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
Method for obtaining BmNPV disease pupae by feeding late-stage 5-year-old virus-inoculated silkworms or puncturing and virus-inoculated silkworms Download PDFInfo
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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
Technical Field
The invention relates to the technical field of silkworm disease-resistant breeding, in particular to a method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding late-stage silkworms with virus inoculation of 5 years or puncturing virus inoculation.
Background
Silkworm hemopus (BmNPV) is a subacute infectious disease of silkworms, is one of the main diseases causing the production reduction of silkworm industry, and is particularly serious in the high-temperature season of subtropical regions in Guangxi. The disease is caused by the infection of silkworm by silkworm nuclear polyhedrosis virus (BmNPV), and the infection modes are mainly food infection and wound infection, so that the pathogen-containing silkworm excrement, silkworm corpses and pathogen-containing environments and articles contacted by the sick silkworms can be the sources of re-infection.
The biological characteristics of the silkworms and the high-density feeding mode of silkworm clusters in a certain space determine that once the disease occurs, the silkworm has the characteristics of large loss and quick infection, and also determine that the prevention and treatment of the silkworm hemopus disease are mainly controlled by the clusters, but not controlled and treated by individuals. So that the disease-resistant breeding of the silkworms is very important. The silkworm breeding for disease resistance generally adopts feeding BmNPV to screen moth areas or individuals, and a large amount of BmNPV is needed in the whole breeding process.
Chinese patent CN108220250a discloses a method for live puncture inoculation of silkworms for virus propagation, which has the following disadvantages: 1. the silkworm body injection is not easy to operate; 2. the silkworms can crawl after being attacked by diseases, and have certain risk on environmental pathogenic pollution; 3. when the diseased silkworms are collected and broken, the skin and silk glands of the big silkworms are not easy to break; 4. the silkworm is still in the mulberry stage, and more impurities exist after the silkworm is crushed, so that pathogen purification is influenced; 5. the silkworm larva can be infected with various virus diseases such as BmNPV, CPV, FV and the like, and the BmNPV propagated by the silkworm larva is difficult to prepare the BmNPV polyhedrosis virus without other virus diseases.
Disclosure of Invention
It is an object of the present invention to address at least the above-mentioned deficiencies and to provide at least the advantages which will be described hereinafter.
The invention also aims to provide a method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding and inoculating 5-instar late-stage silkworm or puncturing and inoculating, which feeds the silkworm with toxic mulberry leaves or inoculated with diseased silkworm blood at the late-stage or pupal stage of the 5-instar silkworm, so that after the silkworm is attacked and cocooning, the wild crawling phenomenon during the silkworm attack at the silkworm stage can not occur, the pupae body is quiet, the operation is convenient and simple when puncturing and inoculating is compared with that of an active silkworm body, the pupae body is easier to crush than the silkworm body, the crushed solution has less impurities than the crushed silkworm at the feeding stage, the virus is easy to extract, the propagation efficiency is high, the cost is low, and the obtained pathogens do not contain other virus pathogens.
To achieve these objects and other advantages and in accordance with the purpose of the invention, the present invention provides a method for obtaining pure BmNPV disease pupae free of other viral pathogens by feeding inoculated late 5-instar silkworms or by stab inoculation, comprising:
the use concentration is 10 5 -10 9 Soaking folium Mori in PIB/ml BmNPV virus solution 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 after the silkworms are fully fed until the living silkworms have a typical disease state, then taking blood of the living silkworms, inoculating the blood to healthy silkworms at the late 5 th instar of the BmNPV sensitive strain, and after the silkworms are mature, mounting cocooning and pupating, and getting the BmNPV diseased pupae after the diseases occur;
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, inoculating the blood to healthy silkworm pupas of the BmNPV sensitive strain which pupate for 24-48 hours, and obtaining the BmNPV disease pupas after disease attack.
According to the characteristics of silkworms, cocooning and cocooning can be performed only after the silkworms need to fully eat and eat before maturing, the cocooning is formed within about 2 days after cocooning, cocooning and pupation are completed within about 4 days, namely, the time from the end of a silkworm in the 5 th instar period to cocooning and pupation is 4-5 days, according to the characteristics, the A, B or the C scheme designed by the applicant can ensure that the silkworm morbidity after cocooning and cocooning occurs does not have a fierce crawling phenomenon in the silkworm morbidity in the stage, the pupa body is quiet, the silkworm pupa body is convenient and simple to operate in a puncture inoculation process compared with an alive silkworm pupa body, the silkworm pupa body is easier to crush than the silkworm pupa body, the crushed solution has fewer impurities than the crushed silkworm in the feeding period, viruses are easy to extract, the propagation expansion efficiency is high, the cost is low, and therefore the problems existing in the traditional silkworm pupa puncture inoculation in the background technology are solved.
In addition, in the virus rejuvenation process of B and C, 5-instar silkworm is selected and fed with virus mulberry leaves, then live silkworm blood is taken, and the 5-instar silkworm has the following characteristics: firstly, the body is large, the blood is sufficient, and the blood is easy to draw; secondly, the feed intake is large, the growth is fast, and the virus-carrying mulberry leaves can be fast taken to cause diseases; second, in the 5 th instar, since the silkworms in the 5 th instar are sensitive to viruses and have low resistance, diseased silkworms are easily obtained.
Preferably, in the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding and virus inoculation of 5-year-old silkworm or virus puncture, the virus concentration of the BmNPV virus solution used in the step A is 10 5 PIB/ml, soaking mulberry leaves in virus liquid for 10 minutes, and then airing to obtain the virus-carrying mulberry leaves;
in B and C, the virus concentration of the virus solution using BmNPV is 10 9 And (3) soaking the mulberry leaves in virus solution for 10 minutes by using PIB/ml, and then airing to obtain the virus-carrying mulberry leaves.
In the above technical scheme, the feed of A is 10 5 The virus liquid with lower virus concentration can ensure that most silkworms take proper amount of virus by combining the normal mulberry leaf feed intake of the silkworms, avoid the situation that the virus intake is too large, cocoons can be formed in the later period but the silkworms cannot pupate, and also avoid the situation that the virus intake is too small and the silkworms cannot pupateThe condition of the disease. Feed applications of B and C10 9 The virus liquid with higher concentration can cause the 5-instar silkworm to rapidly attack the disease, thereby obtaining a large amount of live silkworm blood and improving the efficiency.
Preferably, in the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding late-stage 5-year-old silkworm for virus inoculation or puncturing virus inoculation, the step A is to feed virus-carrying mulberry leaves on the day of or before the maturity of the silkworms, the silkworms are matured and clustered, and immature silkworms continue to feed normal mulberry leaves.
In the conventional silkworm breeding method, mulberry leaves are fed for 1 to 4 times in 1 day, which is also called 1 day to 4 times of recovery in the industry, and 1 day and 2 times of recovery are used in the invention. The amount of the virus mulberry leaves fed by the feed is not different from the amount fed by the conventional feed, and the silkworm can be fed by the feed only by ensuring that the silkworm is full of the virus mulberry leaves. For example, 5-year-old silkworms are fed 6kg of mulberry leaves throughout the silkworm rearing period (on the basis of 6 days) in terms of moth zones (400 silkworms on average in 1 moth zone), so that approximately 0.5kg of leaves are fed to 1 moth zone at a time.
Preferably, in the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding silkworm at the late stage of 5-year virus inoculation or puncturing virus inoculation, in the step B and the step C, in order to ensure that the silkworm eats virus-containing mulberry leaves, after the virus-containing mulberry leaves are fed, waiting for 8-12 hours, and after the virus-containing mulberry leaves are eaten light or are close to the light, feeding the silkworm with normal mulberry leaves.
Preferably, in the method for obtaining pure BmNPV disease pupa without other virus pathogens by feeding the silkworm at the late 5 th of virus inoculation or puncturing and virus inoculation, the live silkworm with virus-bearing mulberry leaves for 5-6 days is fed, the body surface of the live silkworm is disinfected by alcohol, the body surface is dried in the air, the tail is cut to collect blood, and the collected blood is immediately used for inoculation, or the cold storage time is not more than 6 hours.
Preferably, in the method for feeding the virus-inoculated 5-year-old late-stage silkworms or performing puncture virus inoculation to obtain pure BmNPV disease pupae without other virus pathogens, the inoculation method aiming at the 5-year-old late-stage healthy silkworms in B is as follows: taking a sterilized injector to extract the blood of the live silkworm, counting the number of the heads of the silkworm bodies towards the tail, pricking the blood into the body wall of the silkworm from any internode membrane of the 6 th to 9 th links, and inoculating the blood of the live silkworm;
the inoculation method aiming at the healthy silkworm pupae in the step C comprises the following steps: taking a sterilized injector to extract the blood of the sick silkworm, counting the head of the silkworm body towards the tail, slightly puncturing the pupa skin from the 2 nd or 3 rd link of the back of the silkworm, and inoculating the blood of the living silkworm into the silkworm pupa.
Preferably, in the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding and virus-inoculation of 5-year-old late-stage silkworms or performing puncture virus-inoculation, in A and B, after the silkworms are mature and cocooning, the silkworms normally grow under the conditions of 25-26 ℃ and 65% air humidity, after cocooning for 2 days, the silkworms are collected and pupated after 2 days, the cocoons are cut to obtain pupae, the pupae is subjected to pathology after 3-4 days, the disease pupae is collected and stored at-20 ℃;
in the step C, the inoculated silkworm pupae develop for 96-120 hours at the temperature of 25-29 ℃, and the disease pupae appear symptoms in the middle and later period of disease attack, and are collected to obtain the disease pupae which are stored in the temperature of-20 ℃.
Preferably, the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding virus-inoculated 5-year-old silkworms or virus-inoculated puncture comprises the following steps:
taking out BmNPV stored at-20 deg.C, thawing, adding sterile water, resuspending, centrifuging at 4000rpm for 10min to remove supernatant, repeating for 2-3 times, and washing off the medicine added during virus storage; then adding sterile water for resuspension, counting the BmNPV suspension by using a blood counting chamber and an optical microscope, and configuring the suspension into 1X 10 5 PIB/ml virus solution for later use;
soaking folium Mori in the virus solution for 10min, soaking thoroughly, taking out, and air drying to obtain virus-carrying folium Mori;
selecting 5-year-old terminal silkworms of a BmNPV sensitive strain on or one day before the mature days of the silkworms;
feeding the aired virus-carrying mulberry leaves to silkworms, ensuring that the mulberry leaves are added 3 times before maturity, wherein 1 time is virus leaves, the mature silkworms are cocooning, and the immature silkworms are continuously fed with normal leaves;
collecting cocoons after cocooning for 2 days after cocooning of mature silkworms at 25-26 ℃ under the condition of humidity of 65%, then cutting cocoons after pupation for 2 days, flatly paving the pupae in a single layer in silkworm nest, and collecting diseased pupae after symptoms of loose links, soft pupae bodies and easy-to-break body walls after 3-4 days, and storing at-20 ℃.
Preferably, the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding virus-inoculated 5-year-old silkworm or virus-inoculated puncture comprises the following steps:
the preserved BmNPV virus was formulated to have a concentration of 10 9 Soaking folium Mori in virus solution of PIB/ml for 10min, taking out, and air drying to obtain virus-carrying folium Mori;
adding virus-carrying mulberry leaves into a 5-instar silkworm of a BmNPV sensitive strain to ensure that the silkworm is fed with full food, feeding the virus leaves with normal leaves after 8-12 hours of light eating or approaching the light eating, collecting live silkworms with typical symptoms of mania and crawling, swelling of links and internode membranes, oil-immersed spots on air valves and turbid blood after 5-6 days, disinfecting silkworm bodies with 75% alcohol, airing, cutting tail horns and collecting the blood, wherein the blood contains a large amount of virus;
puncture inoculation is carried out by collected blood, the blood is taken immediately for use, or the refrigerated storage time is not more than 6h;
selecting healthy silkworms of the BmNPV sensitive strain at the later 5 th instar; using a 32G syringe, boiling for 30 minutes before use, and then using 75% alcohol by volume fraction for disinfection;
taking a sterilized injector to extract the blood, puncturing the blood into the body wall of the silkworm at any internode membrane of the 6 th to 9 th links in the direction from the head to the tail of the silkworm, and inoculating the blood with BmNPV virus;
after inoculation, mature silkworms are cocooned, normal temperature and humidity protection with the air humidity of 65 percent is carried out at the temperature of 25-26 ℃, cocoons are picked after cocooning is carried out for 2 days, then the cocoons are cut after pupation is carried out for about 2 days, pupa single-layer is laid in silkworm nests, the links are loosened after 3-4 days, pupa bodies are softened, the body walls are easy to break, and then disease pupas can be collected and stored at the temperature of-20 ℃.
Preferably, the method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding virus-inoculated 5-year-old silkworm or virus-inoculated puncture comprises the following steps:
the preserved BmNPV virus is prepared to have a concentration of 10 9 Soaking folium Mori in virus solution of PIB/ml for 10min, taking out, and air drying to obtain virus-carrying folium Mori;
adding virus-carrying mulberry leaves into a 5-instar silkworm of a BmNPV sensitive strain to ensure that the silkworm is fed with full food, feeding the virus leaves with normal leaves after 8-12 hours of light eating or near light eating, collecting live silkworms with typical symptoms of mania and crawling, swelling of joints and internode membranes, oil-immersed spots on air valves and turbid blood after 5-6 days, disinfecting silkworm bodies by using alcohol with volume fraction of 75%, airing, cutting tail horns and collecting the blood, wherein the blood contains a large amount of viruses;
performing puncture inoculation by using collected blood, wherein the blood can be taken immediately for use, or the cold storage preservation time does not exceed 6h;
selecting healthy and tender pupa Bombycis of BmNPV sensitive strain which pupates for 24-48 hours, wherein pupa skin just becomes hard, soaking pupa body in bleaching powder solution with effective chlorine of 1% for disinfection for 30s, washing, and air drying body surface water for later use;
using a 32G syringe, boiling for 30 minutes before use, and then using 75% alcohol by volume fraction for disinfection;
taking a sterilized injector to extract the blood, slightly puncturing pupa skin from the 2 nd or 3 rd link of the back of the pupa in the direction from the head to the tail of the pupa, and inoculating the blood into the pupa;
and (3) the inoculated silkworm chrysalis is placed at the temperature of 25-29 ℃ to develop for 96-120 hours, and the pupa body is loosened in link and begins to soften in the middle and later period of disease occurrence, so that the disease pupa can be collected and stored at the temperature of-20 ℃.
The invention at least comprises the following beneficial effects:
the method has low requirements on equipment and tools and low requirements on the technical level of personnel; the requirement on environmental conditions is low, the phenomenon of environment pollution caused by mania and crawling in the disease attack in the silkworm period can not occur after the disease attack, and the influence on the safety and cleanness of the environment is small; the pupa body is quiet, and the operation is more convenient and simpler than that of an active silkworm body during puncture inoculation; the pupa body is easier to break than the silkworm body, and the broken solution has less impurities than the silkworm in the feeding period, so that the virus is easy to extract; high propagation efficiency and low cost.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Example 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 comprises the following steps:
taking out BmNPV stored at-20 deg.C, thawing, adding sterile water, resuspending, centrifuging at 4000rpm for 10min to remove supernatant, repeating for 2-3 times, and washing off the medicine added during virus storage; then adding sterile water for resuspension, counting the BmNPV suspension by using a blood counting chamber and an optical microscope, and configuring the suspension into 1X 10 5 PIB/ml virus solution for later use;
soaking folium Mori in the virus solution for 10min, soaking thoroughly, taking out, and air drying to obtain virus-carrying folium Mori;
selecting 5-year-old terminal silkworms of a BmNPV sensitive strain on or one day before the mature days of the silkworms;
feeding the aired virus-carrying mulberry leaves to silkworms, ensuring that the mulberry leaves are added 3 times before maturity, wherein 1 time is virus leaves, the mature silkworms are cocooning, and the immature silkworms are continuously fed with normal leaves;
collecting cocoons after cocooning for 2 days after cocooning of mature silkworms at 25-26 ℃ and under the condition of humidity of 65%, then cutting cocoons after pupation for 2 days, flatly paving pupa in a single layer in silkworm nest, collecting diseased pupas after symptoms of loose links, soft pupa bodies and easy-to-break body walls after 3-4 days, and storing at-20 ℃.
Example 2
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 comprises the following steps:
the preserved BmNPV virus was formulated to have a concentration of 10 9 Soaking folium Mori in PIB/ml virus solution for 10min, taking out, and air drying to obtain virus-carrying folium Mori;
the mulberry leaves with the virus are added into silkworms of 5 th instar of BmNPV sensitive strain to ensure that the silkworms are full of food, the silkworms are fed by using normal leaves after the virus leaves are subjected to light or close to light after 8-12 hours, live silkworms with typical symptoms of mania, crawl, swelling of links and internode membranes, oil-immersed spots on air valves and turbid blood are collected after 5-6 days, the silkworm bodies are sterilized by 75% of alcohol, and the blood is collected by cutting tail corners after airing, wherein the blood contains a large amount of virus;
puncture inoculation is carried out by collected blood, the blood is taken immediately for use, or the refrigerated storage time is not more than 6h;
selecting healthy silkworms of the BmNPV sensitive strain at the late 5 th age; using a 32G syringe, boiling for 30 minutes before use, and then using 75% alcohol by volume fraction for disinfection;
taking a sterilized injector to extract the blood, puncturing the blood into the body wall of the silkworm at any internode membrane of the 6 th to 9 th links in the direction from the head to the tail of the silkworm, and inoculating the blood with BmNPV virus;
after inoculation, mature silkworms are cocooning, normal temperature and humidity protection with the air humidity of 65 percent is carried out at 25-26 ℃, cocoons are picked after 2 days of cocooning, pupas are cut after 2 days of pupation, pupa are flatly laid in silkworm nests in a single layer mode, link relaxation occurs after 3-4 days, pupa bodies become soft, the body walls are easy to break, and then diseased pupas can be collected and stored at-20 ℃.
Example 3
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 comprises the following steps:
the preserved BmNPV virus was formulated to have a concentration of 10 9 Soaking folium Mori in virus solution of PIB/ml for 10min, taking out, and air drying to obtain virus-carrying folium Mori;
adding virus-carrying mulberry leaves into a 5-instar silkworm of a BmNPV sensitive strain to ensure that the silkworm is fed with full food, feeding the virus leaves with normal leaves after 8-12 hours of light eating or near light eating, collecting live silkworms with typical symptoms of mania and crawling, swelling of joints and internode membranes, oil-immersed spots on air valves and turbid blood after 5-6 days, disinfecting silkworm bodies by using alcohol with volume fraction of 75%, airing, cutting tail horns and collecting the blood, wherein the blood contains a large amount of viruses;
puncture inoculation is carried out by collected blood, the blood is taken immediately for use, or the refrigerated storage time is not more than 6h;
selecting healthy and tender pupa Bombycis of BmNPV sensitive strain which pupates for 24-48 hours, wherein pupa skin just becomes hard, soaking pupa body in bleaching powder solution with effective chlorine of 1% for disinfection for 30s, washing, and air drying body surface water for later use;
using a 32G syringe, boiling for 30 minutes before use, and then using 75% alcohol by volume fraction for disinfection;
taking a sterilized injector to extract the blood, slightly puncturing pupa skin from the 2 nd or 3 rd link of the back of the pupa in the direction from the head to the tail of the pupa, and inoculating the blood into the pupa;
and (3) the inoculated silkworm chrysalis is placed at the temperature of 25-29 ℃ to develop for 96-120 hours, and the pupa body is loosened in link and begins to soften in the middle and later period of disease occurrence, so that the disease pupa can be collected and stored at the temperature of-20 ℃.
The method for obtaining the silkworm nuclear polyhedrosis virus (BmNPV) by the diseased pupa comprises the following steps: the disease pupae of examples 1 to 3 were crushed, and then the extract was extracted and collected.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to 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.
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