AU2020104304A4 - A lyophilizing method of duck hepatitis virus type 1 - Google Patents
A lyophilizing method of duck hepatitis virus type 1 Download PDFInfo
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Abstract
The invention discloses a lyophilizing method of duck hepatitis virus type 1, which is
prepared by directly lyophilizing materials containing duck hepatitis virus type 1. The material
mentioned above is allantoic fluid of 8-10-day-old chicken embryo which died after being
inoculated with duck hepatitis virus type 1 in allantoic cavity for 24 hours, or sterile
homogenate mixed solution of allantoic fluid, allantoic membrane, amniotic fluid and embryo
body; The method is simple in operation with low cost, it does not need to add freeze-dried
protective agent and freeze-dried diluent. The prepared lyophilized preparation of duck
hepatitis virus type 1 can be stored at 10°C--80°C, with no obvious change in virus titer, low
requirements of storage conditions and long storage time.
Description
PATENTS ACT 1990
A lyophilizing method of duck hepatitis virus type 1
The invention is described in the following statement:-
A lyophilizing method of duck hepatitis virus type 1
The invention belongs to the field of biotechnology, which relates to the virus
lyophilizing method and its preparation.
Duck viral hepatitis, namely, DVH is a highly lethal infectious disease caused by duck
hepatitis virus(DHV), which is characterized by acute onset, short course of disease, high
morbidity and mortality. Duck viral hepatitis can be divided into three independent
serotypes without serological relationship: type 1, type 2 and type 3. Among them, duck
hepatitis virus type 1 (DHV-1), also known as duck hepatitis A virus(DHAV), is the main
duck hepatitis virus prevalent in China at present, which has caused huge economic losses
and threatened the healthy development of duck industry in China.
At present, there are three genotypes of duck hepatitis A virus, namely DHAV-1,
DHAV-2 and DHAV-3. According to reports, DHAV-1 is popular in China. All three
genotypes belong to the genus Avihepatovirus of the family Picornaviridae.The virus
particles are spherical or spheroid-like, and the nucleocapsid is icosahedron symmetrical,
with a diameter of 20-40nm, without capsule and propagating in cytoplasm. The nucleic
acid is a single strand positive strand RNA without segments. The virus has a certain degree
of resistance to ether, chloroform, methanol, pancreatin, ammonium sulfate and common
disinfectants. It can tolerate acidic environment with pH3.0; It also has strong resistance to heat, and some viruses still survive after being heated at 56°C for 1 hour. Duck hepatitis A virus cannot agglutinate red blood cells of any animal, but it can proliferate in chorioallantoic cavity of duck embryo and chicken embryo, chicken embryo liver cells, duck embryo kidney cells, goose embryo kidney cells, etc.
Preservation of viruses is a very important aspect of virus research. For example, it is
necessary to maintain the genetic stability of viruses when conducting long-term, in-depth
studies of the biological characteristics of a virus strain or comparing similarities and
differences between prevalent virus strains of different ages and regions. The preservation
of weak virus vaccines and inactivated vaccines is also closely related to virus preservation.
The methods of virus preservation include refrigerator (ordinary refrigerator: 4-8°C; low
temperature refrigerator: -20°C~ -40°C; ultra-low temperature refrigerator: -85°C), liquid
nitrogen (-196°C) and freeze-vacuum-drying (lyophilizing). Among them, the freeze
vacuum-drying preservation method is a better method of virus preservation because it is
less likely to cause contamination due to liquid leakage and it has many advantages, such
as longer preservation time, simpler preservation conditions, and lower preservation costs
(without consuming liquid nitrogen). In freeze-vacuum drying preservation method, it is
usually necessary to add a lyophilizing protectant before lyophilizing. The function of the
lyophilizing protectant is to protect the preserved object from damage and reduce death
during the lyophilizing process. There are many types of lyophilizing protectants, such as
bovine/horse serum, sterile milk and so on, but the suitable lyophilizing protectants for
different preserved objects are not necessarily the same, and researchers need to use
appropriate lyophilizing protectants according to the characteristics of the preserved
objects in order to obtain the required preservation effect.
In view of this, the purpose of the present invention is to provide a lyophilizing method
of duck hepatitis virus type 1, which is simple and economical, and the obtained lyophilized
preparation has low requirements on preservation conditions with long preservation time.
After research, the invention provides the following technical scheme:
1. The lyophilizing method of duck hepatitis virus type 1 is that the material with duck
hepatitis virus type 1 can be obtained by direct freeze drying; The material containing duck
hepatitis virus type 1 is allantoic fluid of 8-10-day-old chicken embryo which died after
being inoculated with duck hepatitis virus type 1 in allantoic cavity for 24 hours, or sterile
homogenate mixed solution of allantoic fluid, allantoic membrane, anmiotic fluid and
embryo body.
Further, the serotype of duck hepatitis virus type 1 used is duck hepatitis A virus type
1.
Furthermore, the lyophilizing method of duck hepatitis virus type 1 according to claim
1 or claim 2, which is characterized in that the material containing duck hepatitis virus type
1 is allantoic fluid of 9-day-old chicken embryo which died after being inoculated with
duck hepatitis virus type 1 in allantoic cavity for 24 hours, or sterile homogenate mixture
of allantoic fluid, allantoic membrane, amniotic fluid and embryo body.
Additionally, the lyophilizing method of duck hepatitis virus type 1 according to claim
1, which is characterized in that the lyophilizing conditions are as follows: pre-freezing at
4°C for 0.5 hours, pre-freezing at -20°C for 1 hour, freezing at -70°C overnight, and vacuum
drying for 24-48 hours the next day.
2. The lyophilized preparation of duck hepatitis virus type 1 prepared by the method
mentioned above.
Beneficial effect:
The present invention provides a simple and economical method for preparing the
lyophilized preparation of duck hepatitis virus type 1, without adding freeze-dried
protective agent and freeze-dried diluent, only materials containing duck hepatitis virus
type 1 (allantoic fluid of chicken embryo or sterile homogenate mixture of allantoic fluid,
allantoic membrane, amniotic fluid and embryo body) need to be directly freeze-dried, and
the prepared duck hepatitis virus type 1 lyophilized preparation can be stored at 10 °C
°C.
In order to make the purpose, technical scheme and beneficial effects of the present
invention clearer, the preferred embodiments of the present invention will be described in
detail below. The experimental methods without specific conditions in the preferred
embodiment are usually carried out according to the conventional conditions or the
conditions suggested by the reagent manufacturer.
1. Virus strain
CH60 strain of duck hepatitis virus type 1, which is a weakened virus strain isolated
in our laboratory, has applied for patent (application number: 201310011872.3) and has been preserved as biological material (preservation number: CCTCCNO. V201248).
2. Embryo for experiment
9-day-old SPF chicken embryo.
3. Virus proliferation
CH60 strain of duck hepatitis virus type 1 was diluted 5 times with sterilized
physiological saline, then penicillin and streptomycin were added to reach the final
concentrations of 100IU/mL and 100tg/mL respectively. 20 chicken embryos were
inoculated with 0.2ml/embryo CH60 strain via allantoic cavity. 5 chicken embryos were
inoculated 0.2ml/embryo sterilized physiological saline via allantoic cavity.After
inoculation, chicken embryos were sealed into pinholes with paraffin wax, incubated at 37°C
for 6 days, and irradiated twice a day. On the premise that physiological saline negative
control embryos were alive, the dead chick embryos were discarded within 24 hours. The
dead chick embryos after 24 hours were collected and refrigerated at 4°C overnight.
4. Preparation of freeze-dried virus materials
Take the 10 chicken embryos inoculated with CH60 strain of duck hepatitis virus type
1 collected above, allantoic fluid was sucked by conventional method under aseptic
conditions, and mixed evenly to be used as virus freeze-dried material (allantoic fluid virus);
In addition, take the other 10 chicken embryos inoculated with CH60 strain of duck
hepatitis virus type 1 collected above, allantoic fluid, allantoic membrane, amniotic fluid
and embryo body were collected under aseptic conditions by conventional methods, and
homogenized aseptically as freeze-dried materials of virus (mixed virus).
5. Preparation of freeze-dried virus preparation
Taking the freeze-dried materials of the above two viruses, and injecting them into
ml or 10ml penicillin bottles respectively, with each bottle of 1-2ml, and half-stoppered;
Two lyophilized preparations of duck hepatitis virus type 1 were obtained by pre-freezing
at 4°C for 0.5 hours, pre-freezing at -20°C for 1 hour, freezing at -70°C overnight and
vacuum drying for 24 hours the next day.
Example 2: Changes of characteristics and titer of serum lyophilized preparation of
duck hepatitis virus type 1 before and afterlyophilizing
1. Determination of the 50% lethal dose (ELD5 o) of CH60 strain of duck hepatitis
virus type 1 to chicken embryo
The CH60 strain of duck hepatitis virus type 1 was continuously diluted with sterilized
physiological saline at a gradient of 10 times, namely 101, 10-2, 10-, 10- 4 , 10- 5, 10-6, 10- 7
, -8 and 10-9. The virus suspension of each dilution was inoculated into the allantoic cavity
of 9-day-old chick embryos. Five chick embryos were inoculated with 0.2ml/embryo at
each dilution. At the same time, the positive control group and the physiological saline
negative control group were set up. The inoculated chick embryos were sealed with paraffin
wax and incubated at 37 °C for 6 days. The embryos were irradiated twice a day. The dead
chick embryos were discarded within 24 hours. The number of dead chick embryos after
24 hours was recorded. The LD5 o of CH60 strain of duck hepatitis virus type 1 to chicken
embryos was calculated by Reed-Muench method.
2. Characteristics and titer changes oflyophilized preparation of duck hepatitis virus
type 1 before and after lyophilizing
Observing and recording the characteristic changes of the lyophilized preparation of
duck hepatitis virus type 1 obtained in Example 1 before and after lyophilizing, and
determining the median lethal dose of thelyophilized preparation of duck hepatitis virus
type 1 obtained in Example 1 to chicken embryos before and afterlyophilizing according
to the aforementioned method. The results are shown in Table 1. The titer of thelyophilized
preparation of duck hepatitis virus type 1 obtained in Example 1 is not lower than 90% of
the titer before lyophilizing, and the titer drops within 0.5 titer.
Table 1 Characteristics and titer changes of lyophilized preparation of duck hepatitis
virus type 1 before and afterlyophilizing
Before lyophilizing After lyophilizing Titer(recovered Virus freeze drying materials . . to volume Characteristic Titer Characteristic before lyophilizing) An opal yellowish (or A transparent liquid reddish) reticular solid, Allantoic that is yellowish (or 7.17 which is difficult to form 6.75 fluid virus slightly red) (fragile to powder and Serotype I duck herotype iuc small pieces) and easily hepatitisvirus soluble in water C60 strain
Mixed A reddish brown A light reddish brown 7.32 loose solid formed and 6.84 virus opaquepaste soluble in water
Note: 7.17 represents ELD5 o=10-7 1 7/0.2ml, which means that the virus is diluted 10717
times, and inoculation of 0.2ml per embryo can kill 50% of chicken embryos; Other titer
data has the same meaning.
Example 3 Changes of titer of serum lyophilized preparation of duck hepatitis virus type 1 under different storage conditions
Taking the freeze-dried virus preparation described in Example 1 and storing it at
4-10°C, 0°C, -20°C--15°C and -80--70°C for different time, and determining its median
lethal dose to chicken embryo according to the above method. The results are shown in
table 2. The lyophilized preparation of duck hepatitis virus type 1 obtained in example 1
has good preservation effect at -80 -70°C, -20 -15°C and 0°C, and the virus titer has no
obvious decrease and the preservation time is long; It can also be stored for at least 1 year
at 4 to 10°C.
Table 2 Changes of titer of lyophilized preparation of CH60 strain of duck hepatitis
virus type lunder different preservation conditions
Preservation Titer time time Sample -80--70 0 C -20--15 0 C 0°C 4~100 C
Lyophilized preparation of allantoic fluid 6.75 6.75 6.75 6.75 3 months lyophilized preparation of mixed poison 6.84 6.84 6.84 6.84
Lyophilized preparation of allantoic fluid 6.75 6.75 6.75 6.75 6 months lyophilized preparation of mixed poison 6.84 6.84 6.84 6.84
Lyophilized preparation of allantoic fluid 6.75 6.75 6.75 6.5 9 months lyophilized preparation of mixed poison 6.84 6.84 6.84 6.63
Lyophilized preparation of allantoic fluid 6.75 6.75 6.75 6.5 12 months lyophilized preparation of mixed poison 6.84 6.84 6.84 6.63
Lyophilized preparation of allantoic fluid 6.75 6.5 6.5 3.5 15 months lyophilized preparation of mixed poison 6.84 6.63 6.63 3.67
Lyophilized preparation of allantoic fluid 6.75 6.43 6.43 _ 18 months lyophilized preparation of mixed poison 6.84 6.46 6.46 _
Lyophilized preparation of allantoic fluid 6.43 6.43 6.43 _ 24 months lyophilized preparation of mixed poison 6.46 6.46 6.46 _
Note: 6.75 represents ELDso=10-6- 7 5/0.2ml, which means that the virus is diluted 106-75
times, and inoculation of 0.2ml per embryo can kill 50% of chicken embryos; Other titer
data has the same meaning.
Example 4 Immune effect of lyophilized preparation of CH60 strain of duck hepatitis
type Ion ducklings
The effect of lyophilized preparation of CH60 strain of duck hepatitis virus type 1 is
determined by measuring the titer of neutralizing antibody in serum of immunized
ducklings and the resistance of immunized ducklings to DHV-I with 10,000 times ELDo.
20 one-day-old Tianfu meat ducks, Cherry Valley ducks, Sichuan Ma ducks,
Jianchang ducks and Aobaixing ducks (all tested by ELISA without anti-DHV-I antibody)
were randomly divided into two groups, with 10 ducks in each group, and one immune
dose of serotype I duck was injected into each muscle of the first group, while 0.2ml of
sterilized physiological saline was injected into each muscle of the second group as the
blank control, and they were kept in isolation. On the 7th day after immunization, blood
samples were collected to measure the titer of neutralizing antibody against DHV-1. The
results are shown in Table 3.
Table 3 Neutralized antibody titers of CH60 strain of duck hepatitis type 1 against
DHV-1 indifferent ducklings
Tianfu meat Cherry Valley Sichuan Ma Jianchang Aobaixing duck Duck breed duck duck duck duck
Neutralized 6.2 6.0 6.3 6.4 6.1 antibody titer
Then, cherry valley ducks were selected for the virulent challenge experiment,
specifically, each duck was injected with DHV-1 virulent virus 10,000 times of ELD5 o (at
least 8/10 ducks died of duck viral hepatitis within 10 days after inoculation), which are
kept in isolation, and the clinical manifestations were observed and recorded in detail. At
the same time, the pathological changes were observed and recorded by necropsy, and the
microscopic pathological changes were observed and recorded by making liver histological
sections. The results are shown in Table 4.
Table 4 Results of DHV-1 challenge of 1-day-old Cherry Valley ducklings immunized
with lyophilized preparation of CH60 strain of duck hepatitis virus type 1for 7 days
After 7 days of immunization, DHV with 10 thousand times ELD5 o was inoculated, and observing for 7 days
Clinical symptoms and pathological changes of liver Weight
Immunization Control group Immunization Control variation analysis* group group group
Five died, and the rest were in poor condition. There are bleeding foci of 552.3 411.2 The difference is Normal different sizes in the liver, and more extremely significant lymphocytes infiltrate in the lobules.
Note: the analysis refers to the significant analysis of x2 test with normal control
ducks (weight refers to the average weight of living ducks in grams)
It can be seen from Table 3 and Table 4 that the titers of neutralizing antibodies against
DHV in duck serum 7 days were all ' 26-0 after1-day-old ducklings were immunized with
lyophilized preparation of duck viral hepatitis attenuated live vaccine (CH60 strain), and
they were effectively protected after being inoculated with DHV virulent virus 10,000
times of ELD50, without harmful clinical symptoms and normal liver histology. In the non
immune control group, 5 ducks died 7 days after inoculation with virulent virus, and the
rest of them were in poor condition. However, the growth and development of non dead
ducks were significantly affected, and their weight was significantly lower than that of
immunized ducks (Table 4). Therefore, the lyophilized preparation of CH60 strain of duck
hepatitis virus type 1 stored at 4-10 °C for half a year has a good effect.
Example 5 Immune effect of lyophilized preparation of CH60 strain of duck hepatitis
virus type Ion breeding ducks
To determine the effect of lyophilized preparation of CH60 strain of duck hepatitis
virus type 1, the titer of neutralized antibody against DHV-I in serum was determined at
different time after the preparation was immunized against ducks.
The lyophilized preparation of CH60 strain of serotype I duck hepatitis virus type 1
CH60 stored at 0°C for one year prepared in example 1 was used to immunize Tianfu meat
duck, Cherry Valley duck, Sichuan Ma duck, Jianchang duck and Aobaixing duck at the
age of 22 weeks (no anti-DHV-I antibody was detected by ELISA) for the first time, and
the booster immunization was carried out at the age of 24 weeks, each immunization dose
was 1 feather/duck and injected into leg muscle. Ten ducks in each group were randomly
collected on days 15, 45, 75, 105, 135, 165, 195 and 270 after immunization, and the
neutralized antibody titers of anti-DHV-I were measured. Detailed observation and clinical records were made during the period. The results are shown in Table 5.
Table 5 Titers of anti-DHV-1 neutralized antibodies in duck serum at different times
after immunization
Titer of neutralized antibody against DHV-1 in sera of breeding ducks at different time (days) after immunization (average number of 10 ducks) Duck Breed 15d 45d 75d 105 d 135 d 165 d 195 d 270d
Tianfu meat 211.2 2-1.4 210-8 29.6 28-9 28.2 27-9 26.4 duck
Cherry 211.2 211.2 210-6 29-4 28-8 28.4 27.8 26.3
Valley duck
Sichuan Ma 211-4 211-4 210-8 29.2 28-8 28.2 27.8 26.4 duck
Jianchang 211.2 211.2 210-9 29.2 28.6 28.2 27-7 26.5 duck
Aobaixing 211-4 211.4 210-8 29-4 28-9 28.4 27.8 26.6 duck
It can be seen from Table 5 that the lyophilized preparation of CH60 strain of serotype
I duck hepatitis virus type 1 was immunized for the first time at the age of 22 weeks and
strengthened at the age of 24 weeks, and the titer of anti-DHV neutralized antibody in the
serum for 270 days was 263~266, which indicated that the immunization period of
lyophilized preparation of CH60 strain of serotype I duck hepatitis virus type 1 stored at
°C for one year could reach 9 months before immunization. Therefore, the lyophilized
preparation of CH60 strain of duck hepatitis virus type 1 stored at 0°C for one year has a
good effect.
Therefore, the lyophilized preparation of CH60 strain of serotype I duck hepatitis virus type 1 prepared by the invention can be applied for immune control of duck viral hepatitis, and the most economical way of immune control is to immunize breeding ducks, so that the next generation of ducklings can obtain passive immune protection. According to the immune protection characteristics of duck viral hepatitis, the titer of anti-DHV-I neutralized antibody in serum >26 can be protected.
Claims (5)
1. The lyophilizing method of duck hepatitis virus type 1 is characterized in that the
material with duck hepatitis virus type 1 can be obtained by direct freeze drying; The
material containing duck hepatitis virus type 1 is allantoic fluid of 8-10-day-old chicken
embryo which died after being inoculated with duck hepatitis virus type 1 in allantoic
cavity for 24 hours, or sterile homogenate mixed solution of allantoic fluid, allantoic
membrane, amniotic fluid and embryo body.
2. The lyophilizing method of duck hepatitis virus type 1 according to claim 1, which
is characterized in that this duck hepatitis virus type 1 is duck hepatitis A virus type 1.
3. The lyophilizing method of duck hepatitis virus type 1 according to claim 1 or claim
2, which is characterized in that the material containing serotype I duck hepatitis virus is
allantoic fluid of 9-day-old chicken embryo which died after being inoculated with serum
type I duck hepatitis virus in allantoic cavity for 24 hours, or sterile homogenate mixture
of allantoic fluid, allantoic membrane, amniotic fluid and embryo body.
4. The lyophilizing method of duck hepatitis virus type 1 according to claim 1, which
is characterized in that the lyophilizing conditions are as follows: pre-freezing at 4°C for
0.5 hours, pre-freezing at -20°C for 1 hour, freezing at -70°C overnight, and vacuum drying
for 24-48 hours the next day.
5. The lyophilized preparation of duck hepatitis virus type 1 prepared by the method
of any one of claims I to claim 4.
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| AU2020104304A AU2020104304A4 (en) | 2020-12-24 | 2020-12-24 | A lyophilizing method of duck hepatitis virus type 1 |
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