Chikungunya virus in US travelers returning from India, 2006.
ABSTRACT Chikungunya virus (CHIKV), a mosquito-borne alphavirus, is endemic in Africa and Asia. In 2005-2006, CHIKV epidemics were reported in islands in the Indian Ocean and in southern India. We present data on laboratory-confirmed CHIKV infections among travelers returning from India to the United States during 2006.
[show abstract] [hide abstract]
ABSTRACT: Partial E1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus Alphavirus. Tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the Old or the New World, with at least two transoceanic introductions to account for their current distribution. The time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indicated that the nucleotide substitution rate varies considerably across sites within the genome. While most trees showed evolutionary relationships consistent with current antigenic complexes and species, several changes to the current classification are proposed. The recently identified fish alphaviruses salmon pancreas disease virus and sleeping disease virus appear to be variants or subtypes of a new alphavirus species. Southern elephant seal virus is also a new alphavirus distantly related to all of the others analyzed. Tonate virus and Venezuelan equine encephalitis virus strain 78V3531 also appear to be distinct alphavirus species based on genetic, antigenic, and ecological criteria. Trocara virus, isolated from mosquitoes in Brazil and Peru, also represents a new species and probably a new alphavirus complex.Journal of Virology 12/2001; 75(21):10118-31. · 5.40 Impact Factor
The Lancet Infectious Diseases 09/2006; 6(8):463-4. · 17.39 Impact Factor
Article: Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections.[show abstract] [hide abstract]
ABSTRACT: Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae, Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of >/=2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.Journal of Clinical Microbiology 05/2000; 38(5):1823-6. · 4.15 Impact Factor
764 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 5, May 2007
Chikungunya V irus
in US Travelers
Robert S. Lanciotti,* Olga L. Kosoy,* Janeen J.
Laven,* Amanda J. Panella,* Jason O. Velez,*
Amy J. Lambert,* and Grant L. Campbell*
Chikungunya virus (CHIKV), a mosquitoborne alpha-
virus, is endemic in Africa and Asia. In 2005–2006, CHIKV
epidemics were reported in islands in the Indian Ocean and
in southern India. We present data on laboratory-confi rmed
CHIKV infections among travelers returning from India to
the United States during 2006.
associated with acute epidemic polyarthralgia. The virus
is serologically and genetically most closely related to
o’nyong-nyong, Igbo Ora, and, to a lesser extent, Mayaro
and Ross River viruses, all of which are associated with
acute polyarthralgia (1).
CHIKV epidemics have been described in Africa,
the Middle East, India, and Southeast Asia, and may have
caused epidemics in the Caribbean and in the United States
during the early 19th century (2). CHIKV epidemics can
be explosive with large numbers of human cases and rapid
virus dissemination. In the Réunion Island epidemic from
April 2005 to June 2006, ≈270,000 cases were reported,
representing nearly 40% of the population (3). Aedes ae-
gypti is the principal vector; however, in recent epidemics
in Réunion Island and southern India, Ae. albopictus has
been co-implicated (4,5). In Africa, CHIKV is maintained
in an enzootic cycle involving primates, but in Asia and
in recent large epidemics, the human-mosquito cycle pre-
dominates, possibly including mechanical transmission (6).
Symptoms are characterized by acute onset of joint pain,
followed by myalgia, fever, and rash with recovery usually
Laboratory diagnosis of CHIKV infection is accom-
plished by serologic methods, virus isolation, and reverse
transcription–PCR (RT-PCR). A typical serologic algo-
rithm involves testing acute- and convalescent-phase serum
specimens for immunoglobulin M (IgM) and IgG antibody,
followed by a plaque reduction neutralization test (PRNT).
Virus isolation and RT-PCR are normally used with early
hikungunya virus (CHIKV) is a mosquito-transmitted
virus (genus Alphavirus, family Togaviridae) usually
acute-phase specimens (before day 5 post-onset) because
duration of viremia is typically 2–4 days.
Recent CHIKV outbreaks have been reported in sev-
eral islands in the Indian Ocean as well as in southern India,
where >1 million cases were reported in 2006 (4,7). CHIKV
infections have also been documented in travelers returning
from these areas (3,7). We report confi rmed CHIKV infec-
tions among 35 travelers returning from overseas travel;
33 were returning from India and 2 from Réunion Island
Serum samples were received by the Centers for Dis-
ease Control and Prevention (Fort Collins, CO, USA) from
April 2006 to December 2006 as part of routine diagnostic
and reference services available to public health labora-
tories. A total of 106 serum samples were received from
persons returning from regions with epidemics or where
CHIKV is endemic (79 from India and the Indian Ocean
islands and 27 from Africa) with compatible CHIKV ill-
ness and submitted by state public health laboratories. Se-
rum samples were tested for antibodies to several viruses
known to occur in the region of travel and residence by
IgM capture ELISA and a standard IgG ELISA (8,9). The
35 CHIKV IgM- and IgG-positive specimens were tested
by using a PRNT (90% reduction cutoff) with several re-
lated alphaviruses (Sindbis, o’nyong-nyong, and Semliki
Forest viruses) to confi rm specifi city of reactivity (10).
A ≥4-fold neutralizing titer difference between antibody
to CHIKV and antibodies to other alphaviruses indicated
a CHIKV-specifi c antibody response. IgM-positive and
PRNT specifi city–confi rmed specimens were classifi ed as
recent CHIKV infections (Table 1).
All serum specimens were tested by a quantitative,
real-time, fl uorescent probed–based RT-PCR assay for
CHIKV RNA. Two primer probe sets were designed in
unique regions of the viral genome and reacted specifi cally
with CHIKV RNA and not with related or unrelated vi-
ruses (Table 2). Both sets showed an analytical sensitivity
<1 PFU, and CHIKV was detected in virus-spiked serum
samples at a concentration of 10 PFU/mL (75 μL of serum
assayed). Eight serum specimens showed positive results
by the real-time assay; all were acute-phase specimens with
number of days post-onset of illness reported as <6. Viral
titers of these specimens were estimated by quantitative
RT-PCR that used CHIKV quantity standards (determined
by plaque assay) to generate a standard curve. Titers of 8
specimens ranged from 103.9 PFU/mL to 106.8 PFU/mL.
All acute-phase specimens (on or before day 8 poston-
set) were also tested for CHIKV by virus isolation in Vero
cells. Isolation was performed by using a recently developed
protocol in which cells were grown in glass shell vials and
centrifuged to enhance viral infectivity (J.O. Velez, unpub.
*Centers for Disease Control and Prevention, Fort Collins, Colo-
Chikungunya Virus in US Travelers
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 5, May 2007 765
data). Five serum specimens displayed prominent and char-
acteristic cytopathic effect on day 2 postinfection, and virus
was identifi ed as CHIKV by RT-PCR. All virus isolates
were obtained from acute-phase specimens that also were
positive by RT-PCR. Three serum specimens (samples 2, 8,
and 10) showed positive RT-PCR results, but CHIKV was
not isolated from these specimens. In these 3 specimens, in-
ability to isolate virus may have been related to viral titers,
which were lower than most of the virus isolation–positive
samples, or to handling or storage of these samples. All
8 virus-positive specimens (whether positive by RT-PCR,
virus isolation, or both) were collected <7 days post-onset
and were negative for IgM and IgG antibodies to CHIKV.
Nearly all of the specimens collected <7 days post-onset
were positive by 1 of the virus-based tests. The 2 excep-
tions, samples 1 and 9, were positive for IgM and IgG an-
tibodies to CHIKV and had high PRNT titers. These fi nd-
ings indicate that these samples were not true acute-phase
specimens; the true onset or collection date had likely been
To identify the strain of CHIKV in these specimens,
a 2,122-bp fragment from the structural region of the ge-
nome (nucleotide positions 9,648–11,770) was amplifi ed
from all 8 virus-positive specimens by RT-PCR and sub-
jected to nucleic acid sequencing with previously described
primers (11). All 8 sequences showed nucleotide identity
Table 1. Diagnostic test results for 35 travelers infected with chikungunya virus (CHIKV), 2006*
*IgM, immunoglobulin M; PRNT, plaque reduction neutralization test; RT-PCR, reverse transcription–PCR; NA, not applicable; ND, not done
(sample depleted); NS, nonspecific reaction in ELISA.
†Values are patient sample optical densities divided by a negative control optical density; values >3 are positive.
‡Values are 90% plaque reduction neutralization titers.
§Real-time, fluorescence-based assay for detecting CHIKV RNA; positive samples had crossing threshold values <37 with both primer sets.
¶Estimated CHIKV PFU/mL by real-time RT-PCR using a standard curve generated with plaque-titrated/calibrated CHIKV standards.
State of US
Return date, 2006
766 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 5, May 2007
>99.7% (GenBank accession nos. EF451142–EF451149).
BLAST analysis (www.ncbi.nlm.nih.gov/blast) of the 8 se-
quences showed that the highest percentage identity was to
CHIKV strains recently isolated from travelers returning
from Indian Ocean islands (Réunion, Mauritius, and Sey-
chelles). Percentage identity matches between the 8 viruses
and Indian Ocean CHIKV strains were >99.5%, with 5 to
8 mismatches occurring randomly. In comparison, percent-
age identities of the 8 viruses to CHIKV prototype S27 or
to a strain previously isolated from India (Nagpur/653496)
were 95.1% and 94.4%, respectively.
The data reported confi rm that the widespread CHIKV
epidemic in southern India has infected US travelers.
CHIKV infections among international travelers are not
unexpected; in 2005–2006, ≈800 CHIKV infections were
reported in France, primarily in travelers returning from
Réunion Island (3). The more noteworthy observation of
this study with potential public health ramifi cations is that
high levels of infectious virus were detected in returning
travelers. Primary vectors for CHIKV are Ae. aegypti and
Ae. albopictus, which are established in several southeast-
ern coastal states in the United States. Vector competence
studies of Ae. aegypti and Ae. albopictus strains from the
United States, as well as strains from the Caribbean and
South America, showed that a titer of ≈104 PFU/mL in
monkeys resulted in productive infection with virus dis-
semination in these mosquitoes, with Ae. albopictus show-
ing higher infection and dissemination rates (12). The level
of viremia reported in most of these imported CHIKV in-
fections, >104 PFU/mL, could be suffi cient to infect North
American vectors, given the appropriate environmental
conditions. However, the time of year and place of resi-
dence of the returning travelers in this study were not con-
ducive to transmission; only 2 (patients 26 and 32) returned
to US regions (South Carolina and Louisiana) known to
have populations of Ae. albopictus.
Nevertheless, returning travelers with high viremia
levels, who live in areas with established Ae. aegypti and
Ae. albopictus populations, could facilitate local transmis-
sion in the United States. Clinicians should therefore obtain
travel histories from persons with CHIKV-compatible ill-
ness and include CHIKV in differential diagnoses when ap-
propriate. Public health laboratories must carefully moni-
tor CHIKV infections of returning travelers and conduct
surveillance for CHIKV-infected vectors in high-risk areas
to prevent local establishment of a new emerging virus.
Diagnostic laboratory personnel involved in virus isola-
tion protocols must be aware of the potential of isolating
CHIKV (a biosafety level 3 agent) from patients returning
from regions endemic for CHIKV or regions with epidem-
ics and take appropriate safety precautions.
Dr Lanciotti is chief of the Diagnostic and Reference Labo-
ratory in the Arbovirus Diseases Branch at the Centers for Dis-
ease Control and Prevention, Fort Collins, Colorado. His primary
research interests are laboratory diagnosis of arbovirus infections
and diagnostic test development and support for public health
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Table 2. Sensitivity and specificity of chikungunya virus (CHIKV) oligonucleotide primers used in real-time
reverse transcription–PCR assay
Primer Genome position*
CHIKV 874 874–894AAAGGGCAAACTCAGCTTCAC
CHIKV 961 961–942
CHIKV 6856 6856–6879 TCACTCCCTGTTGGACTTGATAGA
CHIKV 6981 6981–6956 TTGACGAACAGAGTTAGGAACATACC
CHIKV 6919-FAM 6919–6941AGGTACGCGCTTCAAGTTCGGCG
*On the basis of CHIKV prototype strain S27, GenBank accession no. NC_004162.
†Absolute no. of PFU detected in triplicate testing.
‡No reactivity was observed with the following viruses: o’nyong-nyong, Ross River, Mayaro, Semliki Forest, Sindbis, western equine encephalitis, eastern
equine encephalitis, and Venezuelan equine encephalitis subtypes 1AB, 1C, 1D, and 1E.
§Primer labeled at the 5′ terminus with 5-FAM and 3′ Black Hole Quencher 1 (Operon Biotechnologies Inc., Huntsville, AL, USA).
GCCTGGGCTCATCGTTATTC 0.3 CHIKV
Chikungunya Virus in US Travelers
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 5, May 2007 767
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Address for correspondence: Robert S. Lanciotti, Diagnostic and
Reference Laboratory, Arbovirus Diseases Branch, Centers for Disease
Control and Prevention, 3150 Rampart Rd, Fort Collins, CO 80521, USA;