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Human transmission of Ebola vi

rus Philip Lawrence1,2, Nicolas Danet1, Olivier Reynard1, Valentina Volchkova1 and Viktor Volchkov1

Ever since the first recognised outbreak of Ebolavirus in 1976,

retrospective epidemiological analyses and extensive studies

with animal models have given us insight into the nature of the

pathology and transmission mechanisms of this virus. In this

review focusing on Ebolavirus, we present an outline of our

current understanding of filovirus human-to-human

transmission and of our knowledge concerning the molecular

basis of viral transmission and potential for adaptation, with

particular focus on what we have learnt from the 2014 outbreak

in West Africa. We identify knowledge gaps relating to

transmission and pathogenicity mechanisms, molecular

adaptation and filovirus ecology.

Addresses 1 Molecular Basis of Viral Pathogenicity, International Centre for

Research in Infectiology (CIRI), INSERM U1111 – CNRS UMR5308,

Université Lyon 1, Ecole Normale Supérieure de Lyon, Lyon 69007,

France 2 Université de Lyon, UMRS 449, Laboratoire de Biologie Générale,

Université Catholique de Lyon – EPHE, Lyon 69288, France

Corresponding author: Volchkov, Viktor ([email protected])

Current Opinion in Virology 2017, 22:51–58

This review comes from a themed issue on Emerging viruses:

intraspecies transmission

Edited by Ron A.M Fouchier and Lin-Fa Wang

For a complete overview see the Issue and the Editorial

Available online 22nd December 2016

http://dx.doi.org/10.1016/j.coviro.2016.11.013

1879-6257/# 2016 Published by Elsevier B.V.

Introduction Filoviruses are enveloped, non-segmented, negative- strand RNA viruses, composed of three genera: Ebola- virus, Marburgvirus and Cuevavirus (Figure 1) [1–3]. Ebo- lavirus and Marburgvirus are together the causative agents of severe disease in human and non-human primates (NHPs) displaying fatality rates reaching 90% [1] (Table 1). There are currently five known, genetically distinct species of Ebolavirus — Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Taı̈ Forest ebolavirus (TAFV), Bundibugyo ebolavirus (BDBV) and the Asian filovirus; Reston ebolavirus (RESTV) [2]. Almost all human cases are due to the emergence or re-emergence of EBOV in Gabon, Republic of the Congo, Democratic Republic of

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Congo (DRC), and most recently in West Africa [4], and of SUDV in Sudan and Uganda [5] (Table 1).

The increase in the number of outbreaks of Ebola virus disease (EVD) in Africa since 2000 (Table 1) has been postulated to result from increased contact between wild- life and humans [6]. The ever increasing encroachment of mankind into previously uninhabited areas will continue to bring not only humans but also potentially susceptible, domesticated animals into contact with unknown patho- gens and their reservoir species [7]. Deforestation and climate change can also be expected to cause certain species to modify their geographic and ecological distri- bution and potentially into greater proximity to human agricultural exploits or settlements. It appears thus urgent to better understand both filovirus ecology and the mech- anisms involved in viral transmission from their natural hosts and between humans.

Retrospective analysis of human outbreaks since the first EBOV epidemic in 1976 and intensive studies performed on animal models have helped to understand both the nature of EBOV pathology and its transmission. However, the 2014 outbreak has again shown that a complete knowledge of EBOV human-to-human transmission mechanisms is still lacking and is in many cases based only on retrospective observations rather than empirical data. Here, focusing on EBOV, we present an overview of our current understanding of filovirus transmission in humans. We also summarise our current knowledge con- cerning the molecular basis of viral transmission and potential adaptation including that gained from the recent outbreak, together with our opinions on knowledge gaps and future directions for research.

Transmission routes In humans, EBOV has been evidenced either directly or via detection of viral RNA in a range of bodily fluids including blood, stool, semen, breast milk and saliva as well as sweat and tears [8,9]. It is generally accepted that contact with such fluids/fomites from an infected and symptomatic, or deceased person is the most likely route of transmission of EBOV. Other than direct or close contact with these fluids, transmission routes proposed for EBOV involve the presence of infectious virus in fomites, droplets and aerosols [10��]. Experiments using NHPs have shown that EBOV is both highly infectious and contagious [11–13].Evidence from NHP studies has confirmed viral infection associated with a variety of administration routes including oral, conjunctival,

Current Opinion in Virology 2017, 22:51–58

52 Emerging viruses: intraspecies transmission

Figure 1

RAVV

EBOV

RESTV SUDVBDBV

TAFV

LLOV

MARV

Ebolavirus Cuevavirus

Marburgvirus

0.1

Filoviridae

Current Opinion in Virology

Phylogenetic relationship for the viral famiy Filoviridae. The family

Filoviridae are enveloped, non-segmented, negative-strand RNA

viruses of the order Mononegavirales, composed of three major

genera: Ebolavirus, Marburgvirus and Cuevavirus. There are currently

five known, genetically distinct species of Ebolavirus — Zaire

ebolavirus (EBOV), Sudan ebolavirus (SUDV), Taı̈ Forest ebolavirus

(TAFV), Bundibugyo ebolavirus (BDBV) and Reston ebolavirus (RESTV).

The genus Marburgvirus comprises one viral species; Marburg

marburgvirus with two current viral members Marburg virus (MARV)

and Ravn virus (RAVV). The genus Cuevavirus currently has one

species member Lloviu cuevavirus (LLOV). 29 filovirus L protein

sequences for the illustrated virus species were obtained from the

ViPR database and aligned using the muscle algorithm. The aligned

sequences served to generate the phylogenetic tree using the

distance method in the Seaview software [80]. The scale bar indicates

evolutionary distance between each node.

submucosal and respiratory routes amongst others. Based on detailed analysis of available data [10��,14] the trans- mission routes for EBOV can be summarised as follows.

Direct contact

As stated above, evidence from outbreaks, epidemiologi- cal data and NHP models have all confirmed direct contact of an individual with contaminated bodily fluids from a symptomatic patient or from a disease victim as the most likely interhuman transmission route. It is interest- ing to note that recent data from an NHP study using the West African outbreak EBOV Makona variant suggest that more natural routes of infection via oral or conjunc- tiva mucosa may require higher doses of EBOV to pro- duce disease [13], although further studies are required in comparison to other EBOV variants to confirm such observations. Since the recent epidemic however it has also become clear that sexual transmission of EBOV presents a certain risk even with patients that are no longer symptomatic for EVD and this many months after remission [15,16��,17,18]. Indeed, infectious EBOV can be detected in semen of survivors at least up to around 500 days [19,20] and sexual transmission has already been linked to the start of new chains of transmission [21,22].

Current Opinion in Virology 2017, 22:51–58

Droplet transmission

By common definition [23] droplet transmission is thought to occur up to a metre from an infected individual depending on the stability of the virus in question and specific environmental conditions. Cases of droplet trans- mission are suspected from epidemiological data for patients where no direct contact was reported [24]. In the case of EBOV, the presence of virus in droplets might arise from a range of infected fluids, including blood, vomit, saliva or diarrhoea or be produced by coughing or during medical intervention.

Fomites

Transmission from fomites involves viral deposition on surfaces that have been in contact with contaminated secretions including disposed medical waste or corpses [10��,14]. Indeed, contamination from disease victims appears common and has been linked to many cases of transmission, highlighting funerals and burial practices as key transmission events [25�]. Viable virus has been detected on solid surfaces and liquids several days to several weeks after contamination and infectious virus has been retrieved from EBOV infected monkeys seven days after death and RNA detected up to 10 weeks [26]. Data is however often lacking concerning the stability of virus on surfaces and in secretions not typically associated with transmission of enveloped RNA viruses, including vomit and diarrhoea.

Aerosols

Experimental data from NHPs have shown that mechan- ical aerosolisation of virus particles can cause disease with even low infectious doses [27] but the relevance of such findings in a natural setting is unclear [10��]. Stability studies would also suggest that aerosolised particles pro- duced in this way are relatively unstable (loss of 99% of particles after 100 min at room temperature and humidi- ty) [28]. As stated above, the majority of transmission cases arise from direct contact and in outbreak settings containment is possible without strict precautions against airborne transmission [10��,14].

EBOV transmission and molecular potential for virus adaptation/evolution Pathogens such as EBOV would appear already intrin- sically able to break the interspecies barrier and both the intrahuman and interhuman barriers, allowing the virus to propagate within the human population. How- ever, as the specific natural host of Ebolavirus is yet to be discovered it is difficult to clearly assess whether EBOV needs any adaptation to successfully infect humans or other species. Nevertheless, evidence of EBOV infection has been reported in various wild species including primates, bats, duikers or domestic pigs [29–32], and thus far, similarly to Marburgvirus, bats are thought to be the most likely natural reservoir for this virus [33�].

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Human transmission of Ebola virus Lawrence et al. 53

Table 1

List of Ebolavirus outbreaks (1976–present day)

Year(s) Country Ebola subtype Reported number

of human cases

Reported

number of

fatalities

Case

fatality

rate

August–November 2014 Democratic Republic of the Congo Ebola virus 66 49 74

March 2014–Present Guinea, Sierra Leone, Liberia and others* Ebola virus 28 616* 11 310* �70** November 2012–January 2013 Uganda Sudan virus 6 3 50

June–November 2012 Democratic Republic of the Congo Bundibugyo virus 36 13 36

June–October 2012 Uganda Sudan virus 11 4 36

May 2011 Uganda Sudan virus 1 1 100

December 2008–February 2009 Democratic Republic of the Congo Ebola virus 32 15 47

November 2008 Philippines Reston virus 6 (asymptomatic) 0 0

December 2007–January 2008 Uganda Bundibugyo virus 149 37 25

2007 Democratic Republic of the Congo Ebola virus 264 187 71

2005 Republic of the Congo Ebola virus 12 10 83

2004 Sudan (South Sudan) Sudan virus 17 7 41

November–December 2003 Republic of the Congo Ebola virus 35 29 83

December 2002–April 2003 Republic of the Congo Ebola virus 143 128 89

October 2001–March 2002 Republic of the Congo Ebola virus 57 43 75

October 2001–March 2002 Gabon Ebola virus 65 53 82

2000–2001 Gulu, Uganda Sudan virus 425 224 53

1996 South Africa Ebola virus 2 1 50

1996–1997 (July–January) Gabon Ebola virus 60 45 74

1996 (January–April) Gabon Ebola virus 37 21 57

1995 Democratic Republic of the Congo Ebola virus 315 250 81

1994 Côte d’Ivoire (Ivory Coast) Taı̈ Forest virus 1 0 0

1994 Gabon Ebola virus 52 31 60

1989–1990 Philippines Reston virus 3 (asymptomatic) 0 0

1990 USA Reston virus 4 (asymptomatic) 0 0

1979 Sudan (South Sudan) Sudan virus 34 22 65

1977 Ebola Ebola virus 1 1 100

1976 Sudan (South Sudan) Sudan virus 284 151 53

1976 Democratic Republic of the Congo Ebola virus 318 280 88

Adapted from [5,79]

* Includes cases from Guinea, Sierra Leone and Liberia only. ** Estimated.

In terms of susceptibility to infection and species tropism, filoviruses have one surface glycoprotein (GP) (Figure 2) that drives binding and entry of the virus through inter- action with multiple cellular surface molecules [34,35]. The cellular endosomal receptor Niemann-Pick C1 (NPC1) has been identified as playing a key role in the fusion process through binding the proteolytically-primed GP. Mapping of the key positions on NPC-1 and EBOV GP responsible for EBOV tropism shows that these residues are shared between both susceptible bat and human cell lines [36�,37]. Such data suggest that EBOV would not require specific adaptation for successful entry into human cells. Likewise, molecular studies performed on human macrophages and a Marburgvirus bat isolate suggest that no further adaptation is necessary for spill- over from bats to the human population [38]. In rodent models however, Ebolavirus infection with wild-type EBOV virus results in an asymptomatic illness [39–41]. Importantly, sequential passaging of wild-type virus in these small animal models can lead to the generation of highly pathogenic variants of the virus that display muta- tions in three viral genes: polymerase (L), nucleoprotein

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(NP) and viral protein VP24, when compared to the initial, wild-type viral sequence [40,42,43]. By generating recombinant viruses containing different combinations of these mutations, it was subsequently shown that a single mutation in VP24 was sufficient for the virus to acquire virulence in Guinea pigs [40,42]. Similarly, an adapted EBOV strain containing mutations in VP24 and NP genes is lethal in immunocompetent mice and hamsters [39,40].

The recent epidemic is the first time that such an out- break has been described in terms of the genetic evolu- tion of the viral genome over the course of an epidemic. Systematic deep sequencing of EBOV positive patients has thus provided new insights into viral spread and transmission chains [4,44–48,49�]. The extent of the 2014 West Africa outbreak lead to numerous concerns about the ability of the EBOV Makona variant to evolve in terms of pathogenicity and/or transmissibility in the human population [44,50]. The emergence of variants with a lower pathogenicity was also feared; as such viruses can potentially establish a long-term endemic presence of the virus in afflicted countries [51]. Initially thought to be

Current Opinion in Virology 2017, 22:51–58

54 Emerging viruses: intraspecies transmission

Figure 2

NP VP35 VP40 sGP/GP VP30 VP24 L3′ Leader

5′ Trailer

Current Opinion in Virology

Schematic representation of Ebola virus genome. The 19 kb negative-sense RNA genome of EBOV and its seven genes give rise to the individual

viral structural and non-structural proteins. The central core of the virion is formed by the genomic RNA molecule encapsulated by nucleoprotein

(NP) and linked to viral inner capsid proteins 30 (VP30) and 35 (VP35) and the RNA-dependent RNA polymerase (L), together with VP24 forming

the viral ribonucleoprotein complex (RNP) that is essential for viral transcription, replication and encapsidation. The two remaining viral proteins,

surface glycoprotein (GP) and VP40 are membrane-associated; VP40 is displayed at the inner surface of the lipid bilayer of the viral envelope and

is linked to the RNP. Through transcriptional RNA editing, three GP gene specific mRNA products are expressed from the GP gene of EBOV,

coding for full-length transmembrane surface GP and the soluble, non-structural proteins sGP and ssGP. Star indicates the position of the GP

gene editing site.

able to evolve more rapidly [44], the whole genome mutation rate for EBOV Makona now appears to be comparable with that observed during other EBOV outbreaks, with a substitution rate estimated at �1.3 � 10�3 nucleotides/site/year [45–48]. Indeed, the molecular data, as well as the epidemiological analysis allowing estimation of parameters such as the basic re- production number R0, so far obtained cannot discrimi- nate EBOV Makona from previous outbreak variants [25�,44,52–55]. In general, EBOV Makona would appear however to have a longer incubation time than most previous EBOV outbreaks, potentially allowing a longer period of dissemination from infected persons between different regions, facilitating propagation of the virus [25�,44,52].

Although both synonymous and non-synonymous muta- tions are detected in all viral genes, the most frequent gene mutations observed during the outbreak were locat- ed in GP, NP, and L [44,45,47,48,49�,56], and interest- ingly this tendency appears conserved between previous outbreaks when such data is available [49�,57,58�]. Anal- ysis of the EBOV Makona GP mucin-like domain has shown it to be more subject to positive selection in several studies, with an acquisition of mutations in B and T cell epitopes [50,54,57,58�]. Additionally for GP, a non- synonymous mutation at amino acid 82 (A82V) appeared to be selected in a region containing the receptor-binding domain [47,49�]. Indeed, recent analyses of this A82V mutation in pseudovirus or reverse genetics systems have highlighted the role of this mutation in adaptation to a human host through a certain refining of receptor binding affinity and associated increase in viral fitness in human cells [59��,60,61]. Whilst GP mutations probably reflect the arms race between the immune system and the virus or differences in receptor binding affinity [57,59��,60–62], mutations in NP, VP30, VP35 and L, the four proteins forming the viral ribonucleoprotein replication complex, might play a role in viral adaptation in the human popu- lation in the processes of viral transcription, replication or encapsidation or in facilitating the interaction of cellular

Current Opinion in Virology 2017, 22:51–58

factors with viral proteins and RNA in a new host envi- ronment, as already shown for Influenza [63]. For EBOV, mutations in L have already been speculated to play a role in both GP editing and viral replication rates [64], al- though experimental evidence for this is currently lack- ing. Several recent studies based on the genetic analysis of Makona variants arising during the recent outbreak have shown that mutations in VP30 and in L [65] or in NP and L [59��] can have implications for virus adaptation and fitness.

As single mutations often occur at the cost of viral fitness, which need to be compensated by other co-mutations, it appears essential to continue to analyse co-occurring mutations to fully understand viral evolution and adapta- tion [59��,65–67]. EBOV co-mutation network analysis shows strong evidence of selection for GP, NP, L and VP40, especially for EBOV Makona, with mutations occurring more frequently in protein interaction domains [58�]. This study suggests that a still understudied coop- eration between viral proteins exists and could play a role in viral adaptation to humans. In addition, several studies reported serial T>C substitutions in viral genomes, sug- gested to be due to specific cellular Adenosine Deami- nase Acting on RNA (ADAR) enzymes [45,46,68]. The role of these substitutions is currently unknown but ADAR modifications have been shown to be involved in replication and pathogenesis for several other viruses including influenza and measles virus and therefore merit further investigation [69].

Another major discovery of sequence analyses was the low percentage (�1%) of viral GP gene specific mRNA encoding for the full-length, transmembrane, surface GP [44]. In fact Ebolavirus is somewhat unique in this respect in that synthesis of its surface GP is dependent on transcriptional RNA editing at a site constituting seven consecutive U residues (editing site) present within the GP gene (Figure 2). Direct expression of the GP gene however results in synthesis of a nonstructural secreted glycoprotein termed sGP [70], which has been proposed

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Human transmission of Ebola virus Lawrence et al. 55

to participate in the immune evasion of EBOV by cap- turing certain antibodies directed against GP [71]. Early reports based on cell culture experiments had indicated a figure of around 1:4 for the ratio of surface GP versus sGP transcripts [70,72]. Discovery of a much lower percentage in patients during the outbreak corroborates recent find- ings indicating that the editing site is also a transcription termination signal and highlights the necessity for a productive viral cycle to minimize surface GP expression, as recently demonstrated [73,74]. Interestingly, these observations resemble those seen in experimental animal models of adaptation in which it was demonstrated that control over surface GP expression is also exerted at the GP editing site at the genomic level [75,76]. On the other hand, the maintenance of the wild-type editing site may indicate that a well-balanced, rationally minimal expres- sion of surface GP vs. synthesis of secreted sGP offers a selective advantage and that this feature is an essential element in the replication and spread of EBOV, playing a role in viral pathogenicity and in counteracting the im- mune system [71,73,77]. In keeping with this idea, another mutation hotspot that was identified during the 2014 outbreak is near the GP tumour necrosis factor-alpha converting enzyme (TACE) cleavage site (Q638R/L) [49�]. This cleavage site is responsible for an additional decrease in expressed membrane-bound GP via its re- moval from the cell surface as a shed form that is proposed to play a role in virus dissemination and pathogenesis [77,78]. However, the biological significance of this mu- tation remains to be tested.

Conclusions and areas for future study Coupled with data from animal models, the outbreak in West Africa has again highlighted the importance of contact with bodily fluids for a successful transmission. However, further characterisation of which fluids are most likely to lead to infection needs to be performed in terms of virus loads and survival rates. In the same vein there is currently very little experimental data on how the virus physically penetrates into the body. Likewise the relative impact and risk associated with the potential of sexual transmission of EBOV should be thoroughly investigated. Based on everything that we have learnt concerning the genomics of the latest outbreak it will be vital to continue to perform molecular studies to assess the importance of the various mutations and polymorphisms consistently detected from epidemiological data in terms of their impact on virus immune escape, receptor binding affini- ties, pathogenicity and transmissibility. In light of recent molecular data based on 2014 EBOV outbreak isolates [59��,60,61,65], it will be of interest to further study and to model such mutations through consecutive passages of initial/early outbreak variants in human cells. It also remains to be seen whether adaptation mutations seen over the course of an outbreak can be in some way preserved, given the unprecedented scale of the epidemic

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and multiple contacts between infected patients, disease victims and the environment.

A surprising feature of some of the recent outbreaks has been the appearance of Ebolavirus species in new loca- tions, including BDBV in DRC and more recently and more devastatingly, EBOV in West Africa [4]. In this respect it seems vital that future studies cover the identi- fication of risk factors linked to the emergence of zoonotic pathogens and include continuing studies of the molecular basis of transmission events that allow such breaches of the animal to human species barrier or that promote efficient human-to-human transfer. Although we are just beginning to understand filovirus ecology, it seems clear given the absence of any current vaccine or proven treatment for EVD and the difficulty in containing outbreaks in coun- tries where access to adapted medical and containment facilities is rare, that for the moment any increased under- standing of filovirus ecology and surveillance may help to minimize the risk of future outbreaks.

Acknowledgements This work was supported by the European Commission (FP7 programme in the framework of the project ‘Antigone — ANTIcipating the Global Onset of Novel Epidemics’, project number 278976) and by Agence Nationale de la Recherche (ANR-14-EBOL-002-01). The sponsor had no role in the collection, analysis and interpretation of data, in the writing of this review; nor in the decision to submit for publication.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:

� of special interest �� of outstanding interest

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