Arch Virol (2014) 159:3239–3247
DOI 10.1007/s00705-014-2188-y
ORIGINAL ARTICLE
First dengue haemorrhagic fever epidemic in the Americas, 1981:
insights into the causative agent
Rosmari Rodriguez-Roche • Yoandri Hinojosa
Maria G. Guzman
•
Received: 21 May 2014 / Accepted: 17 July 2014 / Published online: 5 August 2014
Ó Springer-Verlag Wien 2014
Abstract Historical records describe a disease in North
America that clinically resembled dengue haemorrhagic
fever during the latter part of the slave-trading period.
However, the dengue epidemic that occurred in Cuba in
1981 was the first laboratory-confirmed and clinically
diagnosed outbreak of dengue haemorrhagic fever in the
Americas. At that time, the presumed source of the dengue
type 2 strain isolated during this epidemic was considered
controversial, partly because of the limited sequence data
and partly because the origin of the virus appeared to be
southern Asia. Here, we present a molecular characterisation at the whole-genome level of the original strains isolated at different time points during the epidemic.
Phylogenetic trees constructed using Bayesian methods
indicated that 1981 Cuban strains group within the Asian 2
genotype. In addition, the study revealed that viral evolution occurred during the epidemic – a fact that could be
related to the increasing severity from month to month.
Moreover, the Cuban strains exhibited particular amino
acid substitutions that differentiate them from the New
Guinea C prototype strain as well as from dengue type 2
strains isolated globally.
Introduction
Dengue viruses (DENV) cause the most important arthropod-borne viral disease of humans, with recent estimates of
R. Rodriguez-Roche (&) Y. Hinojosa M. G. Guzman
Department of Virology, PAHO/WHO Collaborating Centre for
the Study of Dengue and its Vector, ‘‘Pedro Kouri’’ Tropical
Medicine Institute (IPK), PO Box 601, Marianao 13, Havana,
Cuba
e-mail: rosmari@ipk.sld.cu
390 million dengue infections per year, of which 96 million
cause disease of any level of severity [4]. DENV, which
belongs to the genus Flavivirus, family Flaviviridae, consists of four antigenically distinct serotypes (DENV-1 to 4).
The genomes of flaviviruses comprise a single-stranded
RNA molecule encoding three structural proteins, the
capsid (C), pre-membrane/membrane (PrM/M), and envelope (E) proteins, and seven non-structural (NS) proteins,
NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [33].
During the past few decades, Latin America has gradually evolved from a low-dengue-endemic to a hyperendemic region with indigenous transmission in most
countries [43]. In Cuba, since the eighteenth to the midtwentieth century, outbreaks clinically compatible with
dengue have been reported. However, a nationwide seroepidemiological survey concluded in 1975 revealed that
only 2.6 % of the adult population had hemagglutinationinhibiting antibodies to group B arboviruses. In 1977, an
epidemic of classical dengue fever (DF) caused by DENV1 affected the country, with more than 400,000 reports.
Serological studies suggest that around 50 % of the Cuban
population was infected [7]. Four years later, in 1981, an
unprecedented major outbreak of DHF/DSS caused by
DENV-2 was recognised. Retrospective epidemiologic
studies suggested that the epidemic had begun at the end of
1980 in three municipalities located in eastern, central and
western Cuba. Cases were reported during the same epidemiologic week in individuals with no history of travel
abroad [19]. Acknowledged as the first laboratory-confirmed dengue haemorrhagic epidemic in the Americas, a
total of 344,203 cases were registered, including 10,312 of
DHF/DSS, resulting in 158 deaths (101 of them being
children) [24]. This explosive epidemic, controlled in
approximately four months, was characterised by rapid
dispersal of the virus throughout the country, with
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extraordinarily high transmission rates. For example, on
the single day of July 6th, 11,400 new cases were identified. According to Armada and Figueredo, the Aedes aegypti house infestation index in early August 1981 was
around 11 % [3].
On the other hand, secondary infection was the most
notable risk factor for the development of severe forms
of the disease [5]. However, at that time it was not ruled
out that the causative strain could have more virulence
potential or at least be different from the virus circulating in the Caribbean and associated with a mild form
of the disease. In addition, the epidemic was characterised by a marked month-to-month increase in clinical severity in patients previously infected by DENV-1
[25].
During the 1990s, some studies revealed that the strain
causing the 1981 Cuban epidemic had great similarity to
the prototype strain New Guinea C (NGC) isolated in 1944
[1, 16, 44]. Indeed, short fragments of the E gene and
E/NS1 gene junction (approximately 240 bp) were
sequenced and compared with only 21 DENV-2 sequences
isolated worldwide using uncomplicated analysis based on
percentage of divergence. Other researchers who in subsequent years detected isolates genetically related to the
NGC strain in Mexico, Haiti, Venezuela and Honduras [9,
12, 34, 35] associated these results with laboratory contamination. To this end, the aim of the present study was to
determine the genetic relatedness of the 1981 Cuban strains
by using nucleotide sequences encoding the entire polyprotein obtained from strains isolated at different time
points during the epidemic. The phylogenetic tree obtained
through a Bayesian approach supported and extended
previous findings, since the Cuban strains are located
within the Asian 2 genotype. In this paper, we report that
viral evolution occurred during the 1981 epidemic,
including non-conserved amino acid substitutions in both
structural and non-structural proteins that could be related
to the increasing clinical severity observed with epidemic
progression.
Materials and methods
Strains
Five DENV-2 strains isolated in suckling mice, corresponding to different time points during the 1981 Cuban
epidemic, were utilised for the molecular characterisation.
These strains were stored for more than 30 years at -80 °C
in the Strain Bank of the National Reference Laboratory of
Virology at ‘‘Pedro Kouri’’ Tropical Medicine Institute.
The original isolates obtained at that time were processed
without passages in any other system (Table 1).
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R. Rodriguez-Roche et al.
Table 1 DENV-2 strains isolated in 1981 utilised for nucleotide
sequencing
Strain
Date of fever
onset
Clinical
classification
Location
Passage
history*
A15
03/06/1981
DF
Havana City
3P
A35
11/06/1981
DHF
Havana City
2P
A115
29/07/1981
DHF/DSS
(Fatal case)
Santiago de
Cuba
3P
A132
11/08/1981
DHF
Havana City
3P
A169
02/10/1981
DF
Havana City
2P
* Number of passages in suckling mice
RNA extraction, RT and PCR
Briefly, viral RNA was extracted from 140 lL of sample
using a QIAamp Viral RNA Mini Kit (QIAGEN, Germany)
and cDNA was synthesized using a Transcriptor High
Fidelity cDNA Synthesis Kit (Roche Applied Science,
Germany) according to manufacturer’s instructions, using a
serotype-specific primer complementary to the 30 UTR as
described previously [36]. An aliquot of 3 ll of cDNA was
subjected to PCR using an Expand High FidelityPLUS PCR
System (Roche Applied Science, Germany) according to
manufacturer’s instructions. Five pairs of primers for each
serotype were utilised, designed to obtain five overlapping
fragments (F1-F5) covering the complete genome [8].
Nucleotide sequencing
PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN, USA). Direct sequencing reactions
were prepared using a CEQ Dye-labeled dideoxy terminator cycle sequencing kit (Beckman Coulter, Germany)
following manufacturer’s instructions. Capillary electrophoresis was carried on a CEQ 8800 Genetic Analysis
System (Beckman Coulter, Germany). The sequences were
assembled into specimen consensus sequences using
Sequencer software version 4.8 (Gene Codes Corporation,
USA). The minimum fold-coverage of the sequences was
at least 3x. The nucleotide sequences reported in this study
are available in GenBank (ID: KF704354-KF704358).
Sequence analysis
Nucleotide sequences encoding the entire polyprotein of each
DENV-2 strain (A15, A35, A115, A132 and A169) obtained
Fig. 1 Phylogenetic analysis of DENV-2 based on the nucleotide c
sequences encoding the entire polyprotein. The evolutionary history
was inferred using Bayesian methods. The analysis involved 93
nucleotide sequences, including five Cuban isolates from 1981. All
horizontal branch lengths are drawn to scale; bar, 0.02 substitutions
per site. The tree is midpoint-rooted for purposes of clarity only
First dengue haemorrhagic epidemic in the Americas, 1981
3241
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R. Rodriguez-Roche et al.
in the present study were aligned using ClustalX [45] together
with relevant sequences retrieved from GenBank (available
from the authors on request) representing all known DENV-2
genotypes. This produced a total data set of 93 sequences
10,173 nucleotides in length. A similar procedure was performed to analyse the E gene. A total of 92 sequences 1485
nucleotides in length were utilised. Phylogenetic trees were
performed using Bayesian analysis in MrBayes v3.1.2 [22],
with a minimum of 20 million generations and a burn-in of
10 %. Stationarity was assessed at effective sample size (ESS
[400) using Tracer v1.4.1 (part of the BEAST package) [10].
All Bioinformatic analyses were carried out on the freely
available Bioportal: www.bioportal.uio.no.
Results
The Bayesian phylogeny obtained for the DENV-2 data set
indicates that the Cuban strains isolated in 1981 group
Table 2 Amino acid
differences among Cuban
strains taking strain A15 as a
reference
Gene
PrM
E
NS1
123
A15
A35
A115
A132
A169
Relevance of the amino acid changes
26
K
N
N
r
r
Neutral change
28
E
G
G
G
G
Could affect interaction Pr-E [28], differentiates
DENV-2 genotypes in its antigenicity [27]
55
F
L
L
r
L
Nearby cysteine 54 and residue 56 involved in the
interaction of PrM-E [13]
43
F
L
L
L
L
Residue F43 is conserved for DENV-2 globally,
located in a non-neutralizing linear epitope
(E37-46) of B and T cells [11]
112
G
S
S
S
S
Close to fusion peptide, site under selection
pressure [46]
158
H
r
r
Y
r
Favoured change only in A132
203
N
D
D
D
D
Located on protein surface, involved in structural
changes that occur when E is converted from a
dimer to a trimer, could affect recognition by
antibodies [26, 31]
403
A
r
r
G
r
The acid residue E403 is conserved for DENV-2
globally; this change could be critical for the
low-pH-induced E protein dimer/trimer
transition [26, 29]
459
I
T
r
T
r
Disfavoured change
174
E
K
r
K
r
An acid residue E174 is uncommon among
DENV-2 strains. Change in NS1 protein have
been associated with intra-epidemic increase
severity [38]
Neutral change only in A132
NS2A
164
T
r
r
A
r
NS3
84
R
K
K
K
K
Neutral change
141
K
r
R
R
R
Neutral change
NS5
r Conserved positions taking
the A15 strain as a reference
Position
within the Asian 2 genotype, together with the prototype
NGC isolated in 1944, and two strains from China isolated
during the 1980s (Fig. 1). All major nodes are statistically
reliable according to the estimates of posterior probability.
Indeed, the Cuban strains are very distant from strains that
belong to the former Jamaica genotype, currently defined
as the Asian/American genotype, which have been circulating in the Americas since the beginning of 1980s.
In addition, all Cuban strains formed an independent
subgroup within the Asian 2 genotype, although there is
noticeable relative genetic diversity among them. A15 and
A169 strains (both isolated from DF cases) were closely
related, while strain A35 was more closely related to strain
A115 (both isolated from DHF cases). Meanwhile, strain
A132 (isolated from a case of DHF) was slightly separated
from the rest of the Cuban isolates.
A comparative analysis of the nucleotide sequences
corresponding to Cuban strains revealed 36/10173 variable
positions taking as reference the strain A15. A total of 16
366
K
R
R
R
R
Neutral change
5
M
r
r
I
r
Favoured change only in A132
252
F
r
r
Y
r
Residue F252 is uncommon among DENV-2
strains, it is located in the methyl transferase
domain [47]
First dengue haemorrhagic epidemic in the Americas, 1981
Table 3 Amino acid
differences among the strains in
the study using the DENV-2
prototype strain NGCp as a
reference
Gene
Position
NGCp
NGCn
A15
A35
A115
A132
A169
Relevance of the amino acid
changes
PrM
28
E
r
r
G
G
G
G
Discussed in Table 2
55
L
F
F
r
r
F
r
Discussed in Table 2
57
R
K
R
R
R
R
R
Favoured change
Both related to neutralization
escape mutants with
monoclonal antibodies [29]
E
NS1
r Conserved positions taking
the NGCp strain as a reference
3243
71
E
D
r
r
r
r
r
124
N
r
I
I
I
I
I
126
E
K
K
K
K
K
K
Described in ref. [6] for the
neurovirulent phenotype. All
strains included in the Asian 2
genotype have K at this
position
277
L
r
V
V
V
V
V
Might affect fusion mediated by
pH changes [26]
360
E
r
G
G
G
G
G
Part of a serotype-specific
peptide that participates in
binding and neutralization,
considered a Th cell epitope
[30, 41]
402
F
I
r
r
r
r
r
All Cuban strains similar to
NGCp
403
E
r
A
A
A
G
A
Discussed in Table 2
105
R
Q
r
r
r
r
r
All Cuban strains similar to
NGCp
174
K
K
E
r
E
r
E
Discussed in Table 2
changes were non-synonymous, producing amino acid
changes in both structural and non-structural genes. Nine
changes were found in structural, and seven in non-structural proteins. E appears to be the most variable protein. In
addition, it is relevant that six mutations that led to amino
acid changes differentiate the first isolate (A15) from the
others obtained during the epidemic. Therefore, changes
located in the PrM 28, E (43, 112, and 203) and NS3 (84,
366) proteins were fixed during the epidemic. Noticeably,
viral evolution occurred during the epidemic. Furthermore,
amino acid changes that could imply structural modifications, with potential impact on viral replication capacity or
immunopathogenic mechanisms, were detected (Table 2).
While the evolution pattern observed during the epidemic constitutes an argument to reject the contamination
hypothesis formulated some years ago, we nonetheless
considered it appropriate to compare the Cuban strains with
two genetic variants of the prototype strain NGC, designed
as parental strain (NGCp) [6] and neurovirulent strain
(NGCn) [23]. The analysis was conducted utilising the
region C-PrM-E-NS1, taking as reference the strain NGCp.
Interestingly, residues E 124, 277, 360 and 403 differentiate the 1981 Cuban strains from the prototype NGC (both
the parental and the neurovirulent variants) (Table 3) as
well as from DENV -2 strains isolated globally.
In this context, it is relevant to mention that the fulllength sequence corresponding to the NGC strain utilised
in the phylogenetic analysis (Fig. 1) corresponds to the
neurovirulent variant (accessible in the GenBank database:
M29095). In addition, a limited number of full-length
sequences belonging to the Asian 2 genotype are available.
In order to include the sequence of NGCp [6] as well as to
increase the number of sequences belonging to the Asian 2
genotype, a Bayesian phylogenetic tree using the E gene
was constructed (Fig. 2). As expected, the NGCp was
located deeper within the Asian 2 genotype. All Cuban
strains formed an independent group within the Asian 2
genotype related to old strains from New Guinea (1944),
Sri Lanka (1969), Taiwan (1981), and China (1987, 1989)
and more recent strains from Mexico (1997), Taiwan
(1998) and the Philippines (1994, 2003).
Discussion
During the last 30 years, several authors have inaccurately
associated the beginning of DHF/DSS epidemics in the
Americas with the introduction of the American/Asian
genotype into Cuba, 1981. Indeed, this genotype was
introduced in the Americas in the 1980s but it was first
associated with a significant DHF/DSS epidemic by 1989,
when Venezuela was severely affected [32]. In fact, all the
Venezuelan isolates collected during the 1990s and more
recently clustered within the American/Asian genotype,
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R. Rodriguez-Roche et al.
1
American Genotype
1
1
Cosmopolitan Genotype
1
1
American/Asian Genotype
1
1
1
1
1
1
1
Asian 1 Genotype
1
1
1
1
1
1
1
0.93
123
1
1
Asian 2 Genotype
First dengue haemorrhagic epidemic in the Americas, 1981
b Fig. 2 Phylogenetic analysis of DENV-2 based on the complete E
gene. The evolutionary history was inferred using Bayesian methods.
The analysis involved 92 nucleotide sequences, including five Cuban
isolates from 1981. All horizontal branch lengths are drawn to scale;
bar, 0.03 substitutions per site. The tree is midpoint-rooted for
purposes of clarity only
suggesting that the former American genotype has been
displaced [39]. Therefore, the phylogenetic position of the
1981 Cuban strains within the Asian 2 genotype does not
substantiate the extensive spreading of the virus from Cuba
to Latin American countries as suspected during the 1980s
[15, 34]. Nevertheless, similar strains grouped within the
Asian 2 genotype were sporadically isolated in countries
such as Venezuela, Mexico [34], Haiti [20] and Honduras
[2] in the same decade. Unfortunately, full-length sequences corresponding to this group of old Latin American
isolates have not been published.
According to Rico-Hesse, low-level nucleotide sequence
differences (\1 %) found with respect to the strain NGC in
1944 are questionable. Indeed, based on the mutation rates
per site per year observed for DENV-2, the virus isolated in
1981 should have at least 2 % genetic divergence relative
to its ancestor [35]. Conversely, Diaz and colleagues, who
detected two isolates in Mexico in 1997 that were closely
related to the NGC strain, considered that the repeated
finding of such sequences in different laboratories from
different countries makes it difficult to believe that the
same mistake has occurred repeatedly. These authors
suggested that introduction and circulation of viruses that
had been stored for years has been reported [14]; therefore,
the old NGC strain could have escaped from one or several
laboratories and started re-circulating in different countries
[9]. Actually, the Asian 2 genotype was circulating as a
prevalent genotype of DENV-2 in the Philippines during
the 1990s [42].
On the other hand, the variability of the Cuban strains
during the course of the 1981 epidemic was noteworthy.
The fact that six amino acid changes were found between
the A15 and A35 strains, both of which were isolated in
June, just one week apart, may suggest the occurrence of a
stochastic phenomenon of genetic drift in the first stage of
the epidemic, where there was a low transmission rate.
Nonetheless, epidemiological data indicate that there was
an increase in severity over time, which indeed might be
associated with an increase in viral fitness during the 1981
epidemic [25]. In addition, if we consider that Aedes aegypti populations differ in vector competence for transmitting the virus or in their susceptibility to infection with
dengue virus, we cannot rule out the possibility that variants with greater fitness for replication in the local vector
could have been selected at the beginning of the epidemic
(December 1980 to May 1981).
3245
Resembling what occurred in the 1981 epidemic, the
month-to-month increasing severity phenomenon was also
observed during the epidemic occurred in Santiago de
Cuba in 1997 [17]. Despite the fact that both epidemics
lasted around 4-6 months and occurred in a relatively
similar context (the same population with the same
sequence of infection, DENV-1/DENV-2), during the 1981
epidemic, the interval between DENV-1/DENV-2 infections was 4 years compared with 20 years in 1997. Based
on the greater variability observed during the 1981 epidemic compared with that of 1997, and especially because
significant amino acid changes occurred in the structural
proteins, whilst in 1997 a few changes were observed only
in the non-structural proteins [36, 37], different mechanisms could explain the increasing clinical severity
observed during these Cuban epidemics, emphasizing that
viral fitness is always context-dependent [40].
Could the increase in severity from month to month
observed during the 1981 epidemic be explained by the
neutralisation escape mutant hypothesis? According to
Guzman et al., [18] the affinity of antibodies against the
homologous serotype increases over time and decreases
against the heterologous serotype. Consequently, antibodies present in individuals who are immune to DENV-1
could generate greater positive selective pressure after a
relatively short time (4 years later) than after a long time
(20 years later). Under this assumption, during short-term
infection with DENV-2, the appearance of escape mutants
would be expected, not only based on the biological
properties of the antibodies (affinity/avidity), but also
because in 1981 more individuals had immunity to DENV1 compared to 1997. During 1981, the circulation of a new
dengue virus serotype in the context of elevated Aedes
aegypti infestation rates and 50 % of the population having
immunity to DENV-1 could play a significant role in
shaping its genetic diversity, since viral lineages that evade
cross-immunity could be at a selective advantage.
The evolution of dengue virus has had a major impact
on the epidemiology of the disease globally [35]. Interestingly, during the 1940s in southern Asia, DENV-2 was
only associated with DF. Indeed, the onset of the modern
pandemic of DHF/DSS was documented as early as 1950 in
Bangkok, Thailand, and the Philippines [21]. Collectively,
the amino acid changes detected in the 1981 Cuban isolates
could explain its wide capacity for dispersion and potential
to cause severe disease in DENV- 1-immune individuals.
Therefore, the impact of such changes on viral fitness and
their association with increasing severity within the epidemic deserve more studies.
Acknowledgments We thank Prof. Ernest A. Gould and Prof.
Marco Vignuzzi for relevant suggestions and useful comments concerning the manuscript.
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R. Rodriguez-Roche et al.
This research was supported by the Cuban Ministry of Public
Health and the EU 7th Framework-Health Programme (Grant
Agreement No. 282378 – DENFREE). The funders had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Conflict of interest
All authors declare no conflicts of interest
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