APA Format Expert, Research Paper

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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.

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