Resurgence of Ebola virus in 2021 in Guinea suggests a new paradigm for outbreaks.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak-between 14 February and 19 June 2021-near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization.

Ebola Virus Transmission Initiated by Relapse of Systemic Ebola Virus Disease.

During the 2018-2020 Ebola virus disease (EVD) outbreak in North Kivu province in the Democratic Republic of Congo, EVD was diagnosed in a patient who had received the recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) (Merck). His treatment included an Ebola virus (EBOV)-specific monoclonal antibody (mAb114), and he recovered within 14 days. However, 6 months later, he presented again with severe EVD-like illness and EBOV viremia, and he died. We initiated epidemiologic and genomic investigations that showed that the patient had had a relapse of acute EVD that led to a transmission chain resulting in 91 cases across six health zones over 4 months. (Funded by the Bill and Melinda Gates Foundation and others.).

Genomic epidemiology reveals multiple introductions of Zika virus into the United States.

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

Retrospective Genomic Characterization of a 2017 Dengue Virus Outbreak, Burkina Faso.

Knowledge of contemporary genetic composition of dengue virus (DENV) in Africa is lacking. By using next-generation sequencing of samples from the 2017 DENV outbreak in Burkina Faso, we isolated 29 DENV genomes (5 serotype 1, 16 serotype 2 [DENV-2], and 8 serotype 3). Phylogenetic analysis demonstrated the endemic nature of DENV-2 in Burkina Faso. We noted discordant diagnostic results, probably related to genetic divergence between these genomes and the Trioplex PCR. Forward and reverse1 primers had a single mismatch when mapped to the DENV-2 genomes, probably explaining the insensitivity of the molecular test. Although we observed considerable homogeneity between the Dengvaxia and TetraVax-DV-TV003 vaccine strains as well as B cell epitopes compared with these genomes, we noted unique divergence. Continual surveillance of dengue virus in Africa is needed to clarify the ongoing novel evolutionary dynamics of circulating virus populations and support the development of effective diagnostic, therapeutic, and preventive countermeasures.

Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus.

BACKGROUND: Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. METHODS: Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction-based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. RESULTS: The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. CONCLUSIONS: This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

Shedding of Marburg Virus in Naturally Infected Egyptian Rousette Bats, South Africa, 2017.

We detected Marburg virus RNA in rectal swab samples from Egyptian rousette bats in South Africa in 2017. This finding signifies that fecal contamination of natural bat habitats is a potential source of infection for humans. Identified genetic sequences are closely related to Ravn virus, implying wider distribution of Marburg virus in Africa.

Complete genomic sequences of Venezuelan equine encephalitis virus subtype IIID isolates from mosquitoes.

Venezuelan equine encephalitis virus (VEEV) is an important pathogen of medical and veterinary importance in the Americas. In this report, we present the complete genome sequences of five VEEV isolates obtained from pools of Culex (Melanoconion) gnomatos (4) or Culex (Melanoconion) pedroi (1) from Iquitos, Peru. Genetic and phylogenetic analyses showed that all five isolates grouped within the VEEV complex sister to VEEV IIIC and are members of subtype IIID. This is the first report of full-length genomic sequences of VEEV IIID.

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.

Ribavirin Had Demonstrable Effects on the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Population and Load in a Patient With CCHF Infection.

The use of ribavirin to treat Crimean-Congo hemorrhagic fever virus (CCHFV) infection has been controversial, based on uncertainties about its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case by deep-sequencing analysis of plasma samples obtained from a CCHFV-infected patient before, during, and after a 5-day regimen of ribavirin treatment. The CCHFV load dropped during ribavirin treatment, and subclonal diversity (transitions) and indels increased in viral genomes during treatment. Although the results are based on a single case, these data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo.

Isolation of Complete Equine Encephalitis Virus Genome from Human Swab Specimen, Peru.

While studying respiratory infections in Peru, we identified Venezuelan equine encephalitis virus (VEEV) in a nasopharyngeal swab, indicating that this alphavirus can be present in human respiratory secretions. Because VEEV may be infectious when aerosolized, our finding is relevant for the management of VEEV-infected patients and for VEEV transmission studies.

Marburg Virus Infection in Egyptian Rousette Bats, South Africa, 2013-20141.

We detected a high seroprevalence of Marburg virus (MARV) antibodies in fruit bats in South Africa; 19.1% of recaptured bats seroconverted. The MARV RNA isolated closely resembled the 1975 Ozolin strain. These findings indicate endemic MARV circulation in bats in South Africa and should inform policies on MARV disease risk reduction.

Genetic Characterization of Enterovirus A71 Circulating in Africa.

We analyzed whole-genome sequences of 8 enterovirus A71 isolates (EV-A71). We confirm the circulation of genogroup C and the new genogroup E in West Africa. Our analysis demonstrates wide geographic circulation and describes genetic exchanges between EV-A71 and autochthonous EV-A that might contribute to the emergence of pathogenic lineages.

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats.

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR-based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000-2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Molecular Evidence of Sexual Transmission of Ebola Virus.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.

High Infection Rates for Adult Macaques after Intravaginal or Intrarectal Inoculation with Zika Virus.

Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.

Isolation of a novel orthobunyavirus from bat flies (Eucampsipoda africana).

The Bunyaviridae family comprises viruses causing diseases of public and veterinary health importance, including viral haemorrhagic and arboviral fevers. We report the isolation, identification and genome characterization of a novel orthobunyavirus, named Wolkberg virus (WBV), from wingless bat fly ectoparasites (Eucampsipoda africana) of Egyptian fruit bats (Rousettus aegyptiacus) in South Africa. Complete genome sequence data of WBV suggests it is most closely related to two bat viruses (Mojuí dos Campos and Kaeng Khoi viruses) and an arbovirus (Nyando virus) previously shown to infect humans. WBV replicates to high titres in VeroE6 and C6-36 cells, characteristic of mosquito-borne arboviruses. These findings expand our knowledge of the diversity of orthobunyaviruses and their insect vector host range.

Microbial Community Functional Potential and Composition Are Shaped by Hydrologic Connectivity in Riverine Floodplain Soils.

Riverine floodplains are ecologically and economically valuable ecosystems that are heavily threatened by anthropogenic stressors. Microbial communities in floodplain soils mediate critical biogeochemical processes, yet we understand little about the relationship between these communities and variation in hydrologic connectivity related to land management or topography. Here, we present metagenomic evidence that differences among microbial communities in three floodplain soils correspond to a long-term gradient of hydrologic connectivity. Specifically, all strictly anaerobic taxa and metabolic pathways were positively associated with increased hydrologic connectivity and flooding frequency. In contrast, most aerobic taxa and all strictly aerobic pathways were negatively related to hydrologic connectivity and flooding frequency. Furthermore, the genetic potential to metabolize organic compounds tended to decrease as hydrologic connectivity increased, which may reflect either the observed concomitant decline of soil organic matter or the parallel increase in both anaerobic taxa and pathways. A decline in soil N, accompanied by an increased genetic potential for oligotrophic N acquisition subsystems, suggests that soil nutrients also shape microbial communities in these soils. We conclude that differences among floodplain soil microbial communities can be conceptualized along a gradient of hydrologic connectivity. Additionally, we show that these differences are likely due to connectivity-related variation in flooding frequency, soil organic matter, and soil N. Our findings are particularly relevant to the restoration and management of microbially mediated biogeochemical processes in riverine floodplain wetlands.