the search for antiviral therapies has been at the forefront of medical research. To date, the 3CLpro inhibitor nirmatrelvir (Paxlovid®) has shown the best results in clinical trials and the greatest robustness against variants. A second SARS-CoV-2 protease inhibitor, ensitrelvir (Xocova®), has been developed. Ensitrelvir, currently in Phase 3, was approved in Japan under the emergency regulatory approval procedure in November 2022, and is available since March 31, 2023. One of the limitations for the use of antiviral monotherapies is the emergence of resistance mutations. Here, we experimentally generated mutants resistant to nirmatrelvir and ensitrelvir in vitro following repeating passages of SARS-CoV-2 in the presence of both antivirals. For both molecules, we demonstrated a loss of sensitivity for resistance mutants in vitro. Using a Syrian golden hamster infection model, we showed that the ensitrelvir M49L mutation, in the multi-passage strain, confers a high level of in vivo resistance. Finally, we identified a recent increase in the prevalence of M49L-carrying sequences, which appears to be associated with multiple repeated emergence events in Japan and may be related to the use of Xocova® in the country since November 2022. These results highlight the strategic importance of genetic monitoring of circulating SARS-CoV-2 strains to ensure that treatments administered retain their full effectiveness.

Sequence Data From a Travel-Associated Case of Microcephaly Highlight a Persisting Risk due to Zika Virus Circulation in Thailand.

Zika virus has been circulating in Thailand since 2002 through continuous but likely low-level circulation. Here, we describe an infection in a pregnant woman who traveled to Thailand and South America during her pregnancy. By combining phylogenetic analysis with the patient's travel history and her pregnancy timeline, we confirmed that she likely got infected in Thailand at the end of 2021. This imported case of microcephaly highlights that Zika virus circulation in the country still constitutes a health risk, even in a year of lower incidence. MAIN POINTS: Here we trace the origin of travel-acquired microcephaly to Thailand, providing additional evidence that pre-American lineages of Zika virus can harm the fetus and highlighting that Zika virus constitutes a health threat even in a year of lower incidence.

The Arenaviridae Family: Knowledge Gaps, Animal Models, Countermeasures, and Prototype Pathogens.

Lassa virus (LASV), Junin virus (JUNV), and several other members of the Arenaviridae family are capable of zoonotic transfer to humans and induction of severe viral hemorrhagic fevers. Despite the importance of arenaviruses as potential pandemic pathogens, numerous gaps exist in scientific knowledge pertaining to this diverse family, including gaps in understanding replication, immunosuppression, receptor usage, and elicitation of neutralizing antibody responses, that in turn complicates development of medical countermeasures. A further challenge to the development of medical countermeasures for arenaviruses is the requirement for use of animal models at high levels of biocontainment, where each model has distinct advantages and limitations depending on, availability of space, animals species-specific reagents, and most importantly the ability of the model to faithfully recapitulate human disease. Designation of LASV and JUNV as prototype pathogens can facilitate progress in addressing the public health challenges posed by members of this important virus family.

Novel Tools for Lassa Virus Surveillance in Peri-domestic Rodents.

Background: Lassa fever (LF) is a rodent-borne disease endemic to West Africa. In the absence of licensed therapeutics or vaccines, rodent exclusion from living spaces remains the primary method of preventing LF. Zoonotic surveillance of Lassa virus (LASV), the etiologic agent of LF, can assess the burden of LASV in a region and guide public health measures against LF. Methods: In this study, we adapted commercially available LASV human diagnostics to assess the prevalence of LASV in peri-domestic rodents in Eastern Sierra Leone. Small mammal trapping was conducted in Kenema district, Sierra Leone between November 2018-July 2019. LASV antigen was detected using a commercially available LASV NP antigen rapid diagnostic test. LASV IgG antibodies against LASV nucleoprotein (NP) and glycoprotein (GP) were tested by adapting a commercially available semi-quantitative enzyme linked immunosorbent assay (ELISA) for detection of mouse-related and rat-related species IgG. Findings: Of the 373 tested specimens, 74 (20%) tested positive for LASV antigen. 40 (11%) specimens tested positive for LASV NP IgG, while an additional 12 (3%) specimens only tested positive for LASV GP IgG. Simultaneous antigen presence and IgG antibody presence was linked in Mastomys sp. specimens (p < 0.01), but not Rattus sp. specimens (p = 1). Despite the link between antigen presence and IgG antibody presence in Mastomys sp., the strength of antigen response did not correlate with the strength of IgG response to either GP IgG or NP IgG. Interpretation: The tools developed in this study can aid in the generation of valuable public health data for rapid field assessment of LASV burden during outbreak investigations and general LASV surveillance. Funding: Funding for this work was supported by the National Institute of Allergy and Infectious Diseases National Institute of Health, Department of Health and Human Services under the following grants: International Collaboration in Infectious Disease Research on Lassa fever and Ebola - ICIDR - U19 AI115589, Consortium for Viral Systems Biology - CViSB - 5U19AI135995, West African Emerging Infectious Disease Research Center - WARN-ID - U01AI151812, West African Center for Emerging Infectious Diseases: U01AI151801.

Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineages.

RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced transmission during the epidemic.

Lassa Virus Genetics.

In a pattern repeated across a range of ecological niches, arenaviruses have evolved a compact four-gene genome to orchestrate a complex life cycle in a narrow range of susceptible hosts. A number of mammalian arenaviruses cross-infect humans, often causing a life-threatening viral hemorrhagic fever. Among this group of geographically bound zoonoses, Lassa virus has evolved a unique niche that leads to significant and sustained human morbidity and mortality. As a biosafety level 4 pathogen, direct study of the pathogenesis of Lassa virus is limited by the sparse availability, high operating costs, and technical restrictions of the high-level biocontainment laboratories required for safe experimentation. In this chapter, we introduce the relationship between genome structure and the life cycle of Lassa virus and outline reverse genetic approaches used to probe and describe functional elements of the Lassa virus genome. We then review the tools used to obtain viral genomic sequences used for phylogeny and molecular diagnostics, before shifting to a population perspective to assess the contributions of phylogenetic analysis in understanding the evolution and ecology of Lassa virus in West Africa. We finally consider the future outlook and clinical applications for genetic study of Lassa virus.

Predicting the evolution of the Lassa virus endemic area and population at risk over the next decades.

Lassa fever is a severe viral hemorrhagic fever caused by a zoonotic virus that repeatedly spills over to humans from its rodent reservoirs. It is currently not known how climate and land use changes could affect the endemic area of this virus, currently limited to parts of West Africa. By exploring the environmental data associated with virus occurrence using ecological niche modelling, we show how temperature, precipitation and the presence of pastures determine ecological suitability for virus circulation. Based on projections of climate, land use, and population changes, we find that regions in Central and East Africa will likely become suitable for Lassa virus over the next decades and estimate that the total population living in ecological conditions that are suitable for Lassa virus circulation may drastically increase by 2070. By analysing geotagged viral genomes using spatially-explicit phylogeography and simulating virus dispersal, we find that in the event of Lassa virus being introduced into a new suitable region, its spread might remain spatially limited over the first decades.

Emergence of an early SARS-CoV-2 epidemic in the United States.

The emergence of the COVID-19 epidemic in the United States (U.S.) went largely undetected due to inadequate testing. New Orleans experienced one of the earliest and fastest accelerating outbreaks, coinciding with Mardi Gras. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large-scale events accelerate transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana had limited diversity compared to other U.S. states and that one introduction of SARS-CoV-2 led to almost all of the early transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras, and the festival dramatically accelerated transmission. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate epidemics.

Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone.

Zika virus (ZIKV) has recently become dispersed throughout the tropics and sub-tropics, causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. In this study, we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared different constructs and confirmed the need to modify the cleavage site between the pre-peptide and prM protein. Genotypic characterization of the chimeras indicated that the emergence of compensatory mutations in the E protein was required to restore viral replicative fitness. Using an immunocompromised mouse model, we demonstrated that mice infected with the chimeric virus produced levels of neutralizing antibodies that were close to those observed following infection with ZIKV. Furthermore, pre-immunized mice were protected against viscerotropic and neuroinvasive disease following challenge with a heterologous ZIKV strain. These data provide a sound basis for the future development of this ZIKV vaccine candidate.

What Does the Future Hold for Yellow Fever Virus? (II).

As revealed by the recent resurgence of yellow fever virus (YFV) activity in the tropical regions of Africa and South America, YFV control measures need urgent rethinking. Over the last decade, most reported outbreaks occurred in, or eventually reached, areas with low vaccination coverage but that are suitable for virus transmission, with an unprecedented risk of expansion to densely populated territories in Africa, South America and Asia. As reflected in the World Health Organization's initiative launched in 2017, it is high time to strengthen epidemiological surveillance to monitor accurately viral dissemination, and redefine vaccination recommendation areas. Vector-control and immunisation measures need to be adapted and vaccine manufacturing must be reconciled with an increasing demand. We will have to face more yellow fever (YF) cases in the upcoming years. Hence, improving disease management through the development of efficient treatments will prove most beneficial. Undoubtedly, these developments will require in-depth descriptions of YFV biology at molecular, physiological and ecological levels. This second section of a two-part review describes the current state of knowledge and gaps regarding the molecular biology of YFV, along with an overview of the tools that can be used to manage the disease at the individual, local and global levels.

What Does the Future Hold for Yellow Fever Virus? (I).

The recent resurgence of yellow fever virus (YFV) activity in the tropical regions of Africa and South America has sparked renewed interest in this infamous arboviral disease. Yellow fever virus had been a human plague for centuries prior to the identification of its urban transmission vector, the Aedes (Stegomyia) aegypti (Linnaeus) mosquito species, and the development of an efficient live-attenuated vaccine, the YF-17D strain. The combination of vector-control measures and vaccination campaigns drastically reduced YFV incidence in humans on many occasions, but the virus never ceased to circulate in the forest, through its sylvatic invertebrate vector(s) and vertebrate host(s). Outbreaks recently reported in Central Africa (2015⁻2016) and Brazil (since late 2016), reached considerable proportions in terms of spatial distribution and total numbers of cases, with multiple exports, including to China. In turn, questions about the likeliness of occurrence of large urban YFV outbreaks in the Americas or of a successful import of YFV to Asia are currently resurfacing. This two-part review describes the current state of knowledge and gaps regarding the molecular biology and transmission dynamics of YFV, along with an overview of the tools that can be used to manage the disease at individual, local and global levels.

Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence.

Yellow fever virus (Flavivirus genus) is an arthropod-borne pathogen, which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow Fever Ap7, by introducing a set of four synonymous and six nonsynonymous mutations into a single subgenomic amplicon, derived from the sequence of the Asibi strain. The resulting strain, Yellow Fever Ap7M, induced a disease similar to that described for Ap7 in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from either Ap7M or Asibi viruses and investigated the role of each of Ap7M nonsynonymous mutations in its in vivo phenotype. This allowed identifying key components of the virulence mechanism in hamsters. In Ap7M virus, the reversion of either E/Q27H or E/D155A mutations led to an important reduction of both virulence and in vivo replicative fitness. In addition, the introduction of the single D155A Ap7M mutation within the E protein of the Asibi virus was sufficient to drastically modify its phenotype in hamsters toward both a greater replication efficiency and virulence. Finally, inspection of the Asibi strain E protein structure combined to in vivo testing revealed the importance of an exposed α-helix in domain I, containing residues 154 and 155, for Ap7M virulence in hamsters.

Molecular epidemiology of dengue viruses in three provinces of Lao PDR, 2006-2010.

Few data on dengue epidemiology are available for Lao PDR. Here, we provide information on the complexity of dengue epidemiology in the country, demonstrating dynamic circulation that varies over space and time, according to serotype. We recruited 1,912 consenting patients presenting with WHO dengue criteria at Mahosot Hospital, Vientiane (central Laos), between 2006 and 2010. Between 2008 and 2010, 1,413 patients with undifferentiated fever were also recruited at Luang Namtha (LNT) Provincial Hospital (northern Laos) and 555 at Salavan (SV) Provincial Hospital (southern Laos). We report significant variations in Dengue virus (DENV) circulation between the three sites. Peaks of DENV infection were observed in the rainy seasons, although 11% of confirmed cases in the provinces and 4.6% in the capital were detected during the dry and cool seasons (between December and February). Four DENV serotypes were detected among the 867 RT-PCR positive patients: 76.9% DENV-1, 9.6% DENV-2, 7.7% DENV-4 and 5.3% DENV-3. DENV-1 was the predominant serotype throughout the study except in LNT in 2008 and 2009 when it was DENV-2. Before July 2009, DENV-2 was not detected in SV and only rarely detected in Vientiane. DENV-3 and DENV-4 were commonly detected in Vientiane, before 2008 for DENV-4 and after 2009 for DENV-3. The phylogenetic analyses of DENV envelope sequences suggest concurrent multiple introductions of new strains as well as active DENV circulation throughout Laos and with neighboring countries. It is therefore of great importance to develop and strengthen a year-round nation-wide surveillance network in order to collect data that would allow anticipation of public health issues caused by the occurrence of large dengue outbreaks.

New reverse genetics and transfection methods to rescue arboviruses in mosquito cells.

Reverse genetics is a critical tool to decrypt the biological properties of arboviruses. However, whilst reverse genetics methods have been usually applied to vertebrate cells, their use in insect cells remains uncommon due to the conjunction of laborious molecular biology techniques and of specific difficulties surrounding the transfection of such cells. To leverage reverse genetics studies in both vertebrate and mosquito cells, we designed an improved DNA transfection protocol for insect cells and then demonstrated that the simple and flexible ISA (Infectious Subgenomic Amplicons) reverse-genetics method can be efficiently applied to both mammalian and mosquito cells to generate in days recombinant infectious positive-stranded RNA viruses belonging to genera Flavivirus (Japanese encephalitis, Yellow fever, West Nile and Zika viruses) and Alphavirus (Chikungunya virus). This method represents an effective option to potentially overcome technological issues related to the study of arboviruses.

G+C content differs in conserved and variable amino acid residues of flaviviruses and other evolutionary groups.

Flaviviruses are small RNA viruses that exhibit genetic and ecological diversity and a wide range of G+C content (GC%). We discovered that, amongst flaviviruses, the GC% of nucleotides encoding conserved amino acid (AA) residues was consistently higher than that of nucleotides encoding variable AAs. This intriguing phenomenon was also identified for a wide range of other viruses, and some non-viral evolutionary groups. Here, we analyse the possible mechanisms underlying this imbalanced nucleotide content (in particular the role of the specific G content and the AA composition in flaviviral genomes) and discuss its evolutionary implications. Our findings suggest that one of the most simple characteristics of the genetic code (i.e., the G or G+C content of codons) is linked with the evolutionary behavior of the corresponding encoded AAs.

Multiple Introductions of Dengue 2 Virus Strains into Saudi Arabia from 1992 to 2014.

INTRODUCTION: Dengue is a significant arboviral infection that represents a major public health concern worldwide. The infection is endemic in most parts of South East Asia, sub-Saharan Africa, and Latin America. Among the four dengue virus (DENV) serotypes, DENV-2 has been reported to be the predominant serotype in Saudi Arabia since 1992. However, virological and epidemiological data of DENV-2 from Saudi Arabia are severely deficient and require further investigations. METHODS: Full genome sequencing of a recent DENV-2 isolate and phylogenetic analysis of all available DENV-2 sequences from Saudi Arabia. RESULTS: Based on full genome and envelope (E) gene sequence, we show that a recent isolate (DENV-2-Jeddah-2014) belongs to the Indian subcontinent lineage of the Cosmopolitan genotype with close similarity to recent strains from Pakistan. Interestingly, the E gene sequence of DENV-2-Jeddah-2014 isolate was slightly divergent from those previously identified in Saudi Arabia between 1992 and 2004 with three to nine amino acid (aa) substitutions. While our data show that the Cosmopolitan genotype is still circulating in Saudi Arabia, they highlight four distinct genetic groups suggesting at least four independent introductions into the Kingdom. CONCLUSIONS: The close clustering of DENV-2 isolates reported from Saudi Arabia between 1992 and 2014 with strains from countries providing the highest numbers of pilgrims attending either Hajj or Umrah pilgrimages (Indonesia, Pakistan, India) clearly suggests a role for pilgrims or expatriates coming from DENV endemic countries in DENV-2 importation into Saudi Arabia. Accordingly, continuous monitoring of the circulation of DENVs in Saudi Arabia must be implemented to undertake effective control and management strategies in the Kingdom. Screening of the pilgrims coming to perform Hajj and Umrah might help prevent the introduction of new DENV strains, which is expected to increase the burden of the disease not only in Saudi Arabia but also in other countries.

A secondary dengue 4 infection in a traveler returning from Haiti confirmed by virus isolation, complete genome sequencing and neutralisation assay: a brief report.

Here we report the clinical and laboratory findings of a dengue 4 virus (DENV) secondary infection in a patient returning from Haiti to France. The diagnostic of acute DEN-4 virus infection was demonstrated by (i) the presence of DEN-4 RNA in two successive serum samples, (ii) the isolation of a DEN-4 virus in Vero cells and subsequent identification of subtype IIb through complete genome sequencing, (iii) the presence of dengue NS1 antigen, (iv) the seroconversion with detection of dengue IgM in the second serum while negative in the first serum. The diagnosis of secondary dengue episode was demonstrated by (i) the presence of dengue IgG in the early serum, and (ii) the demonstration that neutralising antibodies against DEN-3 were present at the acute stage of the disease. Next-generation sequencing has a primary role to play in phylogeographic studies including database sequences, sequences from imported cases, and sequences from autochthonous cases.