Arbovirus (arbovirus) refers to a group of viruses that cause natural epidemic diseases and zoonotic diseases through the bite of sensitive vertebrates by blood-sucking arthropods, including many viral families of different genomes, such as flaviviruses (flaviviridae) flaviviruses (Flavivirus). Lentivirus family (togaviridae) A virus genus (Alphavirus). Bunyaviridae, reoviridae, and orthomyxoviridae. As arthropod-borne viruses, their common characteristic is the ability to reproduce in arthropods, and after a certain external incubation period, the virus is transmitted to a new host by biting and sucking blood. Common arthropod vectors are mosquitoes, cicadas, white ridge, snails, moths. Na, mites, especially mosquitoes and the most important. Birds, bats, primates and domestic animals are the most important vertebrate hosts.
Principle
Epidemic B encephalitis (Japanese encephalitis, JE) is a serious central nervous system, zoonotic natural epidemic disease caused by the B encephalitis virus transmitted by mosquitoes, transmitted between humans or animals, the pathogen was discovered in Japan in 1934, hence the name. Yellow fever (yellow fever, YF) is an acute infectious disease caused by the yellow fever virus and transmitted by mosquitoes, belonging to one of the infectious diseases of international quarantine, mainly endemic in the tropical regions of Central and South America and Africa, with cycles of natural infection occurring periodically between mosquitoes and non-human primates. Dengue fever (DF) is an acute insect-borne infectious disease caused by the transmission of dengue virus through mosquito vectors, with Aedes aegypti mosquitoes as the main vectors, and occurs in tropical and subtropical areas around the world.
Forest encephalitis (tick-borme encephalitis, TBE) is a natural epidemic disease characterized by central nervous system lesions caused by the infection of cicadas-borne encephalitis virus (forest encephalitis virus), known as roach encephalitis in China, and also known as Russian pounding summer encephalitis and Central European encephalitis in foreign countries, with cicadas as the main vector. West Nile fever (West Nile fever, WNF) is an infectious disease caused by the West Nile virus, which was first isolated from the blood of a febrile patient in the West Nile region of Uganda in 1937 and was named after it, and it is a zoonosis. Chikungunya fever (CHIK) is a viral acute infectious disease caused by the Chikungunya virus, transmitted by the bite of the Aedes mosquito, and characterized by fever, rash and severe joint pain." Chikungunya" is phonetically translated from the Swahili vernacular language of Tanzania. As the patient suffers from severe joint pain and is forced to adopt a bending position, the locals describe this position in the vernacular language as "Chikungunya". Mainly endemic in Africa and Southeast Asia, it is a zoonosis. The clinical symptoms of the disease are similar to those of dengue fever, and it is easy to misdiagnose the disease. Although the case fatality rate is very low, it is prone to forming large-scale outbreaks and epidemics in areas with high mosquito vector density.
Operation method
Clinical manifestations and specimen collection of arboviruses
Principle
(a) The incubation period of epidemic encephalitis B is 10-15 days, which can be as short as 4 days or as long as 21 days. Most patients have mild symptoms or asymptomatic latent infection, and only a few develop central nervous system symptoms, which are characterized by acute onset, high fever, headache, projectile vomiting, and various degrees of impaired consciousness after 2-3 days of fever. Headache, projectile vomiting, and various degrees of impaired consciousness occur 2-3 days after the onset of fever. In severe cases, central nervous system symptoms such as convulsions, tonic spasms, or paralysis may occur. The course of a typical case can be divided into the following four stages: 1. initial acute onset, body temperature rises sharply to 39~40 ℃, accompanied by headache, nausea and vomiting, some patients have nursery or mental lethargy, and there is mild cervical ankylosis, with a duration of 1~3 days. 2. extreme stage: body temperature continues to rise, and can reach more than 40 ℃. Initial symptoms gradually aggravate, consciousness is obviously impaired, and drowsiness, lethargy and even coma appear. The deeper the coma, the longer the duration, the more serious the condition. Delirium can occur as early as on the first two days of the disease, but it is more common on the third to eighth days. Severe patients may develop generalized convulsions, tonic spasms or tonic paralysis, and in a few cases, flaccid paralysis. Severe patients may develop central respiratory failure due to parenchymal brain (especially brainstem) lesions, hypoxia, cerebral edema, cerebral herniation, intracranial hypertension, hyponatremic encephalopathy and other lesions, which manifests itself as an irregular respiratory rhythm, double inspiration. Sigh-like breathing, apnea, tidal breathing and jaw breathing, etc., and finally respiratory arrest. Physical examination may reveal signs of meningeal irritation, pupil blunted response to light. Disappearance or pupil dilatation, disappearance of abdominal wall and tilting reflexes, deep reflexes hyperreflexia, and pathologic pyramidal tract signs, such as Bartholomew's sign, can be positive.3. Recovery period, after the pole rats body humidity gradually decreases, mental. Neurological symptoms improve day by day. Severe patients are still slow, dementia, aphasia, dysphagia, facial paralysis, tonic spasm of the limbs or torsion spasm, etc. A few patients may also have flaccid paralysis. After active treatment, most of the symptoms can be recovered within half a year. 4. Sequelae period: A few patients with severe disease still have psychoneurological symptoms after half a year. For sequelae, mainly have cognitive disorders. Dementia, aphasia and limb paralysis, epilepsy, etc. can be recovered to varying degrees with active treatment. Sequelae of epilepsy may last for life. (ii) The incubation period of yellow fever is generally 3~6 days, and the clinical manifestations vary greatly, from mild self-limited to fatal infection. The main clinical manifestations are fever, yellow staining, hemorrhage, etc. In some outbreaks, the case fatality rate can be as high as 20%-40%. Typical clinical course can be divided into the following 4 stages: 1. Viremia stage with acute onset, chills, fever, up to 39-40 ℃, relatively slow pulse. Severe headache, backache, generalized muscle pain, nausea, and vomiting. Conjunctival and facial congestion, rhinorrhea. Proteinuria may be present. Symptoms last for 3-5 days. 2. Remission period: After 3-5 days of illness, there is a remission period of 12-24 hours, which is characterized by a drop in body temperature, disappearance of headache, and improvement of the general condition of the body. During this period, the virus is cleared from the body and non-infectious immune complexes can be detected in the blood. Mild patients can be cured in this phase. 3. Liver and kidney damage phase This phase lasts for 3~8 days, and 15%~25% of patients enter this phase after the remission period. Body temperature rises again, systemic symptoms reappear, vomiting, epigastric pain, etc. Jaundice appears and deepens. Jaundice appears and gradually deepens, bleeding manifestations such as petechiae, ecchymosis, rhinorrhea, extensive bleeding of mucous membranes, and even hemorrhage in the cavity. Renal function is abnormal, with decreased urine output and proteinuria. Cardiac damage can be seen on electrocardiogram with ST-T segment abnormality, and acute myocardial dilatation can be seen in a few cases. Cerebral edema may occur, cerebrospinal fluid protein is elevated but white blood cells are not high. Hypertension, tachycardia, shock, and intractable eructation suggest a poor prognosis. Twenty percent to 50 percent of patients in this phase die within 7 to 10 days of the onset of the disease. 4. Recovery phase This phase is characterized by extreme fatigue and weakness, which may last for 2 to 4 weeks. Deaths have been reported during the recovery phase, partly due to cardiac arrhythmias. Elevated aminotransferases may persist for several months after recovery, usually without sequelae. (C) Dengue fever: hemorrhagic tendency, enlarged lymph nodes, decreased white blood cell count, thrombocytopenia, etc. 1. Dengue fever: fever: the onset of the disease is mostly sudden, with body temperature rapidly reaching 39 ℃ or above, usually lasting for 2~7 days, with irregular heat pattern, and the temperature drops to normal on the 3rd~5th day of the disease, and then rises again a day later, presenting as bimodal fever or oat-shaped fever. In children, the onset of the disease is slower and the degree of fever is lower. The onset of the disease is accompanied by headache, backache, muscle and joint pain, orbital pain, retroocular pain and other systemic symptoms. Gastrointestinal symptoms such as hypersensitivity, nausea, vomiting, abdominal pain, poor appetite, diarrhea and constipation may be present. The face and conjunctiva are congested, and the skin of the neck and upper chest is flushed. Relative bradycardia may occur during the febrile period. (2) Rash: It appears 2 to 5 days after the onset of the disease, and is first seen on the palms of the feet, soles of the feet, or trunk and abdomen. The soles of the feet or trunk and abdomen, gradually extended to the neck and limbs, some patients see in the face, can be maculopapular rash, measles-like rash, scarlet fever-like rash, erythema rash, slightly itchy, but also in the last 1 day of fever or after the fever subsides, in the feet. It may also appear on the last day of fever or after the fever subsides on the feet, back of legs, restlessness. Small petechiae may appear on the back of the wrists and abdominal fossa, and disappear within 1~3 days, with brown spots remaining for a short time, which usually disappear at the same time as the fever. (3) Bleeding: 5-8 days after the onset of the disease, about half of the cases may have different parts of different degrees of bleeding, such as nosebleeds, skin age spots, gastrointestinal bleeding, hemoptysis, hematuria, vaginal bleeding and so on. (4) Enlarged lymph nodes: the lymph nodes of the whole body may be mildly enlarged, accompanied by mild tenderness. (5) Others: there may be liver enlargement. Patients often feel weak after the disease, and full recovery often takes several weeks. Severe dengue fever in the third to fifth day of the course of the disease, headache, nausea, vomiting, impaired consciousness, meningoencephalitis or some manifestations of gastrointestinal hemorrhage and hemorrhagic shock. This type is often due to the rapid development of the disease, death due to central respiratory failure and hemorrhagic shock. 2. Dengue hemorrhagic fever (1) Shock: generally occurs in the 2nd~5th day of the course of the disease, lasts for 12~24 hours, the patient is irritable and restless, the limbs are cold, the face is pale, the skin appears to have patterns, the body temperature drops, the respiration is fast and irregular, the pulse is weak, the pulse pressure shrinks progressively, the blood pressure drops and even can't be found, and cerebral edema may appear in the course of the disease, occasionally coma. Cerebral edema may appear in the course of the disease, occasionally coma. If the patient is not rescued in time, he will die in 4~10 hours. (2) Bleeding: bleeding tendency is serious. There are nosebleeds, massive blood petechiae on the skin, vomiting blood, blood in stool, hemoptysis, hematuria, vaginal bleeding, and even intracranial hemorrhage. (d) The incubation period of handmaiden-borne cerebral moxibustion is 7~14 days on average (1~30 days or longer), and the shorter the incubation period is, the more serious the disease is. The acute stage is 2~3 weeks, and with the course of the disease, it can develop into chronic roach encephalitis. The disease is characterized by high fever. Neurological symptoms (paralysis, pathological reflexes, impaired consciousness, meningeal agitation) and respiratory and circulatory disorders are the main signs. 1. Fever is the essential symptom of the disease, usually between 38.5-41.5 ℃, mostly arrested fever, and partly flaccid fever or irregular fever. It lasts for 5 to 12 days, and a sudden drop in temperature in severe cases signals that death is imminent.2 Neurological symptoms begin to appear 1 to 2 days after the onset of the disease. They include the following: (1) impaired consciousness: lethargy, tameness, and mania often appear 1~2 days after the disease. Mania, severe cases are mostly in a coma state, and if the phenomenon of convulsions occurs, it is a sign of poor prognosis. 90% of the cases will recover gradually with the drop of body temperature. (2) Meningeal irritation: headache, nausea, vomiting and neck stiffness. (3) Symptoms of localized lesions and brain nerve damage: mainly paralysis, mostly muscle flaccid paralysis, mainly in the cervical muscles, followed by the upper limbs, and in a few cases dysphagia, dysarthria and speech disorders. The sequelae of cicada-borne encephalitis disease are characterized by atrophic paralysis of neck and upper limb muscles. (4) Reflex dysfunction: the vast majority of patients present with diminished or absent deep reflexes, and a few present with twitching of the hands and feet, deafness, and paralysis of the masticatory muscles.3. Respiratory and circulatory disordersPart of patients have respiratory and circulatory dysfunction, and may present with myocarditis. Peripheral circulatory failure, decreased blood pressure, altered pulse frequency, and even respiratory failure. Severe patients may die due to complications caused by medullary paralysis or myocardial changes within 3 to 6 days after onset of the disease (or within 1 day after onset of the disease). Most of the patients can be complicated by bronchitis in the acute stage, which further leads to bronchopneumonia, which is also one of the important lethal factors. (e) West Nile Fever The incubation period of West Nile Virus infection is 2 to 15 days (usually 2 to 6 days), which is usually manifested as West Nile Fever. West Nile virus encephalitis, very few cases may also manifest as severe pancreatitis, hepatitis, myocarditis, and recently reported to be similar to polio-like illness. After human infection with West Nile virus, the virus first replicates and proliferates in the peripheral blood, there is a transient viremia process, and then the virus invades the peripheral lymph nodes and the central nervous system and other target organs and tissues, resulting in disease. 1. West Nile fever West Nile virus infected patients with the typical symptomatic manifestations of West Nile fever, accounting for about 1/3 of the infected people. the incubation period is generally in the 1-6 days, the clinical manifestations of fever, The incubation period is usually 1-6 days, clinically manifested as fever, headache, lethargy, fatigue, drowsiness, increased fatigue, sudden onset of symptoms with or without antecedent symptoms, and more than 1/3 of the patients' body temperature can reach 38.3~40 ℃. Some patients may also have severe eye pain, conjunctival edema, congestion, and muscle pain; 80% of patients have self-limited symptoms that last 3-5 days. In West Nile, the population prevalence is high, with 61% of young adults and about 22% of children positive for WNV. In childhood, patients commonly present with unremarkable or unexplained fever. It has been shown that 14.6% of hospitalized cases of febrile children in Alexandria are due to WNV infection, which can lead to lifelong immunity.2 West Nile Virus Encephalitis: 1/300-1/150 WNV-infected patients can develop aseptic meningitis. Encephalitis or meningoencephalitis, generally referred to as West Nile virus encephalitis; WNV encephalitis has an incubation period of 2-14 days and presents clinically with fever, headache, convulsions, impaired consciousness and meningoencephalitis. The incubation period of WNV encephalitis is 2-14 days, and clinical symptoms include fever, headache, convulsions, impaired consciousness, and meningeal irritation symptoms of encephalitis or meningeal brisket inflammation; severe neurological symptoms are rare, and the lesions are mainly concentrated in the thalamus. The midbrain and brainstem and other parts of the two Nile encephalitis patients are mostly in the elderly and immunocompromised or immune damage and cardiovascular and cerebrovascular diseases. Diabetes and other patients. Healing is good with almost complete recovery and residual weakness as well as memory loss can return to normal within a few weeks. Recovery is rapid in children and worse with age. West Nile virus encephalitis has a mortality rate of 3% to 15%, mainly in elderly or immunosuppressed patients. 3. Polio-like syndrome. Jonnthan D. Glass and A. Arturo Leis et al. reported several cases of polio-like syndrome in 2002, which was determined to be caused by West Nile virus infection by laboratory tests. Clinical manifestations include high fever above 39℃, headache, lethargy, chills, night sweats, myalgia, and confusion in the early stages; severe muscle weakness is also a common symptom, with progressive development of bilateral or unilateral upper limb muscle weakness, and weakness or even paralysis of the lower limbs; bladder dysfunction, and acute respiratory distress have also been reported. Physical examination revealed that: deep tendon reflexes were delayed or absent, and myelinated nerves showed demyelination-like changes; the gray matter portion of the spinal cord was a target site for West Nile virus infection. Symptoms are similar in humans and animals. Cerebrospinal fluid testing reveals a 4-fold or greater increase in anti-West Nile virus antibodies in the acute phase, and in the recovery phase. (vi) Chikungunya fever is characterized by fever, rash and severe joint pain. The incubation period is 2~12 days, usually 3~7 days.1. Acute phase (1) Fever: patients often have sudden onset of illness, chills, fever, and temperature up to 39 ℃, accompanied by headache, nausea, vomiting, loss of appetite, and enlargement of superficial lymph nodes. Generally, the fever can subside in 1~7 days, and some patients have a mild fever (bimodal fever) again in about 3 days, which lasts for 3~5 days and returns to normal. Some patients may have cough. Some patients may have cough, pharyngitis and other upper respiratory tract inflammation and conjunctival congestion. Conjunctivitis with mild photophobia. (2) Joint pain and stiffness: At the same time of fever, pain in several joints and spine, swelling of joints, may be accompanied by generalized myalgia. The joint pain is mostly migratory, aggravated with movement, and is worse in the morning. The disease progresses rapidly, often with loss of joint function and immobility within minutes or hours. It mainly involves small joints, such as the hands. Wrist, ankle and toe joints, etc. Large joints such as the knee and shoulder may also be involved.Severe pain and stiffness caused by compression of the wrist joint is characteristic of this disease. Joint effusions are rare. x-rays are normal. The pain usually lasts for a few weeks to a few months and eventually becomes chronic in about 12% of infected patients. (3) Rash: Skin symptoms are usually present after infection. 80% of patients develop a rash on the trunk, extended sides of the limbs, palms, and soles of the feet 2 to 5 days after onset of the disease. Rash appears on the palms of the hands and soles of the feet, which is macular, papular or purpuric, and the skin between the rashes is mostly normal, with itching sensation in some patients, which subsides after a few days, and may be accompanied by slight flaking. The trunk and limbs are the common parts of the maculopapular rash, and the face, palms of the hands and feet are also the most common parts. (4) Others: Meningoencephalitis, hepatic impairment, myocarditis, and skin and mucosal hemorrhage may occur in a very small number of patients. In India and Southeast Asian countries, simple chikungunya fever can be accompanied by hemorrhagic symptoms after fever, which is a hemorrhagic fever not accompanied by shock (mainly seen in children). 2. After the acute stage of recovery, joint pain and stiffness can be completely recovered in the majority of patients. Some patients have persistent joint pain and stiffness for weeks to months, or even 3 years or more. Patients in the recovery phase can be categorized according to their symptoms as follows: (1) complete recovery of joint pain and stiffness; (2) intermittent stiffness and discomfort in the distal joints, which worsens with movement but is normal on X-ray; (3) persistent stiffness; (4) persistent pain and stiffness with swelling in a small number of patients, which is the case for about 90% of patients in the recovery phase (1). Although the majority of patients can eventually recover from joint damage, the recovery is slow. 3. Prognosis: The case fatality rate of chikungunya fever is low, about 0.4%. Infants are often severely ill and occasionally die of circulatory failure. Adults hardly die, but the severe pain and slow recovery obviously affect the normal life and work of the infected. Recently, it has been reported that overlapping infection with dengue/dengue hemorrhagic fever can seriously affect the prognosis, and the mortality rate of children infected with hemorrhagic fever with shock can be as high as 20% in clinical practice.
Materials and Instruments
Materials: blood, liver tissue, feces Move (i) Specimen collection The success of virus detection depends largely on the type, time and quality of specimen collection. Preservation, transportation and specimen processing and other links. According to the type of insect-borne infectious disease the patient is infected with, the corresponding specimen is collected. Comply with biosafety regulations and personal protection during specimen collection. 1. Blood ﹑ In the early epidemiological and morbid stages (i.e., viremia stage), aseptically collect 2-10 nl of venous blood without anticoagulant. Whenever possible, use tubes or glass vials with threaded caps and seal the edges of the caps with sealing film, wax, or other material to prevent leakage of the specimen during transportation. Collected blood is left at room temperature for 1 hour and then at 4 °C for 2 hours to clot the blood. Centrifuge the blood at 3 000 e/min at room temperature, aspirate the serum with a sterile pipette, put it into sterile tubes, and store it at -80 ℃ for spare use, noting that contaminated serum needs to be treated aseptically. In addition, blood samples can also be collected from patients in acute and recovery coral, but attention should be paid to the time of collection, for example, blood samples should be collected as soon as possible from patients in acute coral no later than 7 days after the onset of the disease, while blood samples from patients in recovery should be collected 3~4 weeks after the onset of the disease. Separated serum should be stored at low temperature and transported to the laboratory within 24 hours, and serum specimens can be stored at 4 ℃ for 1 week. Serum specimens can be stored at 4 ℃ for one week. Long-term storage should be at -20 ℃ or below. 2. Cerebrospinal fluid, in the early epidemiological and early morbidity (i.e., viremia stage), aseptically collected cerebrospinal fluid, cerebrospinal fluid virus isolation rate is lower than that of blood, and is not suitable for diagnosis. 3. Tissue specimens (1) B encephalitis and West Nile fever: take several small pieces of brain tissues (cerebral cortex, brainstem, midbrain, hippocampal gyrus and cerebral bridges), put them in sterilized glass vials, and put them in a refrigerator to be sent for examination as soon as possible within 3 hours after the patient's death. Brain tissue can not be immediately sent for examination, should be - 25~30 ℃ refrigerator, or add 50% glycerol saline, 4 ℃ preservation and wait for the examination. (2) yellow fever: take the liver specimens of dead patients or monkeys, put them in sterile test tubes, seal them, and place them in -80 ℃ refrigerator for storage. (3) Dengue fever: take the brain of the patient as soon as possible within 24 hours after death. Liver, spleen, lymph nodes and other tissues, placed in a clean container, frozen at -80 ℃ refrigerator storage, immediately sent to the relevant laboratory for processing. (4) Cicada-borne encephalitis: All patients who die within the 10th day of illness may have the possibility of isolating the virus from their brain tissues, and with the prolongation of the disease, the isolation rate is getting lower and lower. In order to increase the isolation rate, the brain of the patient should be dissected within 12 hours after death, and the brain tissue of the brain cadres should be taken for virus isolation. The specimen should be sent to the laboratory for inoculation and other tests as soon as possible. If the specimen cannot be sent for testing immediately, it should be kept frozen at -70 ℃. 4. Media specimens (1) Mosquitoes: According to the type of vector, collect the suspected mosquitoes that have sucked blood, feed them with 0.50 mol/L (10%) glucose solution, place them at -20 ℃ after the stomach blood has been completely digested, and group them into groups according to mosquito species and capture locations after they die, and put them into sterilized vials or tubes, and store them in liquid nitrogen or ultra-low-temperature freezers. Common mosquito collection methods are as follows: (1) electric mosquito suction device artificial trapping method: choose a variety of mosquitoes handle resting places, such as Culex mosquitoes to human houses, barns. Pig shed as a capture point in 1 ~ 1.5 hours after sunset to start trapping, Aedes aegypti to bamboo forests, banana plantations and other semi-domestic environment-based, in the morning and near dusk with electric mosquito suction traps. (2) Sleeve mosquito magnetic field automatic trapping method: choose a variety of mosquito habitats, place a mosquito trapping magnetic field, liquefied petroleum gas as a gas source and power source, according to the instructions for continuous opening of the mosquito trapping magnetic field automatic trapping mosquitoes, trapping 2 ~ 4 hours or overnight. (3) human tent method: in the vicinity of the mosquito sins, hanging a large mosquito net (specifications: tent 80 cm x 80 cm top corner to the lower edge of the corner of 150 cm. 150 cm distance between the two corners of the lower edge), the top of the tent to support the level of the corners of the four corners of the tent to pull open. The lower edge of the ground 30 ~ 40cm, people in the tent to attract mosquitoes into the tent, to suck mosquitoes continuously catch auger. (4) light trapping method: mosquito trapping lamps hanging in the mosquito breeding grounds, pig farms, cowsheds, settlements around the open space, 1 ~ 1.5 m from the ground, the list of the night to turn on the power for automatic trapping, trapping 2 ~ 4 hours or overnight. (5) Net trapping method: during operation, the collector holds the net handle, straightens the armpit for "α" shape to wave the net, and collects at the rate of 55 times/minute each time. (6) spoon fishing method: choose a variety of mosquitoes in the place (ponds.). Streams, ditches, pools, rice paddies, etc.) with a standard metal fishing spoon (volume of about 400 mml) or water net (made of gauze sewn on the coarse iron wire), in the larvae breeding water, within 1 meter offshore, randomly collect water samples, such as the age of the larvae or pupae, sucked with a thick-mouthed pipette into a wide-mouth bottle. (2) Handmaiden: Collection of handmaiden: collect unsucked roach in the grassland where animals are active, such as the grassland where sheep are grazing or the area where wild animals are active. Sweep a white cloth flag of 1m3 several times on the grass or drag the flag on the grass (flag dragging method), check and collect the roach on the flag with tweezers, and put the wasps in test tubes or small glass vials (with pieces of filter paper dripping with a small amount of water to keep the filter paper humid) and bring them back to the laboratory. Collecting handmaidens in grassy areas should be avoided after rain and in the morning when the ground is wet and at midday when it is hot. Wear protective clothing or white socks and white pants with long white faces, and tie the pants into the socks to prevent roaches from burrowing into the pants. In addition, blood-sucking roaches are also collected from animals. The captured roaches should be sorted and grouped into groups of 10-50 roaches in sterilized vials or test tubes and stored in liquid nitrogen or ultra-low temperature refrigerators.
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