CN114847243B - Method for obtaining BmNPV disease pupae by feeding late-stage 5-year-old virus-inoculated silkworms or puncturing and virus-inoculated silkworms - Google Patents

Abstract

The invention discloses a method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding late stage silkworms inoculated with virus of 5 years old or inoculating virus by puncture, which comprises the following steps: the use concentration is 10 ⁵ ‑10 ⁹ Soaking folium Mori in PIB/ml BmNPV virus solution to obtain virus-containing folium Mori; healthy silkworms of a BmNPV sensitive strain at the late 5 th instar are fed with virus-carrying mulberry leaves, 1 of the mulberry leaves fed before the silkworms are mature is ensured to be fed with virus-carrying mulberry leaves, the silkworms mature, cocooning and pupating, and disease pupation is obtained; or selecting a 5-instar silkworm of the BmNPV sensitive strain, feeding virus-carrying mulberry leaves, taking blood of a live silkworm after the disease occurs, inoculating the blood to a healthy silkworm at the late 5-instar of the BmNPV sensitive strain, and cocooning and pupating the mature silkworm to obtain a disease pupa; or selecting a silkworm of the BmNPV sensitive strain at the 5 th instar, feeding the silkworm with virus mulberry leaves, feeding the silkworm with normal mulberry leaves after the silkworm is saturated, taking live silkworm blood when the disease occurs, inoculating the live silkworm blood to the healthy silkworm pupae of the BmNPV sensitive strain which pupates for 24-48 hours, and obtaining the disease pupae when the disease occurs. The invention has the advantages of convenient and simple operation, less impurities, high propagation efficiency and low cost.

Description

The method of feeding infected silkworms at the end of the 5th instar or puncturing infected pupae to obtain BmNPV diseased pupae

technical field

The invention relates to the technical field of silkworm disease-resistant breeding, and more specifically, the invention relates to a method for obtaining pure BmNPV diseased pupae free of other virus pathogens by feeding infected silkworms at the end of the fifth instar stage or puncturing and inoculating with viruses.

Background technique

Bombyx mori blood pyogenic disease (BmNPV) is a subacute infectious disease of silkworm, and it is one of the main diseases that cause production reduction in sericulture. It is especially serious in the high temperature season in tropical Guangxi. The occurrence of this disease is caused by silkworms being infected by silkworm nuclear polyhedrosis virus (BmNPV). Both the environment and objects can be sources of reinfection.

The biological characteristics of silkworms and the high-density feeding method in silkworm clusters in a certain space determine that once the disease occurs, it has the characteristics of large losses and rapid infection. Rather than individual control and treatment. Therefore, breeding for silkworm disease resistance is particularly important. However, silkworm disease resistance breeding generally uses BmNPV for moth area or individual selection, and a large amount of BmNPV is needed in the whole breeding process.

Chinese patent CN108220250A discloses the method of silkworm live puncture inoculation to carry out virus propagation, and the shortcomings of existence are: 1. Silkworm body injection is not easy to operate; When diseased silkworms are collected and broken, the skin and silk glands of large silkworms are not easy to break; 4. Silkworms are still in the stage of eating mulberry, and there are more impurities after crushing, which affects the purification of pathogens; 5. Bombyx mori larvae can be co-infected with various BmNPV, CPV, FV, etc. A variety of viral diseases, it is difficult to prepare BmNPV polyhedrosis virus free from other viral diseases by using BmNPV that is propagated by larvae.

Contents of the invention

It is an object of the present invention to solve at least the above-mentioned disadvantages and to provide at least the advantages which will be described hereinafter.

Another object of the present invention is to provide a method for obtaining pure BmNPV sick pupae that do not contain other viral pathogens by feeding silkworms at the end of the 5th instar silkworm stage or puncturing and inoculating the virus. Poison mulberry leaves or inoculate diseased silkworm blood, so that the silkworms will not appear manic crawling phenomenon during the onset of silkworms after the onset of silkworms. The pupal body is relatively quiet. The silkworm body is easier to break, and the broken solution has less impurities than the broken silkworm in the eating period. It is easy to extract the virus, the propagation efficiency is high, and the cost is low. The obtained pathogen does not contain other viral pathogens.

In order to realize these objects and other advantages of the present invention, the present invention provides a kind of method that obtains the pure BmNPV sick pupa that does not contain other viral pathogens by feeding the end stage silkworm of the 5th instar of inoculation or puncture inoculation, comprising:

Soaking mulberry leaves with BmNPV virus solution with a concentration of 10 ⁵ -10 ⁹ PIB/ml to obtain virus-carrying mulberry leaves;

A: Choose healthy silkworms of the BmNPV sensitive strain at the end of the 5th instar, and feed them with virus-containing mulberry leaves. Make sure that they are fed with mulberry leaves within 2 days before the silkworms mature. One time, the silkworms are fed with virus-containing mulberry leaves. Afterwards, the morbidity obtains described BmNPV sick pupa;

Or B: select silkworms from the 5th instar of the BmNPV sensitive strain, and feed the virus-carrying mulberry leaves. After the silkworms are full, feed the normal mulberry leaves until the live silkworms show typical symptoms, and then take the blood of the live silkworms and inoculate them into 5-year-old BmNPV sensitive strains. Healthy silkworms at the end of instar stage, after the silkworms are mature, go up to cocoon and pupate, and get the diseased pupa of BmNPV disease;

Or C: choose silkworms from the 5th instar of BmNPV sensitive strains, feed the virus-carrying mulberry leaves, and feed the normal mulberry leaves after the silkworms are full, until the live silkworms show typical symptoms, then take the blood of live silkworms and inoculate them until they pupate 24-48 On the healthy silkworm chrysalis of the BmNPV sensitive strain of the hour, the BmNPV diseased pupae are obtained.

According to the characteristics of silkworms, before silkworms mature, they need to eat enough food before they can mature and make cocoons on the cocoon. The cocoon will be formed within 2 days after being placed on the cocoon, and the cocooning and pupation will be completed in about 4 days after being placed on the cocoon, that is, the fifth instar silkworm. The time from the final stage to cocooning and pupation is 4 to 5 days. According to this characteristic, the above-mentioned schemes A, B or C designed by the applicant can ensure that the silkworm disease occurs after the cocooning in the upper cocoon, and there will be no silkworm stage at this stage. Manic crawling phenomenon at the time of onset, the pupal body is relatively quiet, and it is more convenient and simple to operate than the lively silkworm body during puncture inoculation. The virus is extracted, the multiplication efficiency is high, and the cost is low, thereby solving the problems existing in the traditional live silkworm puncture inoculation mentioned in the background art.

In addition, in the process of virus rejuvenation in B and C, the 5th instar silkworms were selected, fed with virus-carrying mulberry leaves, and then the blood of living silkworms was taken. The 5th instar silkworms have several characteristics: first, they are large in size and blood Sufficient, easy to draw blood; second, large food intake, fast growth, can quickly eat virus-carrying mulberry leaves and cause disease; second, in the 5th instar, silkworms in the 5th instar are more sensitive to the virus and have low resistance. Therefore it is easy to get diseased silkworms.

Preferably, in the method of feeding infected silkworms at the end of the 5th instar stage or puncturing and inoculating to obtain pure BmNPV diseased pupae without other viral pathogens, in the above A, the virus concentration of the BmNPV virus liquid used is 10 ⁵ PIB/ ml, the mulberry leaves were soaked in the virus solution for 10 minutes, and then dried to obtain the virus-carrying mulberry leaves;

In B and C, the BmNPV virus liquid with a virus concentration of 10 ⁹ PIB/ml was used, and the mulberry leaves were soaked in the virus liquid for 10 minutes, and then dried to obtain the virus-carrying mulberry leaves.

In the above technical scheme, the feeding of A uses the virus liquid with a relatively low virus concentration of 10 ⁵ , combined with the normal mulberry leaf feed intake of silkworms, it can ensure that most silkworms take in an appropriate amount of virus and avoid excessive intake of virus , In the later stage, cocoons can be cocooned but cannot pupate, and it can also avoid the situation that the amount of virus ingested is too small to cause the disease. The feeding of B and C uses a higher concentration of 10 ⁹ virus liquid, which can make the 5th instar silkworm disease rapidly, and obtain a large amount of live silkworm blood, which improves the efficiency.

Preferably, in the method of feeding infected silkworms at the end of the fifth instar stage or puncturing and inoculating them to obtain pure BmNPV diseased pupae that do not contain other viral pathogens, in said A, specifically, feeding the silkworms with virus on the day of maturation or the day before Mulberry leaves, mature silkworms go to the stack, and immature silkworms continue to feed on normal mulberry leaves.

In the conventional silkworm rearing method, mulberry leaves are fed 1 to 4 times a day, which is also called 1 to 4 times of breeding in the industry, but in the present invention, 2 times of breeding per day are used. There is no difference between the amount of virus mulberry leaves fed and the amount of conventional feeding, and it is enough to ensure that the silkworms are full. For example, based on the moth area (an average of 400 silkworms in a moth area), the 5th instar silkworm is fed with 6 kg of mulberry leaves throughout the silkworm period (calculated as 6 days), so that a moth area is fed with 0.5 kg of leaves at a time.

Preferably, in the method of feeding silkworms at the end of the 5th instar stage of inoculation or puncturing and inoculation to obtain pure BmNPV diseased pupae that do not contain other viral pathogens, in B and C, in order to ensure that silkworms are full of virus-carrying mulberry leaves, After feeding the virus-carrying mulberry leaves, wait for 8 to 12 hours, and then feed with normal mulberry leaves after the virus-carrying mulberry leaves are eaten up or close to being eaten up.

Preferably, in the method of feeding infected silkworms at the end of the 5th instar stage or puncturing and inoculating them to obtain pure BmNPV diseased pupae without other viral pathogens, live silkworms with typical disease symptoms are collected after feeding mulberry leaves with virus for 5-6 days , Use alcohol to disinfect the body surface of live silkworms. After the body surface is dry, cut the tail to collect blood. The collected blood is used for vaccination immediately, or used within 6 hours after refrigerated storage.

Preferably, in the method of feeding infected silkworms at the end of 5th instar stage or puncturing and inoculating to obtain pure BmNPV sick pupae without other viral pathogens, the inoculation method for healthy silkworms at the end of 5th instar stage in B is: detoxification is good extract the live silkworm blood with a syringe, count from the head to the tail of the silkworm body, penetrate the silkworm body wall from any intersegmental membrane of the 6th to 9th links, and inoculate the live silkworm blood;

The inoculation method for the healthy silkworm chrysalis in C is as follows: the blood of the diseased silkworm is extracted from the sterile syringe, the pupa body is counted from the head to the tail, and the pupa skin is gently punctured from the second or third link of the back of the pupa, and the living Silkworm blood was inoculated into silkworm chrysalis.

Preferably, in the method of feeding silkworms at the end of the 5th instar stage of inoculation or puncture inoculation to obtain pure BmNPV diseased pupae that do not contain other viral pathogens, in A and B, after the silkworms mature and go to the bundle, the silkworms will be fed within 25 to 26 hours. ℃ temperature, 65% air humidity and normal growth. After 2 days in the coop, the silkworms were collected, and after another 2 days, the silkworms pupated, and the cocoons were cut to obtain pupae. After 3 to 4 days, the pupae appeared sick, and the diseased pupae were collected and stored at -20°C. ℃;

In the above C, the silkworm pupae after inoculation develop at 25-29°C for 96-120 hours, and in the middle and late stages of the onset, the pupae appear diseased, and the sick pupae are collected and stored at -20°C.

Preferably, in the method for the pure BmNPV diseased pupae that do not contain other viral pathogens in the described method of feeding the silkworm at the end of the 5th instar stage of inoculation or puncture inoculation, specifically include:

Take out and thaw the BmNPV virus stored at -20°C, add sterile water to resuspend, centrifuge at 4000rpm for 10 minutes to remove the supernatant, repeat 2-3 times, wash away the drugs added when saving the virus; then add sterile water to resuspend , using a hemocytometer and an optical microscope to count the BmNPV suspension, and configure it into a virus solution of 1×10 ⁵ PIB/ml for standby;

Soak the mulberry leaves in the virus solution for 10 minutes, fully soak them evenly, take out the surface moisture of the mulberry leaves and dry them to obtain the virus-carrying mulberry leaves for later use;

Select silkworms at the end of the 5th instar of BmNPV sensitive strains, on the day of silkworm maturity or the day before;

Feed the dried virus-carrying mulberry leaves to the silkworms, ensure that mulberry leaves are added three times before ripening, and one time is virus leaves. The mature silkworms are piled up, and the unripe silkworms continue to feed normal leaves;

After the cooked silkworms are put into the cocoon, under the conditions of 25-26°C and 65% humidity, the cocoons are picked after 2 days in the cocoons, and the cocoons are cut after 2 days after pupation. If the link is loose, the pupal body is soft, and the body wall is easily broken, the diseased pupae can be collected and stored at -20°C.

Preferably, in the method for the pure BmNPV diseased pupae that do not contain other viral pathogens in the described method of feeding the silkworm at the end of the 5th instar stage of inoculation or puncture inoculation, specifically include:

Prepare the preserved BmNPV virus into a virus solution with a concentration of 10 ⁹ PIB/ml, take the mulberry leaves and soak them in the virus solution for 10 minutes, pick up the mulberry leaves and dry them to obtain the virus-carrying mulberry leaves;

Add virus-carrying mulberry leaves to the 5th-instar silkworms of BmNPV-sensitive strains to ensure that the silkworms are full. After 8-12 hours, the virus leaves are eaten or nearly eaten, and the normal leaves are used to feed them. Collect them after 5-6 days. For live silkworms with typical symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked spots on the valves, and cloudy blood, disinfect the silkworm body with 75% alcohol, cut the tail horns after drying, and collect blood. At this time, the blood contains a lot of viruses. ;

Use the collected blood for puncture inoculation, and the blood can be used immediately, or stored in cold storage for no more than 6 hours;

Select healthy silkworms at the end of the fifth instar of BmNPV-sensitive strains; use a syringe with a specification of 32G, boil for 30 minutes before use, and then use 75% alcohol for disinfection;

The blood is extracted from the sterile syringe, and inserted into the body wall of the silkworm from any intersegmental membrane of the 6th to 9th link in the direction from the head to the tail of the silkworm, and inoculated with the blood of the BmNPV virus;

After inoculation, the cooked silkworms are placed in cocoons for protection under normal temperature and humidity at 25-26°C and air humidity of 65%. The cocoons are picked after 2 days in the cocoon, and the cocoons are cut after about 2 days after pupation. During 3-4 days, when the link becomes loose, the pupal body becomes soft, and the body wall is easily broken, the diseased pupae can be collected and stored at -20°C.

Preferably, in the method for the pure BmNPV diseased pupae that do not contain other viral pathogens in the described method of feeding the silkworm at the end of the 5th instar stage of inoculation or puncture inoculation, specifically include:

Prepare the preserved BmNPV virus into a virus solution with a concentration of 10 ⁹ PIB/ml, take the mulberry leaves and soak them in the virus solution for 10 minutes, pick up the mulberry leaves and dry them to obtain the virus-carrying mulberry leaves;

Add virus-carrying mulberry leaves to the 5th-instar silkworms of BmNPV-sensitive strains to ensure that the silkworms are full. After 8-12 hours, the virus leaves are eaten or nearly eaten, and the normal leaves are used to feed them. Collect them after 5-6 days. For live silkworms with typical pathological symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked spots on the valves, and blood turbidity, disinfect the silkworm bodies with alcohol with a volume fraction of 75%, cut the tail horns after drying, and collect blood. At this time, the blood contains a large number of viruses;

Use the collected blood for puncture inoculation, and the blood can be used immediately, or stored in cold storage for no more than 6 hours;

Select the healthy and tender silkworm chrysalis of the BmNPV sensitive strain that has pupated for 24-48 hours. At this time, the pupal skin has just hardened, and the pupal body is soaked and disinfected with a bleach solution of 1% available chlorine for 30 seconds, rinsed, dried, and set aside;

Use a syringe with a specification of 32G, boil for 30 minutes before use, and then use 75% alcohol for disinfection;

The decontaminated syringe is used to extract the blood, and the skin of the pupa is lightly punctured at the second or third link of the back of the pupa from the head to the tail of the pupa, and the blood is inoculated into the silkworm pupa;

Put the inoculated silkworm chrysalis under the condition of 25-29 ℃ for 96-120 hours to develop. In the middle and later stages of the disease, the pupal body will relax and the pupa body will start to soften. Then the sick pupae can be collected. The collected sick pupae Store at -20°C.

The present invention at least includes the following beneficial effects:

The method of the present invention has low requirements on equipment and tools, and low requirements on the technical level of personnel; it has low requirements on environmental conditions, and there will be no phenomenon of manic crawling during the onset of the silkworm stage to pollute the environment after the onset of the disease, and has little impact on the safety and cleanliness of the environment; The pupal body is relatively quiet, and the operation of puncture inoculation is more convenient and simple than the lively silkworm body; the pupa body is easier to break than the silkworm body, and the broken solution has less impurities than the broken silkworm in the feeding period, and it is easy to extract the virus; the propagation efficiency High and low cost.

Other advantages, objectives and features of the present invention will partly be embodied through the following descriptions, and partly will be understood by those skilled in the art through the study and practice of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments, so that those skilled in the art can implement it with reference to the description.

It should be understood that terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

Example 1

The method of feeding the infected silkworm at the end of the 5th instar stage or puncturing and obtaining the pure BmNPV sick pupae without other viral pathogens includes:

Take out and thaw the BmNPV virus stored at -20°C, add sterile water to resuspend, centrifuge at 4000rpm for 10 minutes to remove the supernatant, repeat 2-3 times, wash away the drugs added when saving the virus; then add sterile water to resuspend , using a hemocytometer and an optical microscope to count the BmNPV suspension, and configure it into a virus solution of 1×10 ⁵ PIB/ml for standby;

Soak the mulberry leaves in the virus solution for 10 minutes, fully soak them evenly, take out the surface moisture of the mulberry leaves and dry them to obtain the virus-carrying mulberry leaves for later use;

Select silkworms at the end of the 5th instar of BmNPV sensitive strains, on the day of silkworm maturity or the day before;

Feed the dried virus-carrying mulberry leaves to the silkworms, ensure that mulberry leaves are added three times before ripening, and one time is virus leaves. The mature silkworms are piled up, and the unripe silkworms continue to feed normal leaves;

After the cooked silkworms are put into the cocoon, under the conditions of 25-26°C and 65% humidity, the cocoons are picked after 2 days in the cocoons, and the cocoons are cut after 2 days after pupation. If the link is loose, the pupal body is soft, and the body wall is easily broken, the diseased pupae can be collected and stored at -20°C.

Example 2

The method of feeding the infected silkworm at the end of the 5th instar stage or puncturing and obtaining the pure BmNPV sick pupae without other viral pathogens includes:

Prepare the preserved BmNPV virus into a virus solution with a concentration of 10 ⁹ PIB/ml, take the mulberry leaves and soak them in the virus solution for 10 minutes, pick up the mulberry leaves and dry them to obtain the virus-carrying mulberry leaves;

Add virus-carrying mulberry leaves to the 5th-instar silkworms of BmNPV-sensitive strains to ensure that the silkworms are full. After 8-12 hours, the virus leaves are eaten or nearly eaten, and the normal leaves are used to feed them. Collect them after 5-6 days. For live silkworms with typical symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked spots on the valves, and cloudy blood, disinfect the silkworm body with 75% alcohol, cut the tail horns after drying, and collect blood. At this time, the blood contains a lot of viruses. ;

Use the collected blood for puncture inoculation, and the blood can be used immediately, or stored in cold storage for no more than 6 hours;

Select healthy silkworms at the end of the fifth instar of BmNPV-sensitive strains; use a syringe with a specification of 32G, boil for 30 minutes before use, and then use 75% alcohol for disinfection;

The blood is extracted from the sterile syringe, and inserted into the body wall of the silkworm from any intersegmental membrane of the 6th to 9th link in the direction from the head to the tail of the silkworm, and inoculated with the blood of the BmNPV virus;

After inoculation, the cooked silkworms are placed in cocoons for protection under normal temperature and humidity at 25-26°C and air humidity of 65%. The cocoons are picked after 2 days in the cocoon, and the cocoons are cut after about 2 days after pupation. During 3-4 days, when the link becomes loose, the pupal body becomes soft, and the body wall is easily broken, the diseased pupae can be collected and stored at -20°C.

Example 3

The method of feeding the infected silkworm at the end of the 5th instar stage or puncturing and obtaining the pure BmNPV sick pupae without other viral pathogens includes:

Prepare the preserved BmNPV virus into a virus solution with a concentration of 10 ⁹ PIB/ml, take the mulberry leaves and soak them in the virus solution for 10 minutes, pick up the mulberry leaves and dry them to obtain the virus-carrying mulberry leaves;

Add virus-carrying mulberry leaves to the 5th-instar silkworms of BmNPV-sensitive strains to ensure that the silkworms are full. After 8-12 hours, the virus leaves are eaten or nearly eaten, and the normal leaves are used to feed them. Collect them after 5-6 days. For live silkworms with typical pathological symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked spots on the valves, and blood turbidity, disinfect the silkworm bodies with alcohol with a volume fraction of 75%, cut the tail horns after drying, and collect blood. At this time, the blood contains a large number of viruses;

Use the collected blood for puncture inoculation, and the blood can be used immediately, or stored in cold storage for no more than 6 hours;

Select the healthy and tender silkworm chrysalis of the BmNPV sensitive strain that has pupated for 24-48 hours. At this time, the pupal skin has just hardened, and the pupal body is soaked and disinfected with a bleach solution of 1% available chlorine for 30 seconds, rinsed, dried, and set aside;

Use a syringe with a specification of 32G, boil for 30 minutes before use, and then use 75% alcohol for disinfection;

The decontaminated syringe is used to extract the blood, and the skin of the pupa is lightly punctured at the second or third link of the back of the pupa from the head to the tail of the pupa, and the blood is inoculated into the silkworm pupa;

Put the inoculated silkworm chrysalis under the condition of 25-29 ℃ for 96-120 hours to develop. In the middle and later stages of the disease, the pupal body will relax and the pupa body will start to soften. Then the sick pupae can be collected. The collected sick pupae Store at -20°C.

The method for obtaining Bombyx mori nuclear polyhedrosis virus (BmNPV) through the diseased pupae of the present invention is as follows: after breaking the diseased pupae of Examples 1-3, extracting and collecting the extract.

Although embodiments of the present invention have been disclosed above, it is not limited to the applications set forth in the specification and examples. It can be fully applied to various fields suitable for the present invention. Additional modifications can readily be made by those skilled in the art.

Claims (5)

1. The method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding virus-inoculated 5-year-old late stage silkworms or puncturing virus inoculation is characterized by comprising the following steps:

the use concentration is 10 ⁵ -10 ⁹ Soaking folium Mori in BmNPV virus solution of PIB/ml to obtain virus-containing folium Mori；

A: healthy silkworms of a BmNPV sensitive strain at the late 5 th instar are fed with virus-carrying mulberry leaves, the mulberry leaves are fed within 2 days before the silkworms are mature for 1 time, and after the silkworms are mature, cocooning and pupating are carried out, and the disease of the BmNPV disease pupa is obtained;

or B: selecting silkworms of a BmNPV sensitive strain at the 5 th instar, feeding virus-carrying mulberry leaves, feeding normal mulberry leaves instead after the silkworms are fully fed until the living silkworms have a typical symptom, then taking the blood of the living silkworms, puncturing and inoculating the blood to healthy silkworms at the 5 th instar late stage of the BmNPV sensitive strain, and after the silkworms are mature, mounting cocooning and pupating, and then getting the disease of the BmNPV pupae;

or C: selecting silkworms of BmNPV sensitive strain at the 5 th instar, feeding virus-carrying mulberry leaves, feeding normal mulberry leaves after the silkworms are saturated until the living silkworms have a typical disease state, then taking the blood of the living silkworms, and puncturing and inoculating the blood to healthy silkworm pupas of the BmNPV sensitive strain which pupate for 24-48 hours to obtain the BmNPV diseased pupas.

2. The method for obtaining pure BmNPV disease pupae without other viral pathogens by feeding virus-inoculated 5-year-old silkworm or puncturing virus-inoculation as claimed in claim 1, wherein in B and C, in order to ensure that the silkworm is saturated with virus-infected mulberry leaves, after the virus-infected mulberry leaves are fed, the waiting time is 8 to 12 hours, and after the virus-infected mulberry leaves are eaten light or nearly so, the normal mulberry leaves are fed for the second feeding.

3. The method of claim 2, wherein the pure BmNPV disease pupae without other viral pathogens are obtained by feeding virus-inoculated 5-year-old silkworm or puncture virus inoculation, wherein live silkworms with typical symptoms are collected after 5~6 days of virus-inoculated mulberry leaves are fed, blood is collected by cutting tails, and the collected blood is used for puncture inoculation.

4. The method of claim 1 for obtaining pure BmNPV disease pupae free of other viral pathogens by feeding late 5 th instar vaccinated silkworms or stab vaccination, wherein for late 5 th instar healthy silkworms in B: taking a sterile syringe to extract or an inoculating needle to pick up the blood of the live silkworm, and pricking the blood into the healthy silkworm body wall at the end stage of 5 years old to inoculate the blood of the live silkworm;

for healthy silkworm pupae in C: and (3) taking the sterilized injector to extract the blood of the live silkworm, and puncturing and inoculating the silkworm pupa skin into the silkworm pupa.

5. The method for obtaining pure BmNPV diseased silkworm without other virus pathogens through feeding of a virus-inoculated last-stage 5-year-old silkworm or puncture virus inoculation as claimed in claim 1, wherein in A and B, after the silkworms are matured and clustered, the silkworms are collected after being clustered for 2 days, then the silkworms are pupated after 2 days, cocoons are cut to obtain silkworm pupas, the silkworm pupas have symptoms after 3~4 days, and the diseased silkworm pupas are collected;

and C, after the inoculated silkworm pupas grow for 96 to 120 hours, until the disease occurs in the middle and later stages, the disease pupas appear symptoms, and the disease pupas are collected.