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Latest Research Of Swine Flu |
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| EFFECT OF INFLUCID IN VITRO AGAINST THE PANDEMIC STRAIN 2009 |
| A(H1N1) SWINE (MEXICAN) FLU |
| M. Yu. Eropkin, N. I. Konovalova, V. A. Grigor" eva, D. M. Baibus, T. M. Gudkova |
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| Scientific Research Institute on Flu,Northwestern Branch Russian Academy of Medical Sciences (St. Petersburg) |
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ABSTRACT |
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| The global development of the flu pandemic of the new variant subtype A(H1N1), the so-called "swine' or Mexican" flu, and the absence of reliable anti-flu preparations effective against all multi-form circulating strains make the search
for less narrowly focussed homeopathic and phyto-preparations against flu more urgent. This paper demonstrates the effectiveness in a model system in vitro of Influcid against a standard strain of the pandemic flu A(H1N1). The activity of
the preparation is mainly expressed not in direct anti-viral action, but rather in adaptive action that leads to increasing resistance of the cells to the viral cytopathogenic effect. |
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| The preparation, Influcid, (German Homeopathic
Union - DHU) has the following composition:
100 g of the solution contains: Aconitum D3 - 10 g,
Gelsemium D3 - 10 g, Ipecacuanha D3 - 10 g,
Phosphorus D5 - 10 g, Bryonia D2 - 10 g,
Eupatonum perfoliatum D1 - 10 g. Other ingre -
dients: eucalyptus globules, 96 % ethanol, puri -
fied water. Alcohol content, 45 % by volume.
According to data in the literature, Influcid signi -
ficantly decreases symptoms present in all acute
respiratory diseases such as hyperthermia, pain in
the extremities, cough, mucus membrane hyperimia
and inflammatory processes in the pharynx and
larynx. |
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Formerly, we investigated the anti-viral effect of
Influcid in vitro with respect to a panel of viruses of
type A bird flu (H5N1; H5N2; H7N3; H9N2) and
the standard strains of human flu A(H3N2), A(H1N1) and B. A study was also done on the effect
of the preparation in vivo on mice infected with a
lethal dose of strain A/PR8/34 (H1N1) adapted to
this model. |
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At present, we are witnesses to the spread of the
first pandemic of the XXI century - the pandemic
of so - called "swine" or "Mexican" flu caused by a
qualitatively new strain of a subtype A flu (H1N1)
- a triple reassortment that contains segments of
RNA derived from the North American line of
swine flu (segments HA, NP and NS), the Eurasian
line of swine flu (segments NA and M), the North
American line of bird origin (segments PA and
PB2) and segment PB1 from seasonal flu subtype
H3N2 |
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At the time this paper was written
(07/07/2009), according to WHO data, there were
94,512 laboratory registered cases of "swine flu"
worldwide, of which 429 were fatal. The pandemic
continued mainly in the southern hemisphere
which was at the time at the height of the winter
season (Argentina, Australia, Chile, New Zealand).
However, regardless of the summer season, in
the northern hemisphere, incidence of this new
flu variant continued to increase - most of all in
the US where the number of cases reached almost
34,000, as well as in Mexico, Canada, Korea, Japan
and Thailand |
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According to laboratory studies, the pandemic
strain A(H1N1) is resistant to adamantans
(remantadine and amantadine), but is sensitive
to neuraminidases (oseltamivir and zanamivir).
However, information has already appeared on the
isolation of the first virus of this variant resistant
to oseltamivir (Tamiflu) |
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Experience of recent
years indicates that it is practically impossible to
select an ethiotropic antiviral preparation effective
against all the multiform seasonal viruses that
circulate. Thus, remantadine, in addition to the fact
that it is ineffective against B flu, in recent epid-
seasons did not have any effect on a significant
portion of viruses of the A(H3N2) subtype. On the other hand, viruses of the A(H1N1) subtype
were in most cases sensitive to remantadine, but
rapidly acquired resistance to oseltamivir. All this
constitutes a basis for searching for preparations
directed not toward the virus as such or its inter -
action with the cell but toward stimulating cell
resistance, developing interferons, and immune
protection. With the present unclear prospects of
timely development of a pandemic vaccination
against "swine" flu, the role of such preparations
in the initial phase of a pandemic is especially
important. This prompted us to study the possible
effect in vitro of Influcid with respect to the
standard strain of "swine" flu A/California/07/09
(H1N1) sw1. |
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| A study of the effect of Influcid done with an
MDSK cell culture recommended by the WHO
reference centers and by the Ministry of Public
Health for Flu of the Russian Federation for
isolating and studying flu viruses (3). The strain
of the pandemic "swine flu" A/California/07/09
(H1N1) sw1 was graciously provided to us for the
scientific studies by Prof. A. I. Klimov (Centers for
Control and Prevention of Diseases (CDC), Atlanta,
Georgia, US). |
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| It turned out that for our purposes, the liquid
(drop) form of Influcid was most suitable. As
demonstrated previously, this preparation is cha-
racterized by complete absence of toxicity when
it is dissolved at 1/50-1/400 and is in contact with
cells for 3 days. |
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| Antiviral activity of Influcid was evaluated on
cells placed in standard doses on a 96-well cell
culture plate. From the original virus-containing
allantoic liquid, a series of ten-fold dilutions
from 10-1 to 10-7 was prepared and placed in
corresponding wells with the cell monolayer.
After 48 hours, the results were checked for
hemagglutination reaction (RHA). In the control
and in the experiment, the inverse logarithm of the
greatest dilution of the original virus that could
cause a positive reaction of hemagglutination in a
well was used as the virus titer and was expressed
as 50 % of tissue infectious doses (TID50). The
virus inhibiting effect of the preparation was
evaluated according to the decrease in virus titer in
the experiment as against the control (ΔlgTID50).
A second important indicator was the reaction of cell regeneration in cultures with MTT tetrazol
stain (Thyazolyl blue), the intensity of which
reflects the degree of viability of cells as a result
of reduction of the stain by mitochondrial and
partially by cytoplasmic dehydrogenases. This
test is used frequently in virology to evaluate the
cytopathogenic action of viruses on the cell (8).
Its results can be interpreted as the degree of cell
resistance to the effect of viruses. The microtetrazol
test is also widely used for evaluating the effect on
cells of toxicants, pharmacological preparations
and unfavorable environmental factors (4) and
for this reason, toxicity of the preparations being
tested in vitro can be evaluated simultaneously
with the antiviral effect. The preparation was
added to the cell culture medium 1 hour before it
was infected with the virus ("prophylactic" plan
of administration) and 1 hour after infection with
the virus ("treatment" plan of administration) in
a dilution with a PBS buffer of 0.5 % and 1 %
(concentration in the medium of the original liquid
preparation). |
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| When Influcid was administered according to
the treatment plan (1 hour after infecting the
cells), the preparation moderately suppressed the
production of virus particles which was evident in
the hemagglutination reaction - at a concentration
of 1 %, the decrease of hemagglutinating activity
(ΔlgTID50) was 1.0; with a dose of 0.5 %, it was
0.5, that is, the infectious activity of the virus
dropped by a factor of 10 and 6.7, respectively.
The results of the comparative study of the effect
of the preparation according to the given criterion
with respect to a panel of human and bird flu virus-
es are presented in Table 1. The greatest activity of
Influcid in this study was apparent with respect to
standard A and B strains of human flu virus isolat-
ed in different years and was comparatively low or
absent with respect to a number of mutants of bird
flu. |
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| Evaluation of MTT regeneration disclosed a
significant decrease in cytopathic activity of the
virus in both the "prophylactic" plan (Fig. 1 and
2) and the "treatment" plan (Fig. 3 and 4) of
administering the preparation. In the "prophylactic"
plan, the effect of the preparation was dose-
dependent (Fig. 1) - Influcid in a concentration
of 1 % had a reliably greater effect than at 0.5 %.The effect was best expressed in the zone of high
virus titers (-1 and -2 log or 104 and 103 TID50),
while in concentrations of 1 %, the preparation al-
most completely blocked the cytopathic reaction of
the cells in the culture even at the highest infectious
dose of the virus: 104TID50 (Fig. 2). Influcid also
reliably decreases the cytopathic effect of the virus
in the "treatment" plan of administration (Fig. 3
and 4), and, in this case, the effect of the preparation
in doses of 0.5 % and of 10 % was approximately
the same, but reliably different from the control
(Fig. 4). |
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| When comparing the data obtained with the MTT
method with the results recorded with the RHA
method, it is evident that with a highly pathogenic
infection with the strain of "swine" flu A/Cali-
fornia/07/09sw1, Influcid has a positive effect
mainly by decreasing cytopathic reactions of the
cells to the virus, that is, it increases the resistance
of the cells themselves. In this sense, its effect
may be considered not so much as being directly
antiviral but as being adaptive, and the result of
this is a reinforcement of the resistance of the
organism to the viral infection. |
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| Evaluation of MTT regeneration disclosed a
significant decrease in cytopathic activity of the
virus in both the "prophylactic" plan (Fig. 1 and
2) and the "treatment" plan (Fig. 3 and 4) of
administering the preparation. In the "prophylactic"
plan, the effect of the preparation was dose-
dependent (Fig. 1) - Influcid in a concentration
of 1 % had a reliably greater effect than at 0.5 %.The effect was best expressed in the zone of high
virus titers (-1 and -2 log or 104 and 103 TID50),
while in concentrations of 1 %, the preparation al-
most completely blocked the cytopathic reaction of
the cells in the culture even at the highest infectious
dose of the virus: 104TID50 (Fig. 2). Influcid also
reliably decreases the cytopathic effect of the virus
in the "treatment" plan of administration (Fig. 3
and 4), and, in this case, the effect of the preparation
in doses of 0.5 % and of 10 % was approximately
the same, but reliably different from the control
(Fig. 4). |
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| Comparison of the data on activity of the pre-
paration with respect to various standard strains of
the flu virus shows that its effect on RHA against
the pandemic strain A(H1N1) is relatively slight
- most of the activity seemed to pertain to strains
A(H3N2) and B. The effect against the standard
vaccine flu strain A(H5N1)-NIBRG-14 was also slight (Table 1). It should be noted, however,
that in this comparative study, the method for
evaluating the cytopathic effect of viruses on MTT
regeneration by the infected cells was not used so
all possible aspects of the effect of the preparation
may not have been disclosed. Nevertheless, the
importance even of the relatively slight positive
effect of Influcid against strains A(H5N1) must be
stressed since the threat of an epidemic of highly
pathogenic "bird" flu should not be removed from
the agenda regardless of the onset of the "swine"
flu pandemic since reports of cases of A(H5N1)
flu with a fatality rate exceeding 50 % appear
constantly in developing countries (7). |
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| Finally, regardless of the simplicity and nature
of the models in vitro, what should be noted in
the study of homeopathic preparations, which, of
course, act mainly at the systemic level, is their
limitations. For this reason, earlier we also confirmed
the antiviral effect of Influcid on an animal model -
white mice infected with lethal and sublethal doses
of a widely known strain A/PR/8/34 (H1N1) (2).
The preparation diluted with a physiological solut-
ion was administered to the mice intraventricularly
once a day for 4 days immediately after infection
with a pharmacologically appropriate dose. Accord-
ing to the index of protection (IP) criterion and
considering the demise of experimental animals
in all periods and with both infectious doses of
the virus of 1LD50 and 0.1LD50, we obtained
IP = 41.2 %, and with the infectious dose of the
virus of 0.1LD50, IP = 75 % (the preparation
is considered active if the IP ≥ 40 %). Thus, the |
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| No. |
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Virus strain |
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Antiviral effect in ΔlgTID50 |
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| 1 |
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A/N.Caledonia/20/99 (H1N1)* |
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1.5 |
| 2 |
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A/Victoria/35/72 (H3N2)* |
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2.0 |
| 3 |
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A/Wisconsin/67/05 (H3N2) |
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1.0 |
| 4 |
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B/Malaysia/2506/04* |
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2.0 |
| 5 |
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A/NIBRG-14 (H5N1)*# |
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1.0 |
| 6 |
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A/duck/Potsdam/1402/6/86 (H5N2)+ |
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0.5 |
| 7 |
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A/mallard/NT/12/02 (H7N3)+ |
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0 |
| 8 |
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A/HongKong/1073/99 (H9N2)+ |
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0 |
| 9 |
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A/California/07/09 (H1N1)*## |
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0.72 |
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| Note: *- human flu viruses; *#- vaccine strain A(H5N1); *##- pandemic virus A(H1N1) (virus of "swine" or
"Mexican" flu) causing a flu pandemic developing since April 2009; +- bird flu virus. |
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| Fig. 1. Antiviral effect of Influcid with respect to the
virus of "swine flu" A/California/0709 (H1N1) sw1 in
MDCK cell culture. Preincubation with the preparation
for 1 hour, infection with the virus, assessment of
results after 24 hours. Test method: MTT. |
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| Fig. 2. Antiviral effect of Influcid with respect to
"swine flu" virus A/California/07/09 (H1N1)sw1.
Preincubation with the preparation for 1 hour. Test
method: MTT, Data with a virus dose of 104TID50 . |
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| Antiviral effect of Influcid with respect to
"swine flu" virus A/California/07/09 (H1N1)sw1
with preliminary infection of MDCK cells with virus
(1 hour before administering the preparation). Test
method: MTT. |
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| Fig. 4. Antiviral effect of Influcid on "swine flu" virus
in an MDCK cell culture with preliminary infection
with virus 1 hour before administering the preparation.
Virus dose: 104TID50 . |
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| effectiveness of the preparation was demonstrated
both on the whole and specifically for an 0.1LD50
infectious dose of the virus (titer of the virus 10-2)
when IP >> 40 %. Statistical processing with non-
parametric methods (Wald-Wolfowitz Runs Test,
program Statistica for Windows 6.0) also indicated
the reliable effect of Influcid in vivo (for a virus
dose of 0.1LD50, P = -3.676; p = 0.000237). A
convincing result was also obtained in processing
with the method of regressive analysis (plotting of
linearized dependence of animals surviving on the
time after infection). |
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Garashchenko, T. I., Complex homeopathic
preparations for treating inflammatory diseases
of the upper respiratory organs. Russian Medical
Journal, 2002, Vol. 20, No. 10. |
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Eropkin, M. Yu., Grigor'eva, V. A., Gudkova,
T. M., .Konovalova, N. I., Lobova, T. G.,
Baibus, D. M., Yaglovskaya, I. B., Activity of
the preparation "Influcid" with respect to viruses
in model systems. MEDLINE Express, 2007,
No. 6, 23-26. |
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Sominina, A. A., Burtseva, E. I., Lobova, T. G.,
et al., Introducing flu viruses into cell cultures
and chick embryos and their identification:
Methodology recommendations. St. Petersburg,
2006, 24 pp. |
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Barenfreund, E., Babich, H., Martin-Alguacil, N.,
Comparison of two in vitro cytotoxicity assays,
the neutral red (NR) and tetrazolium (MTT) tests.
Toxicol. in Vitro, 1988. Vol. 2, No. 1, 1-6. |
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Dawood, F. S., Jain, S., Finelli, et al., Emergence
of a novel swine-origin influenza A(H1Т1) virus
in humans. New Engl. J. Med., 2009. Vol. 361,
1-10. |
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http://www.who.int/csr/disease/swineflu/en/
index.html |
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http://www.who.int/csr/disease/avian_influenza/
en/index.html. |
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Watanabe, W., Konno, K., Ijichi, K., et al. MTT
colorimetric assay system for the screening
of anti-ortho-myxo- and anti-paramyxoviral
agents. J. Virol. Methods, 1994. Vol. 48, No. 23,
257-265. |
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| Author's address: |
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D.B.I. Eropkin, M. Yu.
Director of the Laboratory of Evolutionary
Mutability of NII flu Viruses
Northwestern Branch,
Russian Academy of Medical Sciences
198096 Saint Petersburg, a/ya 52
eropkin@influenza.spb.ru |
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