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Long-Lasting Stability of Vaccinia Virus (Orthopoxvirus) in Food and Environmental Samples

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Poxviruses are known to remain infectious in the scabs of patients for months to years. The aim of this study was to investigate viral stability in storm water, food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm water, storm water supplemented with either fetal calf serum (FCS) or potting soil was stored at two different temperatures (refrigerator, room temperature; 4 degrees C/25 degrees C). In addition, we analysed the viability of VACV M1 on the surface of bread, salad, sausages and gauze bandages stored at 4 degrees C. Samples were titrated in MA 104 cells and the presence of viral DNA was demonstrated by orthopoxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds of food, bandage and water samples except for storm water supplemented with potting soil was possible. Viral DNA was detected in almost all samples by PCR. Prolonged experiments with VACV M1-spiked storm water and storm water supplemented with FCS revealed that samples kept at 4.5 degrees C are infectious for up to 166 days. Our data demonstrate that VACV M1 has a longlasting stability in water and food. The results obtained during this study should be taken into account for risk assessment calculations for poxvirus transmission. Implying that variola virus and vaccinia virus behave in a similar way, our data call for sophisticated countermeasures in cases of a variola release in biological warfare.
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ORIGINAL ARTICLE
Long-Lasting Stability of Vaccinia Virus (Orthopoxvirus) in
Food and Environmental Samples
S. Essbauer, H. Meyer, M. Porsch-O
¨zcu
¨ru
¨mez and M. Pfeffer
Bundeswehr Institute of Microbiology, Neuherbergstr. 11, 80539 Munich, Germany
Introduction
All members of the genus orthopoxvirus (OPV) have a
brick-shaped morphology and a size of about
200 ·300 nm. Four species of the genus OPV are known
to infect humans: cowpox virus (CPXV) that is geograph-
ically restricted to Northern Europe, monkeypox
virus (MPXV) found in primates in Africa, variola virus
(VARV), the agent of smallpox and vaccinia virus
(VACV), the type species of the genus OPV. VACV has
been used in eradicating smallpox and as a vector for
genetic engineering (Staib and Sutter, 2003). Due to the
immunological cross reactivity within the genus OPV,
immunization with VACV also protects against infections
with other viral species of this genus, such as CPXV
(Fenner et al., 1989). However, VACV immunization
exhibits potential adverse effects, especially in first-time
vaccines. After the eradication of variola in 1980, small-
pox vaccination was considered no longer necessary. It
was also abandoned due to the potential adverse effects of
VACV, especially in first-time vaccinated people (Breman
and Arita, 1980). As a consequence, the generation com-
ing of age (over 30 years) is now immunologically naive.
In Europe, humans should be aware of the potential of
zoonotic infections caused by poxviruses. Here, CPXV
infections have been detected in a broad range of species,
e.g. cats, elephants, horses and dogs. In some cases, these
infections caused severe illness in animals and men
(Czerny et al., 1991; Essbauer et al., 2002; Wolfs et al.,
2002).
Recently, for political and ethical reasons, the OPV
monkeypox virus and variola virus again attracted the
interest of the public and of research units. Both OPV are
highly contagious: MPXV is classified as BSL-3, while
VARV is a BSL-4 agent. VARV is the causative agent of
smallpox, a severe and often fatal disease in humans.
Keywords:
Environment; food; orthopoxvirus; stability;
tenacity; vaccinia virus
Correspondence:
S. Essbauer. Bundeswehr Institute of
Microbiology, Neuherbergstr. 11, 80539
Munich, Germany. Tel.: +49 89 31683975;
Fax: +49 89 31683292;
E-mail: sandraessbauer@bundeswehr.org
Received for publication June 14, 2006
Summary
Poxviruses are known to remain infectious in the scabs of patients for months
to years. The aim of this study was to investigate viral stability in storm water,
food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm
water, storm water supplemented with either fetal calf serum (FCS) or potting
soil was stored at two different temperatures (refrigerator, room temperature;
4C/25C). In addition, we analysed the viability of VACV M1 on the surface
of bread, salad, sausages and gauze bandages stored at 4C. Samples were titra-
ted in MA 104 cells and the presence of viral DNA was demonstrated by ortho-
poxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds
of food, bandage and water samples except for storm water supplemented with
potting soil was possible. Viral DNA was detected in almost all samples by
PCR. Prolonged experiments with VACV M1-spiked storm water and storm
water supplemented with FCS revealed that samples kept at 4.5C are infectious
for up to 166 days. Our data demonstrate that VACV M1 has a longlasting
stability in water and food. The results obtained during this study should be
taken into account for risk assessment calculations for poxvirus transmission.
Implying that variola virus and vaccinia virus behave in a similar way, our data
call for sophisticated countermeasures in cases of a variola release in biological
warfare.
Zoonoses and Public Health
ª2007 The Authors
118 Journal compilation ª2007 Blackwell Verlag, Berlin Zoonoses Public Health. 54 (2007) 118–124
VARV has been classified as category A agent by the
CDC. VARV was eradicated in 1980. In contrast, MPXV
is still prevalent in Africa. Outbreaks of human monkey-
pox have been reported in the Democratic Republic of
Congo from 1996 to 1998 (Hutin et al., 2001). Human
monkeypox virus is transmitted by close contact with ani-
mals and also by poorly cooked meat from wild animals
(‘bush-meat’) (Meyer et al., 2002). There is ongoing dis-
cussion that the risk for human infections will rise if
MPXV should inhabit the ecological niche of variola
virus. In 2003, MPXV was introduced into the United
States by African rodents. Seventy cases of MPXV infec-
tions via infected prairie dogs have been reported in
humans (Enserink, 2003; Knight, 2003; Maskalyk, 2003).
Data on the stability of OPV mainly stem from investi-
gation of scabs, vesicle and pustule fluids, lymph, and
purulence of smallpox patients from Africa or India during
the late phase of the eradication campaign (MacCallum
and McDonald, 1957a,b; Herrlich et al., 1967). However,
no detailed data exist on stability, i.e. the survival of OPV
on nutrition products or in surface water. Therefore, we
conducted experiments with vaccinia virus as a model to
determine the stability of OPV ‘deposited’ artificially in/on
food or in the environment, e.g. storm water, and stored at
different temperatures.
Material and Methods
Vaccinia virus
A stock of vaccinia virus strain Munich 1 (VACV M1,
passage 7; Czerny et al., 1989) grown in African green
monkey kidney MA 104 cells (ATCC No. CRL-2378) in
minimum essential medium (MEM) containing 2% fetal
calf serum (FCS) was used for all experiments. Three days
after infection cell cultures showed cytopathic effects con-
sisting of plaque formation followed by cell detachment
and cell lysis. Infected cells were freeze-thawed, and the cell
debris was removed by centrifugation for 10 min at 6000 g.
Virus-containing supernatants were subjected to ultrasoni-
cation for 15 s and the titre of VACV M1 was found to be
10
7.5
TCID
50
/ml.
Stability of VACV M1 in environmental water
Experiment 1
Storm water was collected in a rain barrel in Munich (pH
5.3), and homogenous 100 ml aliquots were prepared
without further treatment or pre-sterilization. Three dupli-
cate sets, consisting of (a) one 100 ml aliquot of storm
water, (b) one 100 ml aliquot of storm water supplemen-
ted with 10% FCS (Biochrom-Seromed, Berlin) and (c)
one 100 ml aliquot of storm water supplemented with
10 g potting soil were inoculated with 5 ml VACV M1
virus stock. Sets were stored in closed, opaque plastic
containers (10 cm ·10 cm ·7 cm) at 4.5C or room
temperature (ranging from 19–23C, average 21.5C).
Aliquot samples of 900 ll were taken daily for (a period
of) 14 days. After supplementation of 100 ll FCS, aliquots
were stored at )20C until use for viral titration or PCR.
Experiment 2
The same sets as described in Experiment 1 were prepared
with larger volumes: 300 ml of the respective sample and
consequently 15 ml of VACV M1 were used. Duplicate
sets were again stored in opaque plastic containers
(10 cm ·10 cm ·7 cm) for 120 days (room temperature
with an average of 21.8 and 18C) and 180 days (4.5C)
respectively. Aliquot samples of 900 ll were taken daily
and treated as described in Experiment 1.
Persistence of VACV M1 in food and gauze bandage
Experiment 3
A piece of bread (4 g), a slice of sausage (‘Bierschinken’,
one layer), a piece of salad (2.5 g) and gauze bandage (6-
fold layer, 2 cm ·2 cm) were placed into Petri dishes, and
inoculated with 1 ml VACV M1 virus stock without allow-
ing further to dry. Duplicate sets containing bread, sausage
and salad were stored at 4C for 1, 5, 7, 10 and 14 days,
whereas the gauze bandage was stored for 1 and 14 days.
To determine viral infectivity, specimens were washed with
1 ml MEM containing 10% FCS. The supernatants were
collected by centrifugation and frozen at )20C until use.
Titration of VACV M1
After freeze-thawing, all samples were centrifuged at
1200 gfor 10 min. Following filtration (0.8 lm), samples
(100 ll) were titrated in log 10 steps. For Experiment 1
and Experiment 3, titration was performed on confluent,
24-h-old MA 104 cells in 24-well plates using standard
methods. Briefly, cell culture medium was discarded and
a 100-ll aliquot of the solution was incubated for 1 h at
37C without shaking. Then, 1 ml MEM with 2% FCS
was added and cells were further incubated at 37C. For
Experiment 2, MA 104 cells (1.5 ·10
5
/well, MEM plus
5% FCS) were seeded in/into 96-well plates and simulta-
neously infected with 100 ll filtrated samples. End titres
(calculated as TCID
50
) were determined 6 days post-
inoculation after staining with crystal-violet according to
a standard protocol (Kaerber, 1931).
Extraction of viral DNA and PCR
Viral DNA was prepared from 400 ll of the frozen ori-
ginal samples by using the QIA amp DNA minikit as
S. Essbauer et al. Stability of Vaccinia Virus
ª2007 The Authors
Journal compilation ª2007 Blackwell Verlag, Berlin Zoonoses Public Health. 54 (2007) 118–124 119
recommended by the manufacturer (Qiagen, Hilden,
Germany). DNA of 400 ll aliquots of storm water sup-
plemented with soil was prepared by using the DNA
stoolkit (Qiagen, Hilden, Germany). An OPV-A27L
gene-specific PCR specific primer set (encoding the
14 kDa protein, primer 14-up: 5¢-ATGGACGGAACTC-
TTTTCCC-3¢; primer 14-low: 5¢-TAGCCAGAGA-
TATCATAGCCGC-3¢) was used for screening as
described in detail previously (Chantrey et al., 1999;
Essbauer et al., 2002). In order to determine the sensitiv-
ity of the PCR reaction in comparison with the viral titre
of VACV M1 in cell cultures, serial dilutions of VACV
M1 (10
)1
–10
)12
) were prepared in MEM. One aliquot
was used for the titration of the virus, another aliquot
was used for the isolation of viral DNA as described
above. Viral titre was determined according to the proto-
col by Kaerber (1931) as described above. DNA samples
of the serially diluted virus stock were analysed by PCR.
Detection limits of titration and PCR were calculated on
the basis of inoculum volume and dilution factors and
compared.
In parallel, a real-time PCR specific for the A27L gene
of OPV (Olson et al., 2004) was used for the investigation
of the DNA samples obtained from Experiments 1 to 3.
Here, DNA was amplified in 50 cycles using the fast start
DNA Master SYBR Green I Kit (Roche, Mannheim,
Germany) and the LightCycler (Roche Applied Science,
Mannheim, Germany).
Results
Stability of VACV M1 in environmental water
The first set of experiments revealed that the stability of
VACV M1 depends on the environmental temperature
and is different in storm water, storm water supplemen-
ted with FCS and potting soil. Representative results of
one experiment are shown in Fig. 1 and Table 1. For
storm water kept at 4.5C, the initial virus titre persisted
on the same level over the 2-week period (Fig. 1a). In
contrast, in storm water kept at room temperatures viable
VACV M1 was only detected until day 4 (Fig. 1b). How-
ever, in the specimen taken from storm water supplemen-
ted with potting soil, virus was propagated in MA 104
cells only for 3 days (room temperature) or 6 days
(4.5C) respectively. In comparison, for both tempera-
tures titres of VACV M1 in water supplemented with FCS
were comparable to the initial values.
Besides the determination of viability or infectivity of
VACV M1 by virus titration in cell culture, the presence
of viral DNA was investigated by conventional and light-
cycler PCR in parallel. The presence of OPV DNA yielded
expected 280 bp amplicons by conventional PCR. In
addition, comparison of the detection limit of virus pro-
pagation in cell culture to that of the conventional OPV
PCR revealed that PCR is 200 times more sensitive than
virus titration (data not shown).
For all conducted experiments on the stability of
VACV M1 DNA results of both PCRs were consistent as
summarized in Table 1. DNA isolated from storm water
and storm water with FCS was detected by PCR at all
time-points. Positive PCR results were obtained for all
samples taken at both temperatures and for all duplicates.
Specific DNA amplification also occurred in aliquots
taken from storm water with potting soil and kept at
4.5C. However, DNA was only detected until day 13 in
samples including soil and kept at room temperature
(Table 1).
In order to evaluate the long-term infectivity of VACV
M1 in storm water a further set of experiments was set
up (Experiment 2). In slightly acidic (pH 5.4–5.7) non-
sterile storm water stored at 4C VACV M1 was stable
over a period of 56 days. Viable orthopoxvirus was detec-
ted for up to 166 days in storm water supplemented with
FCS and kept at 4C. The corresponding data varied in
0
01234567891011121314
Room temperature
Day
logTCID50/100 µl
Stormwater (SW)
SW and FCS
SW and soil
4.5°C
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Day
logTCID50/100 µl
Stormwater (SW)
SW and FCS
SW and soil
3.5
3
2.5
2
1.5
1
0.5
3.5
3
2.5
2
1.5
1
0.5
(a)
(b)
Fig. 1. Stability of vaccinia virus-spiked storm water at different tem-
peratures. VACV titres of storm water (SW), storm water supplemen-
ted with fetal calf serum (FCS), storm water supplemented with soil
(soil) have been determined following storage at temperatures 4.5C
(a), and room temperature (b) respectively. Representative results of
one experiment of the duplicate sets are shown.
Stability of Vaccinia Virus S. Essbauer et al.
ª2007 The Authors
120 Journal compilation ª2007 Blackwell Verlag, Berlin Zoonoses Public Health. 54 (2007) 118–124
the duplicate long-term experiments at room tempera-
ture: for duplicate 1 (room temperature at an average of
21.8C) VACV M1 was found for 21 days, in the second
duplicate (average temperature of 18C) virus was detec-
ted in cell culture even at day 50.
In summary, VACV M1 had highest stability in sam-
ples supplemented with FCS, a protein-enriched medium
that may reflect biomass, in comparison with pure or
soil-spiked storm water. Even at low temperatures solid
particles in the water had a negative effect on the viability
of the virus. The environmental temperature had a recip-
rocal influence on the duration of the infection, i.e. at
higher temperatures the virus degraded faster than at 4C.
Persistence of VACV M1 in food and gauze bandage
During the course of the duplicate experiments VACV
M1 remained viable on the surface of sausages, salad and
bread and in gauze bandages at 4C for 14 days. Viral
DNA was detected by both OPV-specific PCRs in all
investigated samples. Resulting representative titres of
VACV M1 in MA 104 cell lines are shown in Table 2.
Discussion
VACV M1 was used as a model to analyse the potential
hazard of the exposure of poxviruses in the environment.
Countermeasures have to focus on finding and elimin-
ating an infectious agent in intentionally contaminated
sources to prevent alimentary or contact transmission of
the pathogen. So far, large community outbreaks due to
intentionally contaminated food have been rarely reported
(Hall et al., 2002). Examples are outbreaks in the US
caused by muffins injected with Shigella dysenteria type 2
(Kolavic et al., 1997) and salad artificially contaminated
with Salmonella typhimurium (Torok et al., 1997).
Poxviruses vectors, e.g. ticks for myxoma virus in rab-
bits, ticks or mosquitoes for fowlpox virus in birds, or
houseflies for VARV, bedding (VARV), scabs (all poxvi-
ruses), etc. might play an important role for transmission
of infection. Two main routes of VARV infection exist:
via the skin, e.g. by face-to-face contact, and via the res-
piratory tract by aerosols. Natural alimentary infection
Table 1. Detection of viable vaccinia virus (VACV) by titration of samples and VACV DNA in storm water, storm water supplemented with fetal
calf serum (FCS), storm water supplemented with potting soil (soil) stored at 4.5C, and room temperature (RT) for 14 days
day 01234567891011121314
Storm water
4.5C
Virus +++++++++++++++
DNA+++++++++++++++
RT
Virus ++++)))))))))))
DNA+++++++++++++++
FCS
4.5C
Virus +++++++++++++++
DNA+++++++++++++++
RT
Virus +++++++++++++++
DNA+++++++++++++++
Soil
4.5C
Virus ++++++)))))))))
DNA+++++++++++++++
RT
Virus + + + ))))))))))))
DNA+++++++++++ + + ))
Table 2. Viability of VACV M1 on food and in gauze stored at 4C
for 14 days. Representative results of viral titres of duplicate sets are
shown. All investigated DNA samples of this set of experiment
revealed positive results in the conventional and real-time PCR
day
Titres in log TCID
50
/100 ll
Sausage Bread Salad Gauze
1 2.8 2.5 2.5 2.8
5 2.8 2.8 2.8 n.d.
7 2.8 1.8 2.3 n d.
10 2.5 2.3 2.3 n.d.
14 2.5 2.3 2.3 1.8
n.d., not determined
S. Essbauer et al. Stability of Vaccinia Virus
ª2007 The Authors
Journal compilation ª2007 Blackwell Verlag, Berlin Zoonoses Public Health. 54 (2007) 118–124 121
with VARV has not been reported but for MPXV in
Africa (Fenner et al., 1989; Meyer et al., 2002).
The results of our investigation may help to estimate
the environmental stability of smallpox virus (VARV) or
MPXV. We demonstrate that VACV M1 in slightly acidic
storm water and on the surface of nutrition products and
tissues is a highly ‘stable’ poxvirus. Our data indicate that
poxviruses in the environment might represent as a con-
tinuing source for human infections. MacCallum and
McDonald (1957a,b) showed that VARV is stable in
crusts of patients for 2–4 months depending on the
humidity of the stored material. Infected tissues in hospi-
tals have been a major cause of new epidemics during the
smallpox era. Stability of variola virus minor, Alastrim,
was reduced in comparison with VARV strain major.
Pustule swabs from VARV patients in India mounted on
slides remained infectious for 8 weeks at 30–35C, and
vesicle content kept in medium was infectious for even
9 months (Herrlich et al., 1967). In comparison, Huq
(1976) showed that infectivity of VARV in scabs stored at
35C and 65–68% relative humidity fell off rapidly
(within 3 weeks). Viability of VARV kept at 26C and
85–90% (<10%) relative humidity was 8 (13) weeks.
Scabs stored at 4C and a humidity of 62% in a desicca-
tor revealed viable VARV for 16 weeks (Huq, 1976). Fur-
ther, ectromelia virus, the mouse OPV, was shown to be
stable in liquids or dry spots most (for 5 days) when sup-
plemented with 10% FCS and incubated at 4C. In dried
spots of blood the agent was infectious for 11 days and in
serum for 4 days, respectively (Bhatt and Jacoby, 1987).
However, all mentioned samples were from patients
and contained host cells and proteins. In comparison,
modified vaccinia virus Ankara (MVA) supplemented
with 1% albumin was stable for 8 months when stored at
4 and 20C (Just and Finke, 1979). Recently, Newman
et al. (2003) showed that the VACV vaccine Dryvax kept
at 4C was infectious for at least 1 year. In contrast, up
to 90% of VACV was degraded at room temperature (21–
24C) after storage for 1 year. Repeated freeze-thawing
(Kline et al., 2005) and temperatures not recommended
for shipping of Dryvax and Aventis Pasteur smallpox
vaccine (Lee et al., 2006) have been evaluated recently.
Viability of VACV in aerosols was investigated by
Harper (1961) for time intervals of up to 23 h. VACV was
highly stable at low temperatures (11C) and low (<50%)
relative humidity. Interestingly, an adverse effect of high
relative humidity was observed at high temperatures.
There have been speculations on a high stability of vac-
cinia virus in the environment for a long time. Recently,
infections of a VACV highly homologous to former vac-
cine strains have been reported from Brazil. Here, the
virus was isolated from a reservoir, wild rodents, 30 years
after the last vaccination against smallpox. Thus,
circulation of poxviruses might be ongoing in reservoirs
(da Fonseca et al., 2003). In Belgium antibodies against
VACV were detected in some wild animals (bank voles
and wood mice) in an area where a VACV-based recom-
binant rabies vaccine was distributed to baits in the field
(Boulanger et al., 1996; Masson et al., 1999; Pastoret and
Brochier, 1999).
Based on results from epidemiological studies in Africa
from 1986 to 1995 the WHO declared that the transmis-
sion of MPXV by contact or bush meat (monkey and
rodents) still is necessary to keep the infectious chain of
MPXV alive (Khodakevich et al., 1987; Esposito and
Fenner, 2001). Our results support this conclusion,
because environmental conditions (high environmental
temperature, high humidity) in the endemic regions of
central Africa might have a negative effect on the tenacity
of MPXV. Investigations of Mahnel et al. (1977) revealed
results comparable to our findings: In drinking water (pH
7.52), lake or river water (pH 8.05) from Bavaria the
initial titre of 10
4
TCID
50
/ml VACV Elstree in samples
stored at 10C after was reduced to 10
2)3
TCID
50
/ml after
200 days. Interestingly, there seemed to be no difference
in the viability of the virus between the samples from dif-
ferent sources although pH ranged from 7.3 to 8.1, tem-
perature from 9 to 15C and ion content of the
investigated water samples was divergent. However, in
contrast to this investigation, the authors used sterile-
filtrated river water samples for the inoculation with
VACV (Mahnel et al., 1977). Our study, for the first time,
provides data on the influence of proteins (biomass) or
soil on the viability/stability of VACV. We noticed an
inactivation of viruses and a disintegration of DNA in
samples of water plus soil that might be explained by the
presence of humid acids in the sample. Adsorption of
virus to soil particles might also be critical for the loss of
viability and may vary with the quality of the soil (Ijzer-
mann et al., 1997; Frostegard et al., 1999; Lewis et al.,
2000).
In Germany, vaccinia virus is used as a standard virus
for the analysis of the efficiency of novel desinfectants
(N.N., 2000, 2005). Generally, desinfection of vaccinia
virus is easily achived with commercial available products
(formalin-, ethanol-, halogen-based; Mahnel and Merlin,
1976). Furthermore, the stability of the virus may be crit-
ically influenced by UV light, pH and other factors. Dur-
ing the course of our experiments the containers with the
virus-supplemented samples were stored in the dark. It is
well known that poxviruses undergo rapid recombination
and mutation events or are inactivated by UV light:
e.g. UV light of 254 nm inactivates VACV in drinking
water within 20 s (von Brodorotti and Mahnel, 1982;
Klein et al., 1994; Tsung et al., 1996; Humlova et al.,
2002). Inactivation will differ depending on pH, virus
Stability of Vaccinia Virus S. Essbauer et al.
ª2007 The Authors
122 Journal compilation ª2007 Blackwell Verlag, Berlin Zoonoses Public Health. 54 (2007) 118–124
concentration and properties of the water (natural water
turbidity, depth, surface, ionic concentration). The com-
plex poxvirus morphology, e.g. amount and folding of
structural proteins of the core, the lateral bodies and the
envelope structure, may also have an influence on the
stability of the virus particles.
In this study we confirmed that even conventional PCR
can be a sensitive read out system for detection of viral
DNA. Molecular methods do not give evidence for viable
virus particles but are rapid and sensitive assays for the
detection of viral DNA in comparison with virus titration.
Recently, fast diagnostic tools – real-time PCRs with high
sensitivity and specificity – for differentiation of VARV,
MPXV and other pathogenic OPV in unusual circum-
stances or suspected outbreaks, have been established
(Meyer et al., 2004; Nitsche et al., 2004; Olson et al., 2004;
Panning et al., 2004).
Data on the model virus vaccinia should be watched
critically and may have limitations in respect of transfer
to other OPV. Nevertheless, the results obtained in this
study should be taken into account for risk assessment
calculations for poxvirus transmission. In case of an out-
break of any human pathogenic poxvirus, tracing and
eliminating the source of infection or interrupting trans-
mission chains will be one crucial step to restrict spread-
ing of the virus.
Acknowledgements
This work was performed at the Institute of Medical
Microbiology, Infectious and Epidemic Diseases, Veterin-
ary Faculty, LMU Munich. We thank Ba
¨rbel Ko
¨ttgen,
Gabriele Platzer and Gudrun Zo
¨ller for the excellent tech-
nical assistance. We thank Dr Heike Gehringer for discus-
sion and proof-reading of the manuscript.
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... Essbauer et al. investigated the persistence of the vaccinia species of the orthopoxviruses in stormwater, bandages, and foods. 61 They reported that vaccinia-spiked stormwater and stormwater supplemented with a protein source (i.e., fetal calf serum) kept at 4.5°C remained infectious for up to 166 days, while no vaccina could be isolated from stormwater supplemented with an organic carbon source (i.e., potting soil) to mimic runoff events after 14 days. 61 Silverman and Boehm 15 evaluated this work and suggested that most of the laboratory experiments were run in the dark, thereby increasing persistence and making the results less realistic. ...
... 61 They reported that vaccinia-spiked stormwater and stormwater supplemented with a protein source (i.e., fetal calf serum) kept at 4.5°C remained infectious for up to 166 days, while no vaccina could be isolated from stormwater supplemented with an organic carbon source (i.e., potting soil) to mimic runoff events after 14 days. 61 Silverman and Boehm 15 evaluated this work and suggested that most of the laboratory experiments were run in the dark, thereby increasing persistence and making the results less realistic. Sunlight is known to enhance inactivation of enveloped viruses, such as vaccinia virus (Poxviridae), in laboratory experiments. ...
... 64 69 for 56 days at low temperature and low relative humidity on glass, galvanized steel, and painted cinder block only achieving 2 log reductions; 67 and for 166 days on gauze, on foods, and in stormwater left at 4.5°C. 61 Overall, the available evidence indicates long survival times of poxviruses on fomites. ...
... Even in aerosols, VACV could survived for 23 h, depending on the temperature and humidity [20]. A high stability in food and in the environment has also been demonstrated for VACV [21]. However, common disinfectants are effective and orthopoxviruses including VARV can be inactivated by 70% ethanol, 50% isopropanol, 0.1-2% sodium hypochlorite, or 1% formaldehyde within one minute or by heating at 65 • C for 15 min [21,22]. ...
... A high stability in food and in the environment has also been demonstrated for VACV [21]. However, common disinfectants are effective and orthopoxviruses including VARV can be inactivated by 70% ethanol, 50% isopropanol, 0.1-2% sodium hypochlorite, or 1% formaldehyde within one minute or by heating at 65 • C for 15 min [21,22]. ...
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In the late 1970s, global vaccination programs resulted in the eradication of smallpox. The Monkeypox virus (MPXV), which is closely related to the smallpox-inducing variola virus, was previously endemic only in Sub-Saharan Africa but is currently spreading worldwide. Only older people who have been vaccinated against smallpox are expected to be sufficiently protected against poxviruses. Here I will summarize current knowledge about the virus, the disease caused by MPXV infections, and strategies to limit its spread.
... We investigated the short-term temperature stability of MVA and TT vaccine vectors, which has previously been demonstrated to be stable long-term under a range of temperature conditions. [54][55][56] Both viruses were stored at À80 C, 4 C, and room temperature for 1 week and then active vaccine particles were quantified by plaque assay. Under these conditions, there was no significant drop in titer TT vectors, but replication of MVA was impaired by storage at room temperature ( Figure 1G). ...
... 68 On the other hand, MVA has been tested as a vaccine vector for many diseases, due its well-characterized safety and immunogenicity. 41,69-75 Both vaccines are stable over a range of temperatures, which ensures easy storage and distribution, 55,56 and VACV can also be freeze-dried and stored for extended periods at 25 C without significant impact on immunogenicity. 54 In our study, the comparative analysis of humoral and cellular immune responses between TT and MVA demonstrated that TT is far more effective at stimulating neutralizing antibody production and alloreactive T cell responses against the SARS-CoV-2 RBD (Figure 2). ...
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... Mucocutaneous pustules have been reported after intratumoral or intravenous attenuated oncolytic VACV injections in patients with cancer 9,11,14,[49][50][51][52] . Environmental viral shedding is also a major issue as VACV can remain infectious for a long period in excreta [53][54][55] . ...
... As previously described, viruses can be opsonized by antibodies Biosafety is a major issue with OV. In particular, VACV is known to remain infectious for a long time in urine, feces or the environment [53][54][55] . Safety studies on beagle dogs receiving TK and VGF deleted oncolytic VACV encoding CD40 ligand reported small amounts of viral DNA, but no infectious virus, in urine and saliva after intravenous injections 61 . ...
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Oncolytic virotherapy is an emerging strategy that uses replication-competent viruses to kill tumor cells. We have reported the oncolytic effects of TG6002, a recombinant oncolytic vaccinia virus, in preclinical human xenograft models and canine tumor explants. To assess the safety, biodistribution and shedding of TG6002 administered by the intravenous route, we conducted a study in immune-competent healthy dogs. Three dogs each received a single intravenous injection of TG6002 at 105 PFU/kg, 106 PFU/kg or 107 PFU/kg, and one dog received three intravenous injections at 107 PFU/kg. The injections were well tolerated without any clinical, hematological or biochemical adverse events. Viral genomes were only detected in blood at the earliest sampling time point of one-hour post-injection at 107 PFU/kg. Post mortem analyses at day 35 allowed detection of viral DNA in the spleen of the dog which received three injections at 107 PFU/kg. Viral genomes were not detected in the urine, saliva or feces of any dogs. Seven days after the injections, a dose-dependent antibody mediated immune response was identified. In conclusion, intravenous administration of TG6002 shows a good safety profile, supporting the initiation of clinical trials in canine cancer patients as well as further development as a human cancer therapy.
... Mucocutaneous pustules have been reported after intratumoral or intravenous attenuated oncolytic VACV injections in patients with cancer 9,11,14,[38][39][40][41] . Environmental viral shedding is also a major issue as VACV can remain infectious for a long period in excreta [42][43][44] . ...
... Biosafety is a major issue with OV. In particular, VACV is known to remain infectious for a long time in urine, feces or the environment [42][43][44] . Safety studies on beagle dogs receiving TK and VGF deleted oncolytic VACV encoding CD40 ligand reported small amounts of viral DNA, but no infectious virus, in urine and saliva after intravenous injections 50 . ...
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Oncolytic virotherapy is an emerging strategy that uses replication-competent viruses to kill tumor cells. We have previously reported the oncolytic effects of TG6002, a novel recombinant oncolytic vaccinia virus, in various preclinical human xenograft models and canine tumor explants. To assess the safety, biodistribution and shedding of TG6002 administered by the intravenous route, we conducted a study in four immune-competent healthy dogs. Three dogs each received a single intravenous injection of TG6002 at 1 x 10 ⁵ PFU/kg, 1 x 10 ⁶ PFU/kg or 1 x 10 ⁷ PFU/kg, and one dog received three intravenous injections at 1 x 10 ⁷ PFU/kg. The injections were well tolerated without any clinical, hematological or biochemical adverse events. Viral genomes were only detected in blood one hour after injection. Post mortem analyses allowed detection of viral DNA in the spleen of one dog. Viral genomes were not detected in the urine, saliva or feces of any dogs. Seven days after the injections, a dose-dependent immune response was identified. In conclusion, intravenous administration of TG6002 shows a good safety profile, a finding that supports the initiation of clinical trials in canine cancer patients as well as further development of TG6002 as a human cancer therapy.
... The probable transmission of CPXV by 1 of the patient's likely infected cats, for which CPXVspecific IgG was found, may have occurred without direct contact. Rather, it may have occurred through contact with a contaminated surface or object because of her atopic dermatitis and the ability of robust orthopoxviruses to survive for an extended time in the environment (43,44). ...
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... As with other poxviruses, sheep-and goat pox is susceptible to sunlight and detergents containing lipid solvents, but in dark environmental conditions, such as contaminated animal sheds, it can persist for many months. The source of environmental contamination occurs from infected animals shedding the virus (Essbauer et al., 2007). (OIE, 2012). ...
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... Similar lesions have been described in dogs receiving a TK-deleted VACV [24]. VACV is known to remain detectable in urine and feces for a long time [25][26][27]. Thus, before the use of TG6002 in pet dogs, evaluations of safety and viral shedding are needed. ...
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... Similar lesions have been described in dogs receiving a TK-deleted VACV [24]. VACV is known to remain detectable in urine and feces for a long time [25][26][27]. Thus, before the use of TG6002 in pet dogs, evaluations of safety and viral shedding are needed. ...
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Full-text available
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Enveloped viruses are characterized by a lipid-containing envelope that encapsulates the virion, and they have been the cause of major outbreaks and pandemics. Some enveloped viruses are excreted in feces and other bodily fluids of infected people and animals, raising the question of their fate in the aquatic environment. Consequently, we conducted a systematic review and meta-analysis of the decay rate constants (k) of enveloped viruses from 12 families (i.e., Coronaviridae, Cystoviridae (specifically Phi6), Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Poxviridae, Retroviridae, Rhabdoviridae, Togaviridae) in environmental waters and wastewater to evaluate their decay kinetics and identify the environmental and virus characteristics that influence k. A total of 812 k that met inclusion criteria were identified in the literature, with the number of k for each family ranging from 0 to 560, and the virus family averaged values of k ranging from 0.11 d-1 and 1.85 d-1. Virus type (i.e., genus, species, subspecies, or subtype), method of virus enumeration (i.e., culture-based or (RT-)QPCR), and experimental water matrix type, temperature and sterility were found to have significant effects on k. Additionally, enveloped viruses were found to have statistically significantly greater k than nonenveloped viruses. Multiple linear regression models that allow prediction of log10k as a function of virus type, enumeration method, water temperature, and water type are provided for six virus families that had sufficient data available for model fitting (i.e., Coronaviridae, Phi6, Herpesviridae, Orthomyxoviridae, Rhabdoviridae, Togaviridae). Compiled log10k and multiple regression models can be used to inform management of human and animal waste, operation of water and wastewater facilities, and exposure risks to treatment plant workers and communities living in regions that lack treatment facilities. Given limited data available for some enveloped virus families with a potential water-related transmission route, there is need for additional data collection to aid academic researchers, public health agencies, and water and wastewater professionals involved in outbreak response.
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Designated as poxvirus consulting laboratory by the Robert-Koch-Institute (Berlin), we provide scientific advice regarding any aspects of poxviruses affecting different animals e.g. cats, elephants, swines, birds, and men. Human smallpox was eradicated in the 1980s, and consequently with diminishing vaccination a generation susceptible for other zoonotic poxviruses grows up. Although the epidemiology of orthopoxvirus infections in Germany remains unclear in the last few years we observed a drastic increase of zoonotic poxvirus infections with three case reports on human «cowpox» infections presented here. In two cases we could only retrospectively trace the source of the orthopoxvirus to cats based on seroconversion. In one case a young cat transmitted the virus to three humans whom all developed clinical pox lesions. Beneath the zoonotic potential of cowpoxviruses (CPXV), these viruses exhibit a broad host range. In the year 2000 two elephants (Elephas maximus) of a German travelling circus revealed a fatal orthopoxvirus infection. The animals exhibited many poxviral lesions and died of this infection. Thus we provide the modified vaccinia virus Ankara (MVA) for vaccination of exotic or expensive animals. Classical virological and serological methods as well as molecular biological techniques including PCR, sequencing and restriction fragment patterns of the newly isolated poxviruses show a very close relationship of the investigated CPXV isolates irrespective of their host species. These findings and our long-term data give evidence of an increase of orthopoxviruses infections in animals and men and underline the importance of further investigations on virus transmission and orthopoxvirus reservoirs.
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Induction of the cytopathic effect (CPE) in cells infected with poxvirus seems ubiquitous in that it has been associated with all different strains and preparations of poxviruses, regardless of the replicating status of these viruses. The study of the mechanisms by which CPE is induced by nonreplicating poxviruses is hampered by the lack of any noncytopathic mutant strains and preparations. In this paper, we report on the patterns of gene expression and induction of CPE by vaccinia viruses treated by limited cross-linking with psoralen and long-wave UV light (PLWUV). We show that treatment of cell-free virus with PLWUV could inactivate viral replication without abolishing the ability of the virus to infect cells. Viral transcription as indicated by reporter genes was generally enhanced and prolonged under early viral promoters and abolished under late promoters. Furthermore, increasing the levels of cross-linking with PLWUV resulted in a decrease and abolishment of viral expression of a large reporter gene and a concomitant loss of the induction of CPE. Cells infected with such a virus were able to express the reporter genes and proliferate. The generation of nonreplicating and noncytopathic recombinant vaccinia viruses may help in studies of the mechanisms of CPE induction by poxvirus and may facilitate the use of poxviral vectors in broader areas of research and clinical applications.
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Die Empfindlichkeit gegenüber UV-Licht von 9 verschiedenen Virusarten in wäßriger Suspension wurde vergleichend unter einheitlichen Versuchsbedingungen untersucht. Mit Abstand am labilsten verhielt sich Vacciniavirus (Stamm Elstree). Bei den anderen Virusarten zeichnete sich ab, daß Viren mit doppelsträngiger Nukleinsäure UV-stabiler sind als die mit einsträngiger, daß sich DNS-Viren insgesamt etwas stabiler verhalten als RNS-Viren und daß die UV-Empfindlichkeit mit der Größe der Virusnukleinsäure zunehmen dürfte.
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The effects of clay, humic acid, u.v. light and shellfish tissue residues on the detection of poliovirus type 2 from environmental samples by culture and RT-PCR were investigated. RT-PCR showed 10–100 times greater sensitivity for PV2 detection in the absence of sample contaminants than did culture by plaque assay in BGM cell monolayers. Bentonite clay (100–1000 mg l−1) and shellfish tissue residues reduced virus detection by plaque assay, but the effect of bentonite was mitigated by simple elution procedures. Bentonite clay, humic acid (5–150 mg l−1) and mussel tissue reduced virus detection by RT-PCR by between 1 and 8 logs, although this was mitigated in part by elution and Sephadex filtration of extracts. Sephadex filtration of samples reduced culturable PV2 by 32–50%. Exposure of PV2 in water to u.v. light reduced culturability of PV2 but not detection by RT-PCR. This study demonstrates that virus detection in environmental samples is strongly influenced by naturally occurring substances and disinfection approaches. The accuracy of results of viral analyses of this nature should be carefully scrutinized with respect to sample constituents.
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Es wird ein Verfahren zur kollektiven Behandlung von Reihenversuchen angegeben, das gestattet, auch bei kleinerem Umfang des Kollektivs (Reihenversuch mit vier bis fnf Gruppen zu je sechs Tieren) und bei starker Streuung zu einem zahlenmigen Ausdruck des Versuchsergebnisses zu gelangen.
Article
The stability of nine viruses, Aujeszky, Sindbis, Vesicular Stomatitis, Newcastle Disease, Vaccinia, FMD, HCC, Reo and Teschen virus in drinking and surface water was investigated comparatively at temperatures of 9 and 15 degrees C as well as the influence of water factors like seasonal difference in temperature, pH value, hardness and sort of water. The results can be summarized as follows: 1. At temperatures of 9 to 15 degrees C the majority of the viruses remained stabil in natural water for an astonishing long time. 2. Starting with virus concentration of about 10(4) infectious units per ml Teschen, Vaccinia, Reo, HCC and ND virus could mostly be demonstrated in water longer than 200 days and FMD, Aujeszky, Vesicular Stomatitis and Sindbis virus for 20 to 50 days on average at 9 degrees C. The stability of the viruses investigated decreased in water in the named turn. 3. Based on these results it can be assumed that under natural conditions with very low virus content of some particles the labile viruses such as Toga, Herpes, Rhabdo and pH labile Picorna remain infectious in water for some days. They should not have any importance as water contaminants. More resistant viruses like Paramyxo may keep infectious for weeks and very stabile viruses such as Entero, Reo, Adeno and Pox viruses several weeks to months. 4. As to factors temperature, pH, hardness and sort of water-within the naturally differing range-only the temperature and only in the case of less resistant viruses showed significant influence on the virus stability in water.