Download Using ICR and SCID mice as animal models for smallpox to assess

Journal of General Virology (2015), 96, 2832–2843
DOI 10.1099/vir.0.000216
Using ICR and SCID mice as animal models for
smallpox to assess antiviral drug efficacy
Ksenya A. Titova,1 Alexander A. Sergeev,1 Alena S. Zamedyanskaya,1
Darya O. Galahova,1 Alexey S. Kabanov,1 Anastasia A. Morozova,1
Leonid E. Bulychev,1 Artemiy A. Sergeev,1 Tanyana I. Glotova,2
Larisa N. Shishkina,1 Oleg S. Taranov,1 Vladimir V. Omigov,1
Evgenii L. Zavjalov,3 Alexander P. Agafonov1 and Alexander N. Sergeev1
Correspondence
Artemiy A. Sergeev
[email protected]
1
Federal Budgetary Research Institution – State Research Center of Virology and Biotechnology
VECTOR, Federal Service for Surveillance on Consumer Rights Protection and Human Well-being,
Koltsovo, Novosibirsk region, Russian Federation
2
State Scientific Establishment – Institute of Experimental Veterinary Science of Siberia and the
Far East Russian Academy of Agricultural Sciences, Krasnoobsk, Novosibirsk Region,
Russian Federation
3
Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Prospekt
Lavrentyeva 10, 630090 Novosibirsk, Russian Federation
Received 22 March 2015
Accepted 9 June 2015
The possibility of using immunocompetent ICR mice and immunodeficient SCID mice as model
animals for smallpox to assess antiviral drug efficacy was investigated. Clinical signs of the
disease did not appear following intranasal (i.n.) challenge of mice with strain Ind-3a of variola
virus (VARV), even when using the highest possible dose of the virus (5.2 log10 p.f.u.). The
50 % infective doses (ID50) of VARV, estimated by the virus presence or absence in the lungs 3
and 4 days post-infection, were 2.7¡0.4 log10 p.f.u. for ICR mice and 3.5¡0.7 log10 p.f.u. for
SCID mice. After i.n. challenge of ICR and SCID mice with VARV 30 and 50 ID50, respectively,
steady reproduction of the virus occurred only in the respiratory tract (lungs and nose).
Pathological inflammatory destructive changes were revealed in the respiratory tract and the
primary target cells for VARV (macrophages and epithelial cells) in mice, similar to those in
humans and cynomolgus macaques. The use of mice to assess antiviral efficacies of NIOCH-14
and ST-246 demonstrated the compliance of results with those described in scientific
literature, which opens up the prospect of their use as an animal model for smallpox to develop
anti-smallpox drugs intended for humans.
INTRODUCTION
According to the requirements of the Russian national control
authority (Scientific Center of Expertise of Medical
Application Products – SC EMAP) as well as the US Food
and Drug Administration (FDA) and the European Medicines
Agency (EMA), in order to assess the efficacies of developed
antivirals at the stages of scientific research and clinical studies
it is important to have at least two animal species simulating
the corresponding infectious disease in humans. Only one
animal species has been chosen for smallpox, cynomolgus
macaque: M. fascicularis, synonym M. irus (Jahrling et al.,
2004; Huggins et al., 2009; Chapman et al., 2010). However,
this animal model is expensive and its application in virological
experiments is labour-intensive, making it difficult to use in
large-scale statistically significant experiments. In addition,
despite the fact that the clinical picture caused by variola
2832
virus (VARV) in these primates is relatively close to that in
humans, they are 1000–100 000 000 times less susceptible to
this pathogen following respiratory challenge (Hahon &
Wilson, 1960; Hahon & McGavran, 1961; Noble & Rich,
1969; LeDuc & Jahrling, 2001; Jahrling et al., 2004) than
humans: less than 1 log10 p.f.u. (Drozdov et al., 1987;
Zamedyanskaya et al., 2014). At the same time, these animals
are used only to assess the therapeutic efficacies of antismallpox drugs. To do this, the animals are challenged intravenously with 8 and 9 log10 p.f.u. (Huggins et al., 2009; Mucker
et al., 2013), whereas the main route of VARV infection in
humans is via the respiratory tract. Moreover, the results of
evaluating the efficacies of therapeutic and prophylactic antismallpox drugs using this animal model are applicable for
humans with a normally functioning immune system. Some
existing and emerging anti-smallpox drugs and live smallpox
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ICR and SCID mice as models for smallpox
vaccines (Marennikova & Shchelkunov, 2005) can be harmful
for individuals with immune system pathologies, and their efficacy can differ from that in individuals without this pathology.
The number of people with suppressed immune system function is growing annually due to deteriorating environmental
conditions, which is associated with increasing volumes of
industrial emissions, the spreading HIV epidemic, an increasing number of cancer patients requiring immunosuppressive
therapy, etc.
which the main primary target cells are respiratory tract
macrophages and epithelial cells.
Previously we discovered that primary lung cell cultures
obtained from outbred immunocompetent ICR mice were
sensitive to VARV and capable of supporting virus replication
(Sergeev et al., 2013). We therefore focused on these mice in
our search for an immunocompetent animal model for smallpox to study anti-smallpox drug activity. Using 10–14 day
outbred ICR mice, we attempted to simulate the infectious
process in infants, who are more frequently affected by smallpox with a more severe course than in adults (Fenner et al.,
1988; Riedel, 2005a, b). In addition, outbred mice to a greater
extent than inbred ones reflect the actual physiological state of
the human population. Among immunodeficient animals,
our attention was drawn to outbred immunodeficient SCID
mice, on the one hand, having a combined immunodeficiency
involving T- and B-lymphocytes and immunoglobulins
(Belizário, 2009), and on the other hand, having no changes
in the cells of the mononuclear phagocyte system (macrophages) and many other cells, which is extremely important
for research with orthopoxviruses (including VARV) for
The sensitivity of mice following intranasal
challenge
Viral load
(log10 p.f.u. per lung)
(a)
In the current study, we investigate the possibility of using
ICR and SCID mice as animal models for smallpox to
assess the efficacy of anti-smallpox drugs.
RESULTS
In the first series of experiments, aimed at the determination
of the sensitivity of ICR and SCID mice to VARV, we
attempted to identify external clinical signs of the disease
in animals challenged with the virus in the maximum dose
of 5.2 log10 p.f.u. Challenged mice were observed up to
21 days post-infection (p.i.) but did not display obvious
clinical symptoms. To investigate the possibility of asymptomatic disease in these animals, the dynamics of the pathogen
accumulation in the lungs were studied in ICR and SCID
mice challenged intranasally (i.n.) with the doses of 4.2
and 5.2 log10 p.f.u., respectively. The results of these studies
are presented in Fig. 1(a, b).
The data in Fig. 1(a, b) show that a more significant
accumulation of the virus was observed 2–4 days p.i. in
the lungs of ICR and SCID mice as compared with that
1 day p.i. More pronounced differences were observed in
ICR mice. The 50 % infective dose (ID50) of VARV was
6
*
5
*
*
3
4
4
3
2
1
0
1
2
5
6
5
6
(b)
6
Viral load
(log10 p.f.u. per lung)
Time p.i. (days)
5
*
*
*
3
4
4
3
2
1
0
1
2
Time p.i. (days)
Fig. 1. Accumulation dynamics of VARV strain Ind-3a in the lungs of (a) ICR and (b) SCID mice (n53) challenged i.n. with
the doses of 4.2 and 5.2 log10 p.f.u., respectively. Mean values were plotted on a log scale. *The virus concentration is higher
than at 1 day after challenge, P,0.05.
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K. A. Titova and others
evaluated by its presence or absence in the lungs of ICR and
SCID mice 3 and 4 days p.i., respectively. It was determined
that the values of this index were 2.7+ 0.4 log10 p.f.u. for
ICR mice and 3.5 + 0.7 log10 p.f.u. for SCID mice.
It can be seen that VARV was detected in the lungs of ICR
mice 1 day p.i. Two days p.i. it was found in high concentrations in the lungs and nasal septum with mucosa, but
by day 10 p.i. the virus was not found in these tissues (Fig.
2a). At 3 and 5 days p.i. VARV was found in relatively low
concentrations in the mouse brain. The virus was not
detected in the cells, serum, trachea, oesophagus, liver,
spleen, kidneys and duodenum at any of the time points of
the study from 1 to 10 days p.i. The highest VARV concentration was recorded in the lungs and nasal septum with
mucosa 2 days p.i. The highest concentrations of VARV
were detected in the lungs and nasal septum with mucosa
2–4 days p.i.
Dynamics of the virus dissemination in infected
mice
Taking into account the relatively high sensitivity of mice to
VARV estimated by recording the pathogen presence in the
lungs, the dynamics of VARV dissemination in different
organs, tissues and serum of ICR and SCID mice following
i.n. infection with the doses of 4.2 log10 p.f.u. (30 ID50) and
5.2 log10 p.f.u. (50 ID50), respectively, were studied. The
results of these experiments are presented in Fig. 2(a, b),
respectively.
The data in Fig. 2(b) show that 1 day p.i. the pathogen was
detected only in the lungs of SCID mice; 2 days p.i. the
pathogen was detected in relatively high concentrations
(a) 5.5
5.0
Viral load (log10 p.f.u. ml–1)
4.5
4.0
Brain
3.5
Nasal septum with
mucosa
Lungs
3.0
2.5
2.0
1.5
Threshold
1.0
Inoculated virus*
0.5
0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
10.0
Time p.i. (days)
(b) 5.0
Viral load (log10 p.f.u. ml–1)
4.5
4.0
3.5
Nasal septum with
mucosa
3.0
Lungs
2.5
2.0
Trachea
1.5
Threshold
1.0
Inoculated virus*
0.5
0
1.0
2.0
3.0
4.0
5.0
7.0
Time p.i. (days)
Fig. 2. Accumulation dynamics of VARV strain Ind-3a in samples of (a) ICR and (b) SCID mice (n54) challenged i.n. with the
dose of 4.2 log10 p.f.u. (30 ID50) and 5.2 log10 p.f.u. (50 ID50), respectively. Virus concentration was detected for organs and
tissue in 10 % (by volume) homogenates. Mean values were plotted on a log scale. *The value was determined in 10 % lung
homogenate 10 min after the virus material inoculation.
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Journal of General Virology 96
ICR and SCID mice as models for smallpox
in the lung and trachea and 3 days p.i. the virus appeared
also in the nose (nasal septum with mucosa) while disappearing in the trachea. For 3 days (days 3–5 p.i.) VARV
was detected only in the lungs and nose, and it was no
longer detectable in the latter 7 days p.i., whereas in the
lungs it was titrated at this time in a low concentration
as compared with that of 2–5 days p.i. The virus was not
detected from 1 to 7 days p.i. in other tested samples
(blood cells, serum, brain, oesophagus, liver, spleen, kidneys and duodenum). The highest VARV concentrations
were detected in the lungs 2–5 days p.i. when their values
were within the range from 3.7 to 4.2 log10 p.f.u.
epithelium were observed in the nasal cavity of infected
mice as compared to control animals (Fig. 4a, b). The
number of cell layers considerably increased, and intercellular spaces seemed to be expanded. Moderate diapedesis
of granulocytes and lymphocytes was sometimes observed.
Noticeable structural changes in the glands, muscle elements,
cartilage and bone components were not identified. Unlike
control animals, examination of paraffin and ultrathin sections of trachea of infected mice during this period revealed
clear signs of vacuolar degeneration of epithelium (Fig. 4c,
d), generally similar to those in the nasal cavity observed in
semithin sections. Moreover, changes in epithelial tissue
were accompanied by pronounced mucosal oedema. All
examined samples of the lungs of ICR and SCID mice
5–7 days p.i. showed pronounced inflammatory changes of
the parenchyma with the predominance of intense perivascular oedema and degenerative changes in the bronchial epithelium. The pathological process equally affected both
bronchial wall and alveoli. The formation of atelectasis
was observed in the lungs of experimental animals, while
these pathological changes were absent in control mice
(Fig. 4e–h). Sometimes a pronounced cellular infiltration
was accompanied by the destruction of lung tissue. Lymph
nodes were randomly identified on the preparations. They
showed no apparent disturbances of morphology. Unlike
SCID mice, local degenerative changes in neurons of the
pyramidal layer were detected only in the cortex of most
ICR mice (Fig. 4i, j). The cells were in the state of
balloon dystrophy and intercellular spaces were expanded
to varying degrees. Inflammatory infiltration was absent.
No pathological changes were recorded in other organs
and tissues of ICR and SCID mice (blood cells, bifurcation
In experiments in vitro using cells of the mononuclear phagocyte system (spleen monocyte-macrophage monolayer
primary culture, SMMMPC) obtained from ICR mice,
infectivity assays revealed a significant increase in the biological concentration of the virus as early as 24, 48, 72 and
168 h p.i. The virus reached the maximal level of accumulation in cell cultures 48 h after challenge with the dose of
0.017 p.f.u. per cell and 168 h after challenge with the dose
of 0.003 p.f.u. per cell (Fig. 3).
Pathomorphological changes in mice challenged
intranasally with the virus
We further studied pathomorphological changes in ICR and
SCID mice after i.n. challenge with the pathogen, using
doses of 4.2 log10 p.f.u. (30 ID50) and 5.2 log10 p.f.u. (50
ID50), respectively.
At the beginning and the height of the infectious process
(3–5 days p.i.), foci of vacuolar degeneration of the surface
6
Viral load (log10 p.f.u. ml–1)
5
SMMMPC 0.017
p.f.u. per cell
SMMMPC 0.003
p.f.u. per cell
Control 0.017
p.f.u. per cell*
Control 0.003
p.f.u. per cell*
4
3
2
Threshold
1
0
1
24
48
72
Time p.i. (h)
144
168
Fig. 3. Accumulation dynamics of VARV strain Ind-3a in SMMMPC of ICR mice challenged with the dose of 0.017 or 0.003 p.f.u.
per cell. Mean values were plotted on a log scale (n54). *The value was determined based on the initial quantity of cells collected
before their destruction. 4The virus concentration is higher than at 1 h after challenge with the corresponding dose, P,0.05.
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K. A. Titova and others
(a)
(b)
5 µm
5 µm
(c)
(d)
50 µm
(e)
(f)
100 µm
200 µm
200 µm
(i)
(j)
(k)
100 µm
5 µm
(g)
2 µm
100 µm
(h)
5 µm
(l)
1 µm
Fig. 4. Light and electron microscopy of internal organs of ICR mice and immunodeficient SCID mice after intranasal challenge with VARV strain Ind-3a in the doses of 4.2 and 5.2 log10 p.f.u., respectively, as well as SMMMPC from ICR mice
3 days p.i. with VARV in the dose 0.017 p.f.u. per cell. (a) Electronogram of nasal mucosa of ICR mouse 3 days p.i.:
degenerative changes in mucosal epithelium – the loss of cilia (white arrows), vacuolization of epithelial cell cytoplasm
(black arrows). (b) Electronogram of nasal mucosa of a control ICR mouse: epithelial cell cilia are not damaged (white
arrows), normal epithelial cell cytoplasm (black arrows). (c) Tracheal mucosa of ICR mouse 3 days p.i.: arrows show epithelial layer with vacuolar degeneration of covering epithelial cells (histological preparation, haematoxylin and eosin staining). (d) Tracheal mucosa of a control ICR mouse: unchanged tissue (histological preparation, haematoxylin and eosin
staining). (e) Lungs of ICR mouse 5 days p.i.: pronounced inflammatory with predominance of atelectasis (arrows) and
intense perivascular oedema (histological preparation, haematoxylin and eosin staining). (f) Lungs of SCID mouse 5 days
p.i.: diffuse oedema, vascular congestion and atelectasis in the area of the large bronchus (histological preparation, haematoxylin and eosin staining). (g) Lungs of a control SCID mouse: no changes (histological preparation, haematoxylin and
eosin staining). (h) Lungs of SCID mouse 5 days p.i.: an area of atelectasis, alveolar spaces are sharply narrowed (white
arrows); type II alveolocyte in the centre (black arrow) and normal lumen of the alveoli at the top (electronogram). (i) Brain
of ICR mouse 5 days p.i.: local degenerative changes in neurons of the pyramidal layer; cells in the state of ballon dystrophy (arrows), intercellular spaces are expanded to varying degrees (histological preparation, haematoxylin and eosin staining). (j) Brain of ICR mouse 5 days p.i.: local degenerative changes in neurons of the pyramidal layer; cells in the state of
ballon dystrophy, intercellular spaces are expanded to varying degrees (electronogram). (k) Mouse spleen monocyte–
macrophage from a primary culture of macrophage-like cells prepared in vitro: numerous viral particles can be seen in the
cytoplasm of the destroyed cell (electronogram). (l) Close-up of an area of monocyte–macrophage cytoplasm (see the previous micrograph) demonstrating a typical ultrastructure of mature viral particles (electronogram).
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Journal of General Virology 96
ICR and SCID mice as models for smallpox
lymph nodes, liver, spleen, pancreas, duodenum, mesenteric
lymph nodes, kidneys, adrenals and skin pieces) collected at
different time points.
In in vitro experiments, electron microscopy revealed signs
of VARV reproduction in cells of the mononuclear phagocyte system (spleen monocytes–macrophages) obtained
from ICR mice (Fig. 4k, l).
Drug efficacy in experiments in mice challenged
intranasally with the virus
The next series of experiments assessed the therapeutic and
prophylactic efficacies of ST-246 and NIOCH-14 in ICR
and SCID mice challenged i.n. with VARV, at doses of
3.7 log10 p.f.u. (10 ID50) and 4.5 log10 p.f.u. (10 ID50),
respectively. Tables 1 and 2 present the results of these
studies.
It was noted (Table 1) that 3 days p.i. the number of infected
ICR mice treated with ST-246 and NIOCH-14 and challenged with VARV was significantly (Pj0.05) lower than
in the control group. Moreover, the drugs reduced VARV
production in mouse lungs 3 days p.i. as compared with
the control.
The data in Table 2 show that the number of SCID mice
treated with NIOCH-14 in which VARV concentration
recorded in the lungs 4 days p.i. was significantly lower
than in the control (Pj0.05); however, this was not the
case for ST-246. At the same time, both drugs caused a significant reduction in virus titres in mouse lungs 4 days p.i.
as compared with the control. NIOCH-14 and ST-246
administered in the same doses did not show significant
differences in their anti-smallpox efficacies.
DISCUSSION
Most researchers studying the susceptibilities of different
mouse lines to search for animal models for monkeypox
have aimed at revealing clinical signs of the disease, including
the lethal effect (Americo et al., 2010; Stabenow et al., 2010;
Earl et al., 2015). It was found that inbred CASA, C57BL/6
stat12/2 and CAST/EiJ mice were highly sensitive to monkeypox virus following i.n. infection (LD505100, 47–213 and
680 p.f.u., respectively). Similarly, in experiments involving
VARV, other scientists also tried to find sensitive mice following i.n. and intraperitoneal infection (Murti & Shrivastav, 1957; Mayr & Herrlich, 1960; Kaptsova, 1967).
However, we did not manage to do it. On the
contrary, our research focused on recording not only external signs of the disease, but also the infectious process
(by the virus presence in the primary target organs), which
significantly reduced the sensitivity threshold of the
Table 1. Preventive activity of drugs against VARV strain Ind-3a 3 days after i.n. challenge of ICR mice with the dose of 3.7 log10
p.f.u. (10 ID50)
Index
Mice treated with drugs
orally once daily 1 day before and 2 days p.i.
NIOCH-14
21
Daily dose of administered drug (mg g )
Number of animals used in experiment
VARV concentration in the lungs (log10 p.f.u. per lung) of each mouse,
3 days after i.n. challenge
Mean virus concentration (log10 p.f.u. per lung, M¡I95) in mouse lungs 3 days p.i.
Virus production suppression index (log10)d in the lungs of infected mice
Number and percentage (%) of infected mice 3 days p.i.
Coefficient (%) of protection|| against challenge
ST-246
Control
60
7
2.4
60
7
2.5
*
7
4.5
2.5
2.1
,1.1
,1.1
,1.1
,1.1
2.3¡0.5D (n53)
1.3
3 (43)§
57
2.3
2.3
,1.1
,1.1
,1.1
,1.1
2.4¡0.3D (n53)
1.2
3 (43)§
57
4.5
4.4
3.5
2.8
2.7
2.7
3.6¡0.8 (n57)
ND
7 (100)
ND
n, Number of animals; M, median value; I95, 95 % confidence level; ND , value was not determined.
*Mice of the control group were administered methylcellulose solution with Tween 80, which was used to suspend NIOCH-14 and ST-246.
DSignificant difference from the control (two-tailed Student’s t-test with equal dispersions).
dDifference between log10 of the virus amount in the control and experimental groups of animals.
§Significant difference from the control (Fisher’s exact test, P one-sided #0.05).
||Difference between the percentages of uninfected animals in the experiment and the control. Values ,1.1 are lower than the sensitivity threshold
(1.1 log10 p.f.u. per lung) of the titration method used.
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K. A. Titova and others
Table 2. Preventive activity of drugs against VARV strain Ind-3a 4 days after i.n. challenge of SCID mice with the dose of 4.5
log10 p.f.u. (10 ID50)
Index
Mice treated with drugs orally once daily once
daily 1 day before and for 3 days p.i.
NIOCH-14
Daily dose of administered drug (mg g21)
Number of animals used in experiment
VARV amount in the lungs (log10 p.f.u. per lung, M¡I95) of each mouse
4 days after i.n. challenge
Mean virus concentration (log10 p.f.u. per lung, M¡I95) in the mouse lungs
4 days p.i.
Virus production suppression index (log10)d in the lungs of infected mice
Number and percentage (%) of infected mice 4 days p.i.
Coefficient (%) of protection|| against challenge
ST-246
Control
50
10
2.8¡2.2
50
9
3.1¡0.1
*
8
3.9¡0.8
2.0¡0.1
3.0¡0.1
2.9¡0.6
3.4¡0.7
,1.1
,1.1
,1.1
,1.1
,1.1
2.8¡0.6D (n55)
2.9¡0.2
3.1¡0.2
2.8¡0.4
2.8¡0.2
3.0¡0.5
3.0¡0.4
,1.1
,1.1
3.6¡0.9
4.3¡0.0
3.8¡0.3
4.3¡0.1
3.7¡0.0
4.0¡0.4
4.0¡0.9
3.0¡0.1D (n57)
4.0¡0.2 (n58)
1.2
5 (50)§
50
1.0
7 (78)
22
8 (100)
ND
ND
n, Number of animals; M, median value; I95, 95 % confidence level; ND , value was not determined.
*Mice of the control group were administered methylcellulose solution with Tween 80, which was used to suspend NIOCH-14 and ST-246.
DSignificant difference from the control (two-tailed Student’s t-test with equal dispersions).
dDifference between log10 of the virus amount in the control and experimental groups of animals.
§Significant difference from the control (Fisher’s exact test, P one-sided #0.05).
||Difference between the percentages of uninfected animals in the experiment and the control. Values ,1.1 are lower than the sensitivity threshold
(1.1 log10 p.f.u. per lung) of the titration method used.
method used to estimate the susceptibility of these animals
due to the opportunity of revealing the asymptomatic
course of the disease. This allowed us to obtain the
indices of susceptibility of ICR and SCID mice to VARV,
estimated by the development of an infectious process
(Table 3). Moreover, these dose values were 10–1000 000
times lower than those obtained in the animal model
for smallpox (M. cynomolgus) and 100 000–1 000 000
times lower than those actually used to challenge these
primates with VARV for assessing the efficacies of the developed anti-smallpox drugs (Huggins et al., 2009; Mucker
et al., 2013).
Table 3. Data on human and animal susceptibilities to variola virus
Evaluated indices
Presence of pox-like symptoms
Presence of infectious process
Method
In vivo
In vivo
In vitro
ID50 as log10 p.f.u. (M¡I95) for macroorganisms
Humans
SCID mouse
,1.0*
ND
.5.2
3.5¡0.7
20.02¡0.23d
ND
ICR mouse
.5.2
2.7¡0.4
1.3¡0.5d
M. cynomolgus
4.7–9.0D
ND
ND
ND , No data.
*Value was determined previously (Drozdov et al., 1987).
DValue was obtained from previously presented data (Hahon & Wilson, 1960; Hahon & McGavran, 1961; Noble, 1970;
LeDuc & Jahrling, 2001; Jahrling et al., 2004).
dValue was determined previously (Zamedyanskaya et al., 2014).
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ICR and SCID mice as models for smallpox
Our expectations based on the information about increased
susceptibility to VARV of mice artificially immunosuppressed by Co60 radiation treatment (Shafrikova, 1970)
were not justified in experiments on immunodeficient
SCID mice, which had neither clinical signs of the disease
nor increased (as compared with ICR mice) maximal concentration of the virus in the lungs. Moreover, ID50 value
in SCID mice was, though insignificantly, higher than in
mice ICR. This discrepancy of the obtained results may be
due to the difference in immunodeficiency levels of SCID
mice with severe combined immunodeficiency (deficiency
of T- and B-lymphocytes and immunoglobulins) (Belizário,
2009) and Co60-treated mice.
Noteworthy is the fact that, in addition to the brain and respiratory tract, we could find VARV in virtually none of the
samples from mice during the entire period of observation,
indicating the absence or extremely low sensitivities of cells
of many organs of experimental animals to the virus. This circumstance resulted in limited development of an infectious
process in mice which steadily affected only the primary
target organs of respiratory tract. However, the pathogen
was twice found in very low concentrations in the brains of
ICR mice only. The reduction of VARV concentration to
undetectable levels in the lungs and nasal septum of ICR
mice 10 days p.i. could be due to the influence of the factors
of immune response of these animals.
The fact of the virus replication in the respiratory tract of ICR
and SCID mice following respiratory infection with VARV is to
a certain extent consistent with that in humans and the known
animal model (Hahon & Wilson, 1960; Sarkar et al., 1973a, b;
Jahrling et al., 2004; Marennikova & Shchelkunov, 2005).
Researchers found the maximal virus concentrations in pharyngeal swabs of humans [3–5 log10 pock forming units ml21]
(Sarkar et al., 1973a) and M. cynomolgus (2.0–4.5 log10 p.f.u.
ml21) (Jahrling et al., 2004), as well as in homogenates of nostrils and nasopharynx of M. cynomolgus (2.0–3.3 log10 pock
forming units ml21) (Hahon & Wilson, 1960), similar to our
data on the virus accumulation in the noses of ICR and
SCID mice. However, the duration of the virus presence in
the upper respiratory tract of primates and humans (Hahon
& Wilson, 1960; Marennikova & Shchelkunov, 2005) was
considerably longer (i11 days) than in i.n. challenged mice
(3–6 days). The viral titres (3.8–5.5 log10 pock forming units
ml21) in the lungs of the model animal (M. cynomolgus) following aerosol challenge with the dose of 4.7 log10 pock forming units ml21 were similar (Hahon & Wilson, 1960) to those
obtained by us in ICR and SCID mice challenged i.n. with similar doses of 4.2 and 5.2 log10 p.f.u., respectively. There were no
significant differences in the duration of infectious processes in
the lungs of primates and mice (8–11 days). It is also interesting to note that in experiments involving another representative of orthopoxviruses (ectromelia virus strain Moscow) for
which the range of sensitive animals is restricted exclusively
to mice, a number of researchers infected groups of control
A/NCr mice i.n. (10 and 100 LD50) to assess ST-246 efficacy
(Yang et al., 2005; Quenelle et al., 2007). The maximum
values of the virus concentration in lung homogenates
http://vir.sgmjournals.org
(4.3 and 4.7–5.5 log10 ml21), similar to those obtained by us
in ICR and SCID mice following i.n. challenge with 30 and
50 ID50 of VARV, were recorded (Fig. 2a, b).
Unlike man and the animal model (M. cynomolgus), in
which generalized disease course with the corresponding
symptoms was observed following respiratory infection,
the infectious process in ICR and SCID mice was asymptomatic and was restricted mainly to primary respiratory
organs. This excludes the possibility of using these animals to evaluate the therapeutic efficacy of the developed
anti-smallpox drugs, but supports the possibility of their
use to study their preventive (extreme preventive) activity.
This is due to the fact that preventive (extreme preventive) administration of drugs aims at the virus control
in primary target organs, which are located in human
and mouse respiratory tracts following respiratory
infection.
The conducted comparative analysis of literature regarding
pathological changes in humans and the animal model
(M. cynomolgus) after challenge with VARV via respiratory
tract (Councilman et al., 1904; Lillie, 1930; Bras, 1952;
LeDuc & Jahrling, 2001; Cann et al., 2013) and the results of
our studies in i.n. challenged ICR and SCID mice evidenced
their great similarity regarding the inflammatory destructive
effect on respiratory tract organs.
We attempted to identify the primary target cells for VARV
when carrying out electron microscopy of organs and tissues
of i.n. challenged ICR and SCID mice. The main focus was
placed on the study of the organs of the mouse respiratory
tract given that these organs are the main and almost the
only primary site of the virus reproduction in these animals.
However, these studies were unsuccessful, which could most
likely be explained by insufficiently high concentrations of
the virus in the examined organs and tissues, that in none
of the cases exceeded 5 log10 p.f.u. ml21 of homogenate.
However, signs of VARV reproduction in these cells were
revealed with virological and electron microscopic methods
in experiments in vitro using the cells of the mononuclear phagocyte system (spleen monocytes–macrophages)
obtained from ICR mice (Figs 3 and 4k, l). The results of
these experiments can be extrapolated to SCID mice, given
the fact that severe combined immunodeficiency created in
these mice affected only the T- and B-cell component and
did not cause any changes in other cells of the organism,
including the mononuclear phagocyte (macrophage) systems. VARV accumulation was revealed following titration
of homogenates of trachea and nasal septum with mucosa
in i.n. challenged ICR and SCID mice at 2 and 3 days p.i.
(Fig. 2), which together with the data on inflammatory and
destructive changes in respiratory tract mucosa of these animals indicated the involvement of epithelial cells in the infectious process. In the scientific literature, VARV replication in
cells of the mononuclear phagocyte (macrophage) system
and epithelial cells of the respiratory tract was also noted in
the animal model for smallpox following aerosol and intravenous (i.v.) challenge (Hahon & Wilson, 1960; Jahrling
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2839
K. A. Titova and others
et al., 2004; Wahl-Jensen et al., 2011). At the same time, some
scientists conducting research involving the use of another
representative of orthopoxviruses (vaccinia virus) on
mouse monocyte–macrophage cell culture (J774.G8 cell
line) observed an abortive infection with the infectious process blocked at an early stage (Humlová et al., 2002). This discrepancy between these findings and our results obtained in
mouse monocytes–macrophages can be explained by differences in the types of viruses and cell cultures used.
Almost all indices of interaction of ICR and SCID mice with
VARV, under criteria associated with the search for animal
models to be used for assessment of only prophylactic (emergency and prophylactic) efficacy of anti-smallpox drugs,
are consistent with or similar to those in humans and the
known animal model, cynomolgus macaque. Primates
(M. cynomolgus) are currently used as animal models for variola only to evaluate therapeutic activities of drugs tested in
experiments on i.v. injection of VARV in huge doses (8 and
9 log10 p.f.u.) (Huggins et al., 2009; Mucker et al., 2013).
This is due to the fact that these animals, to a certain extent,
reproduce only the second and the third stages of the infectious
process observed for smallpox in humans: active delivery (and
replication) of the virus via the haematogenous route to secondary target organs (Stage 2) and the virus replication in
secondary target organs (Stage 3). Pathological changes occurring in secondary target organs of these primates were similar
to those observed in humans who died of smallpox. On the
contrary, ICR and SCID mice reproduce only the first stage
of the infectious process (virus replication and pathomorphological changes in respiratory organs; virus replication in the
corresponding target cells), and do not reproduce the second
and third stages. This excludes the possibility of using these
animals to evaluate the therapeutic efficacy of the developed
anti-smallpox drugs, but creates the possibility of their use to
study drug preventive (extreme preventive) activity, which is
not possible with intravenously challenged primates. In this
regard, the studies on the antiviral activity of NIOCH-14
being developed in Russia in comparison with the US drug
ST-246 were conducted in order to verify the adequacy of
results of using such models. The study of the prophylactic
activity levels of these drugs in ICR and SCID mice using
VARV confirmed the presence of a positive effect observed
previously by us and many researchers (Nalca et al., 2008;
Jordan et al., 2009; Smith et al., 2009, 2011; Sergeev et al.,
2015a, b), which indicates the adequacy of animal models
used by us for this purpose, including immunodeficient
SCID mice reflecting the expressed immunosuppressive condition in humans.
Thus, despite the fact that the ICR and SCID mice challenged
i.n. with VARV did not reproduce the clinical picture of a poxlike disease, their susceptibilities to this virus were relatively
high (by recording the pathogen presence in the lungs), and
they simulated the first component of the smallpox infectious
process in humans (or the animal model, cynomolgus macaque), including the virus replication and pathological changes
in respiratory tract and the types of the primary target
cells, as well as adequately demonstrated that the tested
2840
drugs produced antiviral effects following i.n. challenge with
VARV. All this makes possible the actual use of these animals
in experiments involving the use of VARV to assess the prophylactic (emergency and prophylactic) action of the developed anti-smallpox drugs. The study of the therapeutic
activity levels of anti-smallpox drugs can be carried out
using VARV in the previously known model (cynomolgus
macaque).
METHODS
All experiments involving the use of live VARV were conducted at
FBRI SRC VB Vector (Vector), in a maximum containment facility
(BSL-4) using insulating pneumatic suits.
Animals. The studies involved the use of outbred male and female
10–14 day ICR mice (weighing 8–10 g) obtained from Vector’s nursery.
These animals are similar to HA(ICR) mice from Jackson laboratory
and originated from Swiss Webster mice previously purchased by the
Soviet Union. The study also involved male and female 18–21 day
immunodeficient SCID mice (Hairless Outbred SHO-Prkdc scidHr hr
Mouse, weighing 12–14 g) obtained from the SPF-vivarium of
the Institute of Cytology and Genetics (RFMEFI61914|0005 and
RFMEFI62114|0010) where this line came from Charles River
Laboratories in 2012.
Experimental animals were kept on a standard diet with enough water
in accordance with the veterinary legislation and requirements for
humane animal care and use in experimental studies (National
Research Council, 2011). Research and manipulations on animals
were conducted with the approval of Vector’s Bioethics Committee
no. 1-01.2014 dated January 28, 2014.
Cell culture. A continuous Vero cell culture obtained from the Cell
Culture Collection of Vector was used to produce a virus-containing
suspension and to titrate various samples. Vero cell monolayer was
grown in DMEM medium (OJSC BioloT) in the presence of 10 % FBS
(HyClone) supplemented with penicillin (100 IU ml21) and streptomycin (100 mg ml21). The same medium supplemented with 2 % FBS,
penicillin (100 IU ml21) and streptomycin (100 mg ml21) was used as a
supporting one for the virus cultivation.
Virus. The study involved the use of VARV strain Ind-3a from the
State Collection of infectious viral and rickettsial agents of Vector.
Using this strain, a virus-containing suspension having bioconcentrations of 6.7+ 0.1 log10 p.f.u. ml21 was prepared in the cultivation
medium. Virus-containing material was packaged in individual vials
and stored at 270 uC.
Virus infecting doses for mice and preparation of samples.
Upon anaesthesia with inhaled isoflurane, the mice were challenged
i.n. with a total of 0.03 ml of virus-containing fluid to both nostrils.
The animals were challenged with a single dose of 5.2 log10 p.f.u. using
six animals per dose in experiments to assess the sensitivity of ICR
and SCID mice to VARV based on the detection of the disease clinical
signs. To study the dynamics of the virus accumulation in the lungs,
ICR and SCID mice were challenged with infectious doses of 4.2 and
5.2 log10 p.f.u., respectively, and (after euthanasia by cervical dislocation) their whole lungs were taken 1, 2, 3, 4, 5 and 7 days p.i. from
each individual animal using three mice per each time point, and
preparing 10 % homogenates by mechanical destruction with a pestle
in a mortar with river sand.
In experiments to assess the sensitivity of animals to VARV based on the
virus detection in the lungs, ICR and SCID mice were challenged with
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Journal of General Virology 96
ICR and SCID mice as models for smallpox
the virus doses (1.2–5.2 log10 p.f.u.) diluted with 1 log10 steps using six
and four animals per dose, respectively; 3 and 4 days p.i. respectively,
whole lungs were taken after euthanasia to prepare homogenates from
each animal. Data on the presence/absence of VARV in the lungs 3 and
4 days p.i. were used to calculate ID50 of VARV for mice.
In experiments to study the dynamics of the virus accumulation in
organs, tissues and serum, ICR mice were challenged i.n. with a single
dose of the virus (4.2 log10 p.f.u.) followed by collecting samples 1, 2,
3, 4, 5, 6, 7 and 10 days p.i. and using eight animals per each time
point. In similar experiments involving the use of SCID mice, the
latter were also challenged with a single dose of 5.2 log10 p.f.u. followed by collecting samples 1, 2, 3, 4, 5 and 7 days p.i. and using eight
animals per each time point. Before euthanasia, blood samples were
collected from the retroorbital venous sinus of ICR and SCID mice
and used to prepare serum and a clot of formed elements by centrifugation for the subsequent preparation of homogenate. Moreover,
after euthanasia, samples (nasal septum with mucosa, brain, trachea,
bifurcation lymph nodes, lungs, liver, spleen, pancreas, duodenum
and kidneys) were taken from infected animals. Organs and tissues,
including clots of blood cells from each of four individual animals
collected at the corresponding time points, were homogenized for the
subsequent virological investigation.
In experiments to study the dynamics of the virus accumulation in
mice, samples of organs and tissues were collected simultaneously
(blood cells, nasal septum with mucosa, brain, trachea, bifurcation
lymph nodes, lungs, liver, spleen, pancreas, duodenum, mesenteric
lymph nodes, kidneys, adrenals and skin pieces) from four ICR and
SCID mice per each point to study pathological changes.
To study drug efficacy, ICR and SCID mice were challenged with the
doses of 3.7 and 4.5 log10 p.f.u., respectively, and 3 and 4 days p.i.,
respectively, their whole lungs were taken after euthanasia to prepare
homogenates from each individual animal.
Production of primary monolayer cultures of mouse spleen
monocyte– macrophage and infecting them with VARV. To
obtain SMMMPC, the spleen of ICR mice euthanized by cervical
dislocation was homogenized in a test-tube with a glass pestle and
washed with RPMI 1640 medium (Vector). One microlitre of cell
suspension prepared in growth medium (RPMI 1640 medium containing 10 % FBS and 80 mg gentamicin ml21) was placed into each
well of a 24-well culture plate and incubated in an atmosphere containing 5 % CO2 at the temperature of 37 uC for 24 h. After washing
twice with RPMI 1640 medium, the remaining adherent monocytes–
macrophages were infected with VARV at an m.o.i. of 0.003 and 0.017
p.f.u. per cell and cultivated for 3 days in the same medium though
supplemented with 2 % FBS and 80 mg ml21 of gentamicin (supporting medium) under other conditions described above. At each
time point 1, 24, 48, 72, 144 and 168 h p.i. four samples were taken
from four wells after triple freezing and thawing to determine the
biological concentration of the virus. VARV-infected cell monolayers
were washed and removed with a rubber crusher, and samples were
prepared for electron microscopic examination.
As a negative control, we used a suspension of cell debris produced by
triple freezing and thawing of SMMMPC (by cell number) in supporting medium followed by testing for the absence of viable cells
using light microscopy and inoculation onto a culture plate.
Administration of chemical compounds to mice challenged
with VARV. The study involved the use of chemical compounds with
known anti-othopoxvirus activity levels (Yang et al., 2005; Mucker et al.,
2013; Sergeev et al., 2015a, b): NIOCH-14 {7-[N9-(4-trifluoromethyl
benzoyl)-hydrazinecarbonyl]-tricyclo[3.2.2.02,4]non-8-en-6-carbonic acid}
and ST-246 {4-trifluoromethyl-N-[3,3a,4,4a,5,5a,6,6a-octahydro-1,
3-dioxo-4,6-etheno-cycloprop[f ]isoindol-2-(1H-yl)benzamide]}
http://vir.sgmjournals.org
synthesized for the study according to a described technique (Jordan
et al., 2005). Both these drugs had similar mechanisms of antiviral
action, which were associated with blocking the process of formation
of the virion envelope in infected cells negatively influencing the
formation of a pathogenetically significant extracellular enveloped
virus (Jordan et al., 2010; Shishkina et al., 2015).
ICR mice were given orally 0.2 ml of suspension of these compounds
at the dose of 60 mg (g body weight)21 once daily 1 day before
challenge, 2 h, 1 and 2 days after challenge and SCID mice received
the dose of 50 mg (g body weight)21 once daily 1 day before challenge,
2 h, 1, 2 and 3 days after challenge with VARV. As a placebo, we
injected 0.2 ml of a solution containing 0.75 % methylcellulose and
1 % Tween-80 used to prepare NIOCH and ST-246 suspensions.
Virological analysis of samples. Viable virus concentrations in
sera and organ and tissue homogenates of infected animals were
determined with the traditional method of plaque quantification by
inoculating serial dilutions of samples into Vero cell monolayer
(Leparc-Goffart et al., 2005). The minimum amount of the virus that
could be detected by the titration method used was 0.4 p.f.u. ml21
and 1.1 log10 p.f.u. per lung for organ and tissue homogenates, and
0.4 log10 p.f.u. ml21 and 1.1 log10 p.f.u. per lung for serum, while for
samples obtained from the virus cultivation in SMMMPC and the
control, it was 0.7 log10 p.f.u. ml21.
Electron microscopy. Sample preparation and recording of the
results of the study were also conducted as described previously
(Sergeev et al., 2015a).
Statistical treatment of results. Statistical treatment of results was
carried out with standard methods (Zaks, 1976) using the software
package Statistica 6.0 (StatSoft 1984–2001) with assessment of significant differences for Pj0.05 (I95). ID50 doses were calculated using the
Spearman–Karber formula. The proportions of animals with the virus
in the lungs 3 or 4 days p.i. were taken into account in each group of
mice infected with different doses of VARV. In experiments to evaluate
the therapeutic and prophylactic effects of drugs, the comparison of the
proportions of infected animals in groups was conducted by the x 2 test,
while the U-Mann–Whitney test and the Student’s t-test were used to
compare VARV titres in mouse lungs (Zaks, 1976).
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