See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/232531471

Assessment of plaque assay methods for alphaviruses

Article  in  Journal of virological methods · October 2012

DOI: 10.1016/j.jviromet.2012.09.026 · Source: PubMed

CITATIONS READS

6 95

5 authors, including:

Diana Juarez Kanya Long

U.S. Naval Medical Research Unit No. 6 World Bank
55 PUBLICATIONS   33 CITATIONS    45 PUBLICATIONS   611 CITATIONS   

SEE PROFILE SEE PROFILE

Eric Halsey
U.S. Naval Medical Research Unit No. 6
279 PUBLICATIONS   1,771 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Rodent borne virus View project

Mortality Rate and Predictors in Children Under 15 Years Old Who Acquired HIV from Mother to Child Transmission in Paraguay View project

All content following this page was uploaded by Kanya Long on 29 January 2020.

The user has requested enhancement of the downloaded file.

 Journal of Virological Methods 187 (2013) 185–189

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

Short communication

Assessment of plaque assay methods for alphaviruses

Diana Juarez a,∗ , Kanya C. Long a,b , Patricia Aguilar c , Tadeusz J. Kochel d , Eric S. Halsey a
a
U.S. Naval Medical Research Unit No. 6, Lima, Peru
b
University of California, Davis, USA
c
Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA
d
U.S. Naval Medical Research Center, Silver Spring, MD, USA

a b s t r a c t

Article history: Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human
Received 1 June 2012 health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, iden-
Received in revised form tification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent
19 September 2012
samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque
Accepted 28 September 2012
reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recog-
Available online 17 October 2012
nition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid
method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated.
Keywords:
Alphavirus
Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis
Plaque assay virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method
Agarose showed consistently greater sensitivity than the semisolid method. Also, a “semisolid-variant method”
Carboxymethylcellulose using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the
same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifi-
cations in plaque assay conditions affect significantly results and therefore evaluation of the performance
of each new assay is needed.
Published by Elsevier B.V.

Alphaviruses circulate widely in the world and cause dis-
ease, often affecting either the musculoskeletal or the central
nervous system. In South America, the main alphaviruses that cir-
culate are Venezuelan equine encephalitis virus (VEEV), Eastern
equine encephalitis virus (EEEV), Western equine encephalitis virus
(WEEV), Mayaro virus (MAYV), Una virus (UNAV) and Trocara virus
(TROCV) (Aguilar et al., 2004; Forshey et al., 2010; Powers et al.,
2006; Travassos da Rosa et al., 2001; Turell et al., 2005). While VEEV
subtype IC and IAB are mainly associated with major epidemics
that reach high rates of mortality in equines, enzootic subtype ID
has been isolated repeatedly from humans and mosquitoes recently
(Aguilar et al., 2009). MAYV produces dengue-like acute febrile ill-
ness including arthralgia and arthritis. Serosurveys suggest that
MAYV is endemic across the Amazon Basin, and isolates have been
obtained from humans, monkeys and mosquitoes (Forshey et al.,
2010; Lavergne et al., 2006; Powers et al., 2006; Tesh et al., 1999).
Despite the lack of apparent human cases of EEEV, WEEV, UNAV and
TROCV in South America, these viruses have been isolated repeat-
edly from mosquitoes, birds and other vertebrates, remaining in an
∗ Corresponding author at: U.S. Naval Medical Research Unit No. 6, American
Embassy 3230, Lima Pl., Washington, DC 20521-3230, USA. Tel.: +1 51 1 614 4158;
fax: +1 51 1 614 4174.
E-mail address: diana.juarez@med.navy.mil (D. Juarez).
often poorly understood, natural-cycle (Aguilar et al., 2007; Powers
et al., 2006; Travassos da Rosa et al., 2001).
Several studies have described arbovirus distribution, vector
transmission, host–pathogen interaction, and public health impact
of arboviruses (Forshey et al., 2010; Gould et al., 2010; Morrison
et al., 2008; Turell et al., 2005; Weaver and Reisen, 2010). Studies
such as these often use plaque assays or plaque reduction neutral-
ization tests (PRNTs) to measure the amount of virus or antibodies
present in a sample. Both plaque assays and PRNTs are consid-
ered gold standards in virus and antibody detection, respectively;
however, these tests should be standardized and evaluated for per-
formance and reproducibility for specific viruses, especially when
data are shared between laboratories.
Since the first description of plaque assays (Dulbecco, 1952),
modifications to the original approach have been made, including
the use of different substances in the overlay media and differ-
ent cell preparations (Hotchin, 1955; Matrosovich et al., 2006;
Morens et al., 1985; Schulze and Schlesinger, 1963). Agar or
agarose have been used widely in standard plaque assays as a
solid overlay (Aguilar et al., 2004; Beaty et al., 1989; Henderson
and Taylor, 1959). However, an alternative semisolid method
using carboxymethyl cellulose (CMC) has been reported to possess
advantages over the traditional agar overlay including: (1) easy
medium removal and (2) prevention of monolayer peel off. Con-
sequently, this approach has been adopted for many viral assays
(Morens et al., 1985; Rapp, 1963; Schulze and Schlesinger, 1963).

0166-0934/$ – see front matter. Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.jviromet.2012.09.026
186 D. Juarez et al. / Journal of Virological Methods 187 (2013) 185–189
Table 1
Viruses used in the study.
Virus strain Passage history
MAYV – TRVL 15537 2 passages in Vero cells
UNAV – PE10800 TVP5402 3 passages in Vero cells
VEEV – TC83 83 passages in Guinea pig heart cell/and three
passages in Vero cells
WEEV – VR70 2 passages in Vero cells
Any modification of the plaque assay and its possible impact on
virus titration or neutralizing antibody titers should be established
carefully. Modifications in assay conditions have been shown to
impact flavivirus results significantly (Thomas et al., 2009); how-
ever, the extent plaque assay conditions affect alphavirus diagnosis
is unknown. In an attempt to provide information that could aid
researchers in selecting appropriate alphavirus plaque assay and
PRNT conditions, a series of experiments using VEEV, MAYV, UNAV
and WEEV were conducted to evaluate the variability of viral titer
and titer of neutralizing antibody under two standard methods:
a solid method using cell monolayer under agarose overlay and
a semisolid method using a cell suspension under CMC overlay.
Finally, a semisolid-variant method was evaluated for examining
the effects of cell preparation from the type of overlay medium
used.
Both Vero-76 and BHK-21 cells are used commonly for
alphavirus plaque assay and plaque reduction neutralization tests.
In this study, Vero-76 cells were selected because plaque mor-
phology was defined clearly compared to those in BHK-21 cells
allowing a reliable counting of plaques (data not shown). Vero-76
cells were propagated at 37 ◦ C in Eagle’s minimum essential media
(EMEM) supplemented with 10% of heat-inactivated fetal bovine
serum (FBS) and 1% penicillin/streptomycin. The study was per-
formed using the alphavirus strains listed in Table 1. All cell lines
had an initial passage number of 40.
A description of the main steps in the plaque assay methods
are shown in Fig. 1. For the solid method, cell suspension was
Fig. 1. Three methods used for alphavirus and antibody titration.
seeded with 3 × 105 cells in a 12-well plate and incubated at 37 ◦ C
in 5% CO2 . Upon reaching ∼80% confluence, growth media was
removed and 100 ␮l of 10-fold virus dilutions from 101 to 109
were added and incubated for 1 h, with rocking every 15 min to
allow virus adsorption. A 3 ml overlay consisting of 0.4% agarose in
EMEM (supplemented with 2% of heat-inactivated FBS and 1% peni-
cillin/streptomycin) was added, and the plates were incubated at
37 ◦ C for 48 (UNAV, VEEV and WEEV) or 72 h (MAYV). After incuba-
tion, agar plugs were removed, and the cells were fixed and stained
with 0.1% napthol blue black, 1.6% sodium acetate in 6% glacial
acid acetic for 30 min. For the semisolid method, 3 × 105 cells were
seeded to 12-well tissue culture plates and 100 ␮l of 10-fold virus
dilutions from 101 to 109 were added immediately to the cells, with
no rocking. Virus inoculation was performed simultaneously from
the same virus dilution under each of the conditions. Cells were
incubated for 3 h to allow virus adsorption and, after incubation,
a CMC overlay media consisting of 0.6% carboxymethylcellulose,
EMEM without phenol red, 10% FBS, 0.075% NaHCO3 and peni-
cillin/streptomycin was added. Plates were incubated at 37 ◦ C for
48 (UNAV, VEEV and WEEV) or 72 h (MAYV). After incubation, cell
overlays were removed, and the cells were fixed and stained as
describe above. For the semisolid-variant method, plates contain-
ing 80% confluent Vero monolayers were used as described in the
solid method, but with the addition of a CMC overlay media. After
incubation, cell overlays were removed, and the cells were fixed
and stained as described above.
For use as positive controls in PRNT, hyperimmune mouse ascitic
fluid (HMAF) was obtained by immunizing adult mice with virus
mixed with Freud’s adjuvant and then injecting them with sar-
coma cells. Two sets of human serum were analyzed to determine
antibody neutralization titers under solid and semisolid condi-
tions. First, nineteen paired acute and convalescent serum samples
were obtained through a febrile surveillance study. All paired
samples were from patients that had MAYV isolated from their
acute sera. All convalescent samples were collected between one
week and three months after the onset of symptoms, as described
previously (Forshey et al., 2010). Second, serum samples were
 D. Juarez et al. / Journal of Virological Methods 187 (2013) 185–189
Fig. 2. Comparison of alphavirus titers by plaque assay method.
(A) Titers have significant differences by method (p < 0.001 by ANOVA) except for VEEV solid vs. semisolid-variant (p = 0.972 by Student’s t-test). Error bars represent ± two
standard deviations. (B and C) Number of antibody positive samples at twofold dilutions using solid and semisolid methods. Samples tested against MAYV = 149 (B) and
VEEV = 30 (C).
collected from healthy participants residing in urban and rural
zones in and around Iquitos, Peru, between 2006 and 2008, as part
of a VEE prevalence study described previously (Morrison et al.,
2008). All study protocols were reviewed and approved by the
Naval Medical Research Center (NMRC; Silver Spring, MD) Insti-
tutional Review Board (NMRCD.2000.0006 and NMRCD.2000.0008)
and by the Naval Medical Research Unit Six (NAMRU-6; Lima, Peru)
Institutional Review Board (PJT.NMRCD.001, PJT.NMRCD.038, and
NMRCD.2008.0003).
For the PRNTs, HMAF and patient sera were heat-inactivated
at 56 ◦ C for 30 min. Twofold sera dilutions (from 1:10 to 1:320
for human sera, and additional dilutions to obtain the end titer
for HMAF) were prepared and mixed in equal volume with 40
plaque forming units (PFUs) of each virus and incubated at 4 ◦ C
overnight. One hundred microliters of the virus-serum dilution
mixtures was inoculated into Vero-76 cells and incubated at 37 ◦ C
for 1 or 3 h (solid or semisolid, respectively) before adding the solid
or semisolid overlay. Plates were incubated for 48 (UNAV, VEEV and
WEEV) or 72 h (MAYV). Cells were fixed and plaques were counted
as described above. PRNT titer was expressed as the reciprocal of
the serum dilution that reduced the number of plaques by 80%
(PRNT80 ).
Plaque assay titrations were carried out 10 times for MAYV,
UNAV and WEEV and 7 times for VEEV. For the statistical analysis,
the results were expressed in log10 PFU/ml ± 2SD. Statistical anal-
yses were performed using SPSS statistics software version 17.0
(SPSS Inc., Chicago, IL). The ANOVA (for comparison of all meth-
ods) or t-test (for solid vs. semisolid comparison) were calculated
to assess the significance of differences between methods, with
two-tailed p values <0.05 considered significant.
187
Comparison of solid and semisolid plaque assays were found
to yield a different number of plaques under the two condi-
tions: the solid method provided higher log10 virus titers than the
semisolid method for UNAV (7.2 ± 0.2 vs. 6.3 ± 0.3), MAYV (7.1 ± 0.1
vs. 6.3 ± 0.4), WEEV (6.9 ± 0.3 vs. 6.5 ± 0.4), and VEEV (9.1 ± 0.5 vs.
8.6 ± 0.5) with a p < 0.001 (Fig. 2A).
To clarify better whether the cell suspension or the CMC over-
lay was responsible for the lower virus titers, a semisolid-variant
method, using a cell monolayer overlaid with CMC was evalu-
ated. The time of virus absorption was 1 h, equivalent to the solid
method. For the semisolid-variant method, the MAYV and UNAV
titers increased compared with the semisolid method but were still
less than the solid method (p < 0.001). For WEEV, the titer by the
semisolid-variant was higher than the solid and semisolid method
(p < 0.001). For VEEV, the titer was equivalent with the semisolid-
variant and solid methods (p = 0.972); however, both were better
than the semisolid method (p = 0.02). For the semisolid-variant
method, the averages ±2SDs are also shown in Fig. 2A (UNAV
6.9 ± 0.2; MAYV 6.9 ± 0.1; WEEV 7.4 ± 0.3; VEEV 9.1 ± 0.5).
To evaluate if the choice of method could influence PRNT results,
HMAF raised against all four of the alphaviruses under study was
tested. There was a sixfold difference between solid and semisolid
methods for both MAYV (10,240 vs. 320) and UNAV (640 vs. 10). In
contrast, the VEEV and WEEV titers under the solid and semisolid
conditions varied only by one dilution (Table 2).
Next, an evaluation of 19 paired samples from febrile patients
with confirmed MAYV infections was performed. No MAYV neu-
tralizing antibodies were detected in the acute samples using either
solid or semisolid methods. In the convalescent samples, 17 of 19
demonstrated PRNT seroconversion, with titers equal to or greater
188 D. Juarez et al. / Journal of Virological Methods 187 (2013) 185–189

Table 2
Comparison of hyperimmune mouse ascitic fluid (HMAF) antibody titers by solid
and semisolid plaque reduction neutralization testing methods.
Antibody titer
HMAF Solid method Semisolid method
MAY 10,240 320
UNA ≥640 10
VEE 1280 640
WEE 40,960 20,480
Number of replicates per assay = 4.
than 40 using the solid method. In contrast, all convalescent sam-
ples were negative using the semisolid method (Table 3). In order
to explore further the effect of PRNT methods on neutralizing anti-
body results, PRNT assays were performed on 145 samples positive
for anti-MAYV antibodies by IgG ELISA from a survey of healthy
residents of Iquitos. Using the solid method, 17% of samples had
titers <20 and 83% of samples had titers ≥20; the semisolid method
yielded 37% of samples with titers <20 and 63% of titers ≥20
(Fig. 2B). For the samples with anti-VEEV antibodies by IgG ELISA
(Morrison et al., 2008), using the solid method, 30% had titers <20,
and 70% had titers of ≥20. Similarly, for the semisolid method, 33%
of the samples had titers <20 and 67% had titers ≥20 (Fig. 2C).
Plaque assays and PRNTs are methods used commonly in the
field of virology and remain the gold standard for virus quantitation
and serology. Over the years, modifications to the initial proto-
col have been made to try to improve reliability and accuracy of
results. However, the effect of these modifications on final results
in alphavirus testing has not been performed rigorously. Two meth-
ods were compared in this study: a solid method using agarose as
an overlay and a semisolid method using CMC as an overlay.
The results showed that despite inoculation of identical doses,
the number of plaques was different when tested by the two meth-
ods, with a six and eightfold difference in MAYV and UNA titers
and a three and twofold difference in VEEV and WEEV titers. One
of the possible explanations could be that an amount of virus
remains in the cell suspension/CMC overlay because of less contact
between virus and free-floating cells and, thus, a lower propor-
tion of cells become infected. It is important to realize that in the
semisolid method, the protocol specified to dilute the virus 1:10
in the cell suspension as compared to direct inoculation onto the
monolayer in the solid method. However, this dilution and the fre-
quency of contact with free-floating cells vs. cells in a monolayer
cannot account for the difference between methods entirely, as
shown by results of the semisolid-variant method. These experi-
ments demonstrated the direct effect of overlay on virus titer for
MAYV and UNAV.
PRNT was also evaluated to determine whether antibody titers
might be impacted by choice of assay method, considering that in
the semisolid method the dose of virus was higher than the solid
method in order to obtain an equivalent number of plaques by
both methods. Again, the results indicated outcomes dependent
on both method and virus. It was observed that: (1) in UNAV, false-
negatives possibly due to binding and wasting antibodies because
of the excess of virus with the semisolid method; (2) in MAYV,
underestimation of antibody titers, even in 6 twofold dilutions; and
(3) in VEEV and WEEV, no significant effects.
Results from the 19 paired MAYV samples showed no neu-
tralizing antibodies against MAYV in the convalescent samples
with semisolid method. By contrast, 17 positive samples were
obtained under solid conditions. Why the semisolid method was
unable to detect neutralizing antibody to MAYV in the 19 sam-
ples remains unclear. Nevertheless, the semisolid method yielded
positive results using samples collected in a serosurvey, although
consistently with lower titers compared with titers obtained using
the solid method. The two methods resulted in a 20% difference in
PRNT confirmation of IgG ELISA positive samples.
When 30 samples were tested for VEEV antibodies, only a 3% dis-
agreement between solid and semisolid methods was found. These
results were consistent with the results observed when testing
VEEV–HMAF.
Attempts to develop an alternative system that combines the
sensitivity observed when using a cell monolayer and the easier
media removal provided with the CMC overlay indicated that a
semisolid variant could be utilized in VEEV and WEEV assays. Nev-
ertheless, the experiments demonstrated that the solid method
remains the method of choice for sensitivity in MAYV and UNAV
titration.
Obtaining the most accurate and reliable results is the prior-
ity of any investigation, and evaluating how assay modifications
can alter results is essential. Enhancing this evaluation will facili-
tate the comparison of results between laboratories and improve
knowledge of specific virus characteristics.

Table 3
Comparison of antibody titers in human sera by plaque reduction neutralization testing (PRNT) method.

No. Acute IFA PRNT MAYV Convalescent PRNT MAYV

Solid Semisolid Solid Semisolid

1 IQE7750 MAYV <1/20 <1/20 IQE7831 1/160 <1/20

2 OBS6225 MAYV ND ND OBS6497 1/320 <1/20
3 FSB0309 MAYV ND ND FSB0310 1/160 <1/20
4 FSB0279 MAYV ND ND FSB0280 1/640 <1/20
5 FSB0311 MAYV ND ND FSB0312 1/40 <1/20
6 FSB0319 MAYV ND ND FSB0320 1/40 <1/20
7 IQD2668 MAYV <1/20 <1/20 IQD2827 >1/640 <1/20
8 IQE2975 MAYV <1/20 <1/20 IQE3232 1/320 <1/20
9 IQE3288 MAYV <1/20 <1/20 IQE3468 1/640 <1/20
10 IQE7564 MAYV <1/20 <1/20 IQE7669 1/40 <1/20
11 DEF0533 MAYV ND ND DEF0538 1/320 <1/20
12 MFI0231 MAYV <1/20 <1/20 MFI0243 1/80 <1/20
13 FSC0497 MAYV ND ND FSC0507 1/160 <1/20
14 FSC0498 MAYV <1/20 <1/20 FSC0508 1/320 <1/20
15 FSL1307 MAYV <1/20 <1/20 FSL1326 1/160 <1/20
16 FSL1610 MAYV <1/20 <1/20 FSL1615 <1/20 <1/20
17 FSL1707 MAYV <1/20 <1/20 FSL1730 ≤1/20 <1/20
18 FSL3058 MAYV <1/20 <1/20 FSL3098 1/320 <1/20
19 FMD0970 MAYV <1/20 <1/20 FMD1119 1/320 <1/20

ND, not done due to insufficient serum.

IFA, immunofluorescent antibody.
 D. Juarez et al. / Journal of Virological Methods 187 (2013) 185–189
Funding
This study was partially funded by the United States Department
of Defense Global Emerging Infections Systems Research Program.
WORK UNIT NUMBER: 847705.82000.25GB.B0016.
Acknowledgments
We would like to thank Brett Forshey and Julia S. Ampuero for
their critical review of this manuscript and Alicia Rosas, Elizabeth
Castillo and Zonia Rios for their laboratory support.
The views expressed in this article are those of the authors
and do not necessarily reflect the official policy or position of the
Department of the Navy, Department of Defense, or the U.S. Gov-
ernment.
Eric S. Halsey and Tadeusz J. Kochel are U.S. military service
members, and Diana Juarez is an employee of the U.S. Govern-
ment. This work was prepared as part of their official duties. Title
17 U.S.C. §105 provides that ‘Copyright protection under this title
is not available for any work of the United States Government.’ Title
17 U.S.C. §101 defines a U.S. Government work as a work prepared
by a military service member or employee of the U.S. Government
as part of that person’s official duties.
References
Aguilar, P.V., Adams, A.P., Suarez, V., Beingolea, L., Vargas, J., Manock, S., Freire, J.,
Espinoza, W.R., Felices, V., Diaz, A., Liang, X., Roca, Y., Weaver, S.C., Kochel, T.J.,
2009. Genetic characterization of Venezuelan equine encephalitis virus from
Bolivia, Ecuador and Peru: identification of a new subtype ID lineage. PLoS Negl.
Trop. Dis. 3, e514.
Aguilar, P.V., Greene, I.P., Coffey, L.L., Medina, G., Moncayo, A.C., Anishchenko, M.,
Ludwig, G.V., Turell, M.J., O’Guinn, M.L., Lee, J., Tesh, R.B., Watts, D.M., Russell,
K.L., Hice, C., Yanoviak, S., Morrison, A.C., Klein, T.A., Dohm, D.J., Guzman, H.,
Travassos da Rosa, A.P., Guevara, C., Kochel, T., Olson, J., Cabezas, C., Weaver,
S.C., 2004. Endemic Venezuelan equine encephalitis in northern Peru. Emerg.
Infect. Dis. 10, 880–888.
Aguilar, P.V., Robich, R.M., Turell, M.J., O’Guinn, M.L., Klein, T.A., Huaman, A., Guevara,
C., Rios, Z., Tesh, R.B., Watts, D.M., Olson, J., Weaver, S.C., 2007. Endemic eastern
equine encephalitis in the Amazon region of Peru. Am. J. Trop. Med. Hyg. 76,
293–298.
Beaty, B.J., Calisher, C.H., Shope, R.E., 1989. Arboviruses. In: Schmidt, N.J., Emmons,
R.W. (Eds.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infec-
tions. American Public Health Association, Washington, pp. 797–855.
Dulbecco, R., 1952. Production of plaques in monolayer tissue cultures by single
particles of an animal virus. Proc. Natl. Acad. Sci. U. S. A. 38, 747–752.
Forshey, B.M., Guevara, C., Laguna-Torres, V.A., Cespedes, M., Vargas, J., Gianella, A.,
Vallejo, E., Madrid, C., Aguayo, N., Gotuzzo, E., Suarez, V., Morales, A.M., Bein-
golea, L., Reyes, N., Perez, J., Negrette, M., Rocha, C., Morrison, A.C., Russell,
View publication stats
189
K.L., Blair, P.J., Olson, J.G., Kochel, T.J., 2010. Arboviral etiologies of acute febrile
illnesses in Western South America, 2000–2007. PLoS Neg. Trop. Dis. 4, e787.
Gould, E.A., Coutard, B., Malet, H., Morin, B., Jamal, S., Weaver, S., Gorbalenya, A.,
Moureau, G., Baronti, C., Delogu, I., Forrester, N., Khasnatinov, M., Gritsun, T.,
de Lamballerie, X., Canard, B., 2010. Understanding the alphaviruses: recent
research on important emerging pathogens and progress towards their control.
Antiviral Res. 87, 111–124.
Henderson, J.R., Taylor, R.M., 1959. Arthropod-borne virus plaques in agar overlaid
tube cultures. Proc. Soc. Exp. Biol. Med. 101, 257–259.
Hotchin, J.E., 1955. Use of methyl cellulose gel as a substitute for agar in tissue-
culture overlays. Nature 175, 352.
Lavergne, A., de Thoisy, B., Lacoste, V., Pascalis, H., Pouliquen, J.F., Mercier, V., Tolou,
H., Dussart, P., Morvan, J., Talarmin, A., Kazanji, M., 2006. Mayaro virus: complete
nucleotide sequence and phylogenetic relationships with other alphaviruses.
Virus Res. 117, 283–290.
Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H.D., 2006. New low-viscosity
overlay medium for viral plaque assays. Virol. J. 3, 63.
Morens, D.M., Halstead, S.B., Repik, P.M., Putvatana, R., Raybourne, N., 1985. Sim-
plified plaque reduction neutralization assay for dengue viruses by semimicro
methods in BHK-21 cells: comparison of the BHK suspension test with standard
plaque reduction neutralization. J. Clin. Microbiol. 22, 250–254.
Morrison, A.C., Forshey, B.M., Notyce, D., Astete, H., Lopez, V., Rocha, C., Carrion,
R., Carey, C., Eza, D., Montgomery, J.M., Kochel, T.J., 2008. Venezuelan equine
encephalitis virus in Iquitos, Peru: urban transmission of a sylvatic strain. PLoS
Negl. Trop. Dis. 2, e349.
Powers, A.M., Aguilar, P.V., Chandler, L.J., Brault, A.C., Meakins, T.A., Watts, D., Rus-
sell, K.L., Olson, J., Vasconcelos, P.F., Da Rosa, A.T., Weaver, S.C., Tesh, R.B., 2006.
Genetic relationships among Mayaro and Una viruses suggest distinct patterns
of transmission. Am. J. Trop. Med. Hyg. 75, 461–469.
Rapp, F., 1963. Variants of herpex simplex virus: isolation, characterization, and
factors influencing plaque formation. J. Bacteriol. 86, 985–991.
Schulze, I.T., Schlesinger, R.W., 1963. Plaque assay of dengue and other group B
arthropod-borne viruses under methyl cellulose overlay media. Virology 19,
40–48.
Tesh, R.B., Watts, D.M., Russell, K.L., Damodaran, C., Calampa, C., Cabezas, C., Ramirez,
G., Vasquez, B., Hayes, C.G., Rossi, C.A., Powers, A.M., Hice, C.L., Chandler, L.J.,
Cropp, B.C., Karabatsos, N., Roehrig, J.T., Gubler, D.J., 1999. Mayaro virus disease:
an emerging mosquito-borne zoonosis in tropical South America. Clin. Infect.
Dis. 28, 67–73.
Thomas, S.J., Nisalak, A., Anderson, K.B., Libraty, D.H., Kalayanarooj, S., Vaughn, D.W.,
Putnak, R., Gibbons, R.V., Jarman, R., Endy, T.P., 2009. Dengue plaque reduction
neutralization test (PRNT) in primary and secondary dengue virus infections:
How alterations in assay conditions impact performance. Am. J. Trop. Med. Hyg.
81, 825–833.
Travassos da Rosa, A.P., Turell, M.J., Watts, D.M., Powers, A.M., Vasconcelos, P.F.,
Jones, J.W., Klein, T.A., Dohm, D.J., Shope, R.E., Degallier, N., Popov, V.L., Russell,
K.L., Weaver, S.C., Guzman, H., Calampa, C., Brault, A.C., Lemon, A.P., Tesh, R.B.,
2001. Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from
mosquitoes in the Amazon Basin. Am. J. Trop. Med. Hyg. 64, 93–97.
Turell, M.J., O’Guinn, M.L., Jones, J.W., Sardelis, M.R., Dohm, D.J., Watts, D.M., Fer-
nandez, R., Travassos da Rosa, A., Guzman, H., Tesh, R., Rossi, C.A., Ludwing,
V., Mangiafico, J.A., Kondig, J., Wasieloski Jr., L.P., Pecor, J., Zyzak, M., Schoeler,
G., Mores, C.N., Calampa, C., Lee, J.S., Klein, T.A., 2005. Isolation of viruses from
mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru. J.
Med. Entomol. 42, 891–898.
Weaver, S.C., Reisen, W.K., 2010. Present and future arboviral threats. Antiviral Res.
85, 328–345.