Micromodification of Virus Neutralization Assay with Vital Staining in 96 Well Plate and Its Use in Diagnosis of Ťahyňa Virus Infections
Virus neutralization test (VNT) is considered to be the gold standard for arbovirus serology because of its high specificity and sensitivity. Its micromodification in 96-well plate with vital staining of cell cultures was developed in the National Reference Laboratory (NRL) for Arboviruses of the Czech Republic and is used for the detection of specific antibodies against various viruses, mainly arboviruses. The test procedure is described for the Ťahyňa virus micromodified neutralization assay in 96-well plate. Results of an anti-Ťahyňa antibody survey conducted among the population of northern Moravia using the VNT assay are presented. The overall anti-Ťahyňa seroprevalence among 1001 tested persons was 3.80 %. The highest positivity rate was found in persons aged over 59 years (17.53 %) comparing to children with the seroprevalence rates of 0.00 % and 0.56 % in the age groups 0-5 years and 6-14 years, respectively.
Virus-neutralizační test (VNT) je v sérologii arbovirů považován za zlatý standard pro svou vysokou specificitu a senzitivitu. V Národní referenční laboratoři České republiky (NRL ČR) pro arboviry byla vyvinuta jeho mikromodifikace na mikrotitračních deskách s 96 jamkami s vitálním barvením buněčných kultur. Metoda je využívána pro detekci specifických protilátek proti různým virům, zejména arbovirům. Je popsán postup provedení VNT v mikromodifikaci pro virus Ťahyňa. Jsou prezentovány výsledky vyšetření protilátek antiŤahyňa u obyvatelstva v Moravskoslezském kraji dosažené pomocí VNT. Celková séroprevalence u 1001 vyšetřovaných osob byla 3,80 %. Nejvyšší podíl séropozitivních osob byl zjištěn u populace starší 59 let (17,53 %). Naopak u vyšetřovaných dětí do 5 let byla séroprevalence 0,00 % a ve věkové skupině 6–14 ůet 0,56 %.
Klíčová slova: virus Ťahyňa – arboviry – neutralizace viru – vitální barvení – buněčná kultura
Ťahyňa virus (TAHV) is a member of the
California serogroup of the genus Bunyavirus,
Principal vectors of Ťahyňa virus are culicine mosquitoes,
predominantly of the genus Aedes.
Principal hosts of Ťahyňa virus are hares, rabbits, hedgehogs and
small rodents.[2, 8]
Ťahyňa virus causes in humans so-called Valtice fever,
a self-limiting febrile illness occurring in summer and early
autumn. The rate of seropositivity depends on the location and age,
highest rates (30-60 %) are found in elderly persons living close to
frequently flooded areas with increased populations of mosquitoes [3,
4, 7]. At least 200 documented cases of Valtice fever have been
published in Slovakia and Moravia since 1963 but many cases have
remained probably unnoticed [1, 5, 7]. The disease also occurs in
other European and non-European countries. The closely related La
Crosse virus is the main cause of arbovirus encephalitis in children
in the USA .
Serologic examination in human population for specific
anti-Ťahyňa antibodies is usually performed with the
hemaglutination-inhibition (HIT) or plaque-reduction neutralisation
tests (PRNT). No commercial assay for serologic diagnostics of Ťahyňa
infections is available. Micromodification of virus-neutralisation
assay in 96-well plates with vital staining of the cell cultures for
detection of neutralizing anti-Ťahyňa antibodies was developed in
the NRL for arboviruses of the Czech Republic.
of virus-neutralisation test with vital staining of the cell cultures
Specific structures on the virus surface are blocked by
virus-neutralisation antibodies, thereby the infectivity of the virus
is neutralised. Manifestation of the virus infectivity may occure in
different forms, depending on the selected host system, way of
inoculation etc. Use of cytopathic effect on cell cultures is the
best suited and most practical method for the routine diagnostics in
virus-neutralisation test. It belongs to the most specific serologic
reactions and in the case of arboviruses, presence of
virus-neutralisation antibodies predicates directly the antiinfective
immunity of the organism. The virus-neutralisation test is considered
to be the golden standard for arbovirus serology.
There are three posibilities of quantitative arrangement
of the virus-neutralisation assay: 1. Serially diluted virus and
nondiluted serum (antibodies) – mostly used for identification of
unknown virus isolate. 2. Nondiluted virus and serially diluted serum
(antibodies) - the most convenient arrangement for the purpose of
antibodies detection from clinical samples. 3. Serially diluted virus
and serially diluted serum (antibodies) – also known as so-called
box-titration – used only for special purposes.
Micromodification of VN assay in 96-well plate with
vital staining was developed in the NRL for arboviruses for the
purpose of serologic diagnostics of many arboviral infections,
Ťahyňa virus including. Neutral red dye is used for the final
visualisation of the reaction.
Vital staining differenciates the viability of the cell
culture. Living cell culture is able to take in all the dye from
diluent in the microplate well. The neutral red dye is absorbed in
the cytoplasm and the whole cell culture gets conspicuously coloured
in the form of clusters, islets or continuous red area on the bottom
of the well. The cell cultures damaged by the virus are not able to
absorb the dye which remains diffused in the diluent and the damaged
cells don’t get coloured. The differences in the colour of the
diluent and especially the morphology and colouring of the cell layer
on the bottom of the wells are very distinct and observable
macroscopically. Macroscopic differentiation of the cytopathic effect
is possible even in the case of partially nonspecifically degenerated
cells when the microscopical evaluating of cell cultures is no more
The detailed test procedure and results of anti-Ťahyňa
serosurvey in the area of Northern Moravia are presented.
of the test procedure
Required number of tissue
culture 96-well plates is prepared and marked (1 row for each
sample, 8 serum samples can be tested in 1 plate). Two rows for
negative controls and 2 rows for positive controls are used (negative
and positive human sera). Universal diluent is MEM (minimal essential
medium) buffered with 0.02 mol/l TRIS-HCl pH 8,1 (stock solution 2
mol/l TRIS-HCl pH 8,1 is diluted 1:100) + 7,5% bovine fetal serum.
This mixture is called MEM-TRIS. Standardized volume of 0.025 ml
per well of each component is used: 0.025 ml of serum in the
respective dilution, (from 1:4 in serial double dilutions), 0.025 ml
of diluent (MEM-TRIS), 0.025 ml of Ťahyňa virus (prototype
strain TAH-92 cultivated on intracerebrally infected suckling mice)
in working dilution (=100 TCID50,
After 10s vortexing the covered plates are incubated for 18 hours in
+4 °C, then warmed up for 1 hour in 37 °C. 0.025 ml of
cells suspension (pork kidney stable cell line = PS cells) in working
dilution (e.g. 600 000 cells/ml i.e. 15 000 cells/well) is
added. After 10s vortexing the covered plates are incubated for 3-4
days in humide incubator with 37 °C. 0.025 ml of neutral red
dye in working dilution (1:10 000) is added (1 part of stock 0.2%
aqueous solution of neutral red and 19 parts of MEM-TRIS) and
vortexed for 10 sec. Covered plates are incubated for 1 day in humide
incubator with 37 °C. It is possible to follow up the dynamics
and continue incubation with neutral red. After disposal of the whole
content of wells the results are read definitely.
First 3 wells of each row serve
as serum controls, instead of the virus is added identical volume of
pure diluent (MEM-TRIS) and only first 3 dilutions of serum are used:
1:4, 1:8 and 1:16.
of the results
Only live cells are stained with neutral red dye and
turn red in the end of the VNT. Dead cells damaged by the virus
(cytopathic effect) are not able to be stained with the neutral red
dye and remain colourless. First 3 wells of each row (serum control
without added virus) must remain vital and macroscopically red
coloured. If they are not vital (colourless), it means that the
sample was toxic for the cells and the reaction is not specific up to
the sample-dilution of more than 50 % damaged cells. Results of the
VNT are expressed in the form of virus-neutralisation (VN) titer
which is inverted value of the highest dilution of the sample which
is able to neutralise the cytopathic effect of the virus for more
than 50 % in the respective microwell.
Plate with completed virus-neutralisation test is showed
in Fig. 1.
Rows 1, 2, 3, 4 and 7 show positive results with VN
titers 64, 32, 16, 64 and 16 respectively, whereas rows 5, 6 and 8
reveal negative results.
Titration of the working virus dilution should be
proceeded simultaneously with each run of virus-neutralisation test
in a separate plate.Working dilution of virus is further diluted
in 10-fold dilutions. Plate for virus titration is prepared and
marked. Two whole columns of the plate are used for each dilution of
the virus. Working dilution of the virus is marked as 10,
further dilutions as 10-1,
and 10-4. Last two
columns serve as cell control (pure diluent instead of virus).
Procedure of virus titration is identical with the proper
virus-neutralisation test, only different dilutions of virus are used
(pure diluent instead of virus in the cell controle columns) and
instead of diluted serum is added only pure diluent.
For the VNT, 100 infectious doses of the virus (100
TCID50) should be
used, it means 100x higher dilution of the virus which causes more
than 50 % cytopathic effect.
To estimate the risk for Ťahyňa virus infections,
human population in the region of Nortehrn Moravia (northeast part of
the Czech Republic) was examinated for specific anti-Ťahyňa
antibodies with virus-neutralisation assay in 96-well plate with
vital staining of the cell cultures. From November 2006 to June 2007
1125 serum samples from 1001 persons tested for various reasons for
other arboviruses in NRL were colected. For establishment of acute
infection, in addition to positive case history, seroconversion or
4-fold increase of VN-titer in paired serum is required. For
establishment of seropositivity without acute infection positivity in
anti-Ťahyňa VNT is necessary.
of anti-Ťahyňa survey
No case of seroconversion or significant increase of
VN-titer was found in paired sera. 41 samples from 38 persons were
positive for anti-Ťahyna antibodies. There were 25 males and 13
females positive in the age from 6 to 78 years. Age distribution of
anti-Ťahyňa positive persons divided to 10-years age intervals is
shown in Fig. 2.
Virus-neutralisation titer of anti-Ťahyňa antibodies
in positive samples ranged from 4 to 512, the most common titer was
16. Values of the titers and numbers of persons with respective
titers: titer 4 (4 persons), titer 8 (7 persons), titer 16 (12 p.),
titer 32 (4 p.), titer 64 (4 p.), titer 128 (2 p.), 256 (2 p.), 512
Total seroprevalence among 1001 examinated persons was
3.80 % (38 persons). The rate of seropositivity was figured out and
related to the commonly used basic epidemiologic age groups (0-5
years, 6-14 years, 15-59 years and ≥60 years). The highest
positivity rate was found in persons older than 59 years – 17 of 97
persons were positive (17.53 %), in children, on the other hand, the
seroprevalence was very low: no case of 31 examinated children in the
age group 0-5 years and only 1 case of 180 children (0.56 %) in the
age group 6-14 years. 20 seropositive persons of 693 (2.89 %) were
found in the age group 15-59 years. Detailed data is demonstrated in
Virus-neutralisation assay for anti-Ťahyňa detection
in 96-well plate with vital staining of the cell cultures developed
in the NRL for arboviruses of the Czech Republic was shown as
a simple, useful, high sensitive and specific tool for detection
of anti-Ťahyňa antibodies in human blood. The same method is
routinely used also for serologic diagnostics, serosurveys and
confirmation of many other antiviral antibodies, arboviruses above
all (tick-borne encephalitis virus, West Nile virus, yellow fever
virus, chikungunya virus and others). In addition, this kind of assay
may be used also for examination of all warm-blooded animals. Vital
staining of cell cultures for detection of neutralisation antibodies
is known in PRNT (plaque-reduction neutralisation test). VNT
modification in 96-microplates with vital staining was firstly
described by Januška et.al.  for tick-borne encephalitis virus
The micromodification with vital staining for the final
visualisation of the reaction enables macroscopic reading of the
results, hence tedious microscopic reading can be totally get rid of.
This modification is more comfortable and precise and affords better
reproducibility of the results comparing to PRNT or classical
virus-neutralisation assay with only microscopic reading.
Additionally, vital staining allows also observation of the dynamics
of cytopathic effects.
Results of serosurvey focused on anti-Ťahyňa
antibodies among popuation in the region of Northern Moravia showed
the total seroprevalence of 3.80 % with the increasing positivity
rate depending on the age but no case of acute Ťahyňa infection was
Northern Moravia was proved as endemic territory for
Ťahyňa virus although the ascertained seroprevalence is lower
comparing to earlier studies from the Breclav area during 1997 floods
(53.8 % seropositivity)  and from the river basins in Central
Bohemia during 2002 floods (16,5 %) [4, 5]. The disparity can be
explained by the difference of environmental factors, the
seropositivity is always higher in wetlands and in areas of
frequently ocuring heavy rains and floods which support
mosquito-vector populations. No of these circumstances occured in
Northern Moravia during recent period with the exception of floods in
1997. Any significant increase of mosquito population wasn’t
noticed in this territory. The dependence of seropositivity rate on
the age is correspponding with the results of other surveys in the
Czech Republic [3, 4, 5, 7].
The study was supported by the Grant Agency of the AS
CR - Academy of Sciences of the Czech Republic [IAA 600930611
(Re)emerging mosquito-borne diseases].
redakce došlo 8. 4. 2008-06-19
MUDr. Hana Zelená
NRL pro arboviry - Zdravotní
702 00 Ostrava
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Hygiena a epidemiologie