Infection of Adenovirus

Infection Diagnosis and Treatment of Adenovirus

Meaning of Adenovirus Infection: Adenovirus is a kind of particles with a diameter of 70 to 90 nm without an envelope. It consists of 252 shell particles arranged in a physahedron. Each shell particle has a diameter of 7-9 nm. The capsid is a linear double-stranded DNA molecule containing approximately 4.7 kb with an inverted repeat of approximately 100 bp at each end. Since the 5 ‘end of each DNA strand is covalently bonded to a protein molecule with a relative molecular mass of 55 × 103 Da, a circular structure of double-stranded DNA can occur.

Name: Adenovirus

Attributes:70-90 nm diameter particles without coating

Make up:252 crusts are arranged in a physahedral arrangement

Number of types:52 known

Structure:Genome length about 25-45kb

Content:Contains 13% DNA and 87% protein

 

Table of Content

1 Brief history of research

2 Composition

3 Categories

4 Induced diseases

▪ Respiratory infections

▪ Eye infections

▪ Gastroenteritis

▪ Other diseases

5 Life cycle

6 Identification method

▪ Morphological examination

▪ Serology

▪ Antigen detection

7 Prevention principles

8 Genetic research

9 Communication characteristics

10 Prevention methods

 

Brief History of Research

There are 52 known human adenoviruses, named adl – ad52, and the most detailed study is ad2. The adenovirus genome is transcribed to produce mRNA. There are at least five known transcription units: the EⅠ region is located on the left side of the viral genome and can be further divided into EⅠA and EⅠB.

Adenovirus

EⅡ region encodes a DNA-binding protein involved in virus replication.

EⅢ region encodes a glycoprotein appearing on the surface of host cells.

EIV region is located at the right end of the ad2 genome and is regulated by the DNA-binding protein encoded by EII region.

The fifth The transcription unit synthesizes ad2 protein IV in the middle of viral infection.

Adenoviruses are carcinogenic to rodents, or can transform rodent cells cultured in vitro. Only a part of the adenovirus genome is required for cell transformation. These genes are located at the left end of the genome and account for about 7% to 10% of the entire genome. Although adenoviruses are widely distributed, they are not carcinogenic to humans. Human cells are a type of permissive cells, that is, these cells allow the infected virus to replicate and proliferate in the cell, and finally the cells lyse and die to release a large number of progeny viruses.

Adenovirus particles have not been detected in a variety of human tumor cells cultured in vitro, but there is an adl2 integration site on human chromosome 1, which means that human cells may also be non-permissive cells for adenoviruses, that is, such cells After a cell is infected by a virus, the virus cannot replicate and proliferate within the cell, but can integrate into the genome of the infected cell. These cells were transformed by the virus, their phenotype changed, and they could be passaged indefinitely in vitro.

 

Constitution of Adenovirus

Adenoviruses have a spherical structure without capsules, and their viral particles are often arranged in a lattice pattern in the nucleus of infected cells. Each virus particle contains a 36 kb line.

Sexual double-stranded DNA has an inverted terminal re-peat (ITR) of 100 to 600 bp at each end. The inner side of the ITR is a virus packaging signal and is a cis-acting element required for virus packaging.

The genome contains early-expressed E1-E4 genes related to adenovirus replication and late-expressed L1-L5 genes related to adenovirus particle assembly.

The linear double-stranded DNA and core protein form a pulp core with a diameter of 60-65 nm and are wrapped in the capsid.

The capsid is symmetrical in icosahedron, and consists of 252 shell particles with a diameter of 8 to 10 nm.The shell particles are arranged on a triangular surface with 6 on each side, of which 240 are hexons (non-apex shell particles).

Twelve are penton bases (apex crusts). Each hexon is a homotrimer of a hexon protein.The trimeric hexon molecule has a triangular spire and a pentahedron base. The tower area is composed of 4 loops, namely loop1, loop2, loop3, Loop4. The base contains two regions P1, P2.

The epitope on the hexon is a diagnostic criterion for different serotypes. It includes the antigenic component of the mammalian adenovirus genus and is the most sensitive part of the virion to the pressure of immune selection.

Each penton base is bound with 1 (mammalian adenovirus) or 2 (avian adenovirus) fiber protrusions with a length of 9 to 77.5 nm, and these fibers protrude from the capsid surface based on the penton protein.

The head of the fiber forms the ganglion region. The fibrous process is serum-specific and contains species-specific epitopes responsible for hemagglutination in vitro.

Adenovirus contains 13% DNA and 87% protein, and the virion has a molecular weight of about 175 × 106. The virus genome is a linear double-stranded DNA, which contains about 35kb to 36kb.

Among the DNA components of adenovirus types 12, 18, and 31, G + C mol% is the lowest (48% to 49%), which is a gene that is highly carcinogenic to animals type.

The G + C mol% of adenovirus types 1, 2, 4, 5, and 8 was higher (61%), but carcinogenicity was low or absent. This is a standard for grouping human adenovirus isolates, and human adenoviruses are divided into 6 groups such as A to F according to their genetic homology.

The genome of the adenovirus exists as a linear double-stranded DNA, surrounded by protein VII and a small protein called mu tightly around it, playing a histone-like role. Another protein V joins this DNA-protein complex and connects to the viral capsid via protein VI.

A specialized structure called DNA terminal protein (pTP) complex (DNA-TPC) is covalently bonded to the 5 ′ ends of the two strands, which are closely related to adenovirus replication. The adenovirus genome has a 100 bp inverted terminal repeat (ITR) at each end of the adenovirus genome, which is the origin of replication. On the 3 ′ side of the left ITR, a 300 bp long packaging signal (ψ) mediates the packaging of the adenovirus genome into the viral capsid.

For adenovirus, only about 0.5 kb of the sequence including the ITR at both ends and the packaging signal (ψ) is a cis-acting element, which means that it must be carried by the adenovirus vector itself, and more than 30 other proteins can pass Helper virus (or cell) trans complement.

There are about 11 kinds of viral proteins (TP and PⅠ- PX), of which 4 kinds of proteins (viral peptides PⅤ, PⅦ, terminal protein TP, enzyme protein PXX) and virus genes constitute the viral core. Peptide PⅦ is the main core protein. The protein encapsulates the viral DNA.

There are about seven proteins that make up the viral capsid. Peptide PⅡ is the most abundant and main component in the viral capsid. Hexon is composed of three PⅡ molecules closely linked. The peptides PⅥ and PⅧ form a connecting bridge between the hexon and the virus core, and together with the peptide PⅨ stabilize the lattice arrangement of the hexon molecule.

Five molecular peptides PⅢ are connected to form the base protein of the penton. PIIIa is a surrounding protein of the penton and also participates in the capsid composition. The penton is connected to the virus core through PInfection Diagnosis Treatment. Polypeptide PIV mainly constitutes a virus trimer fibrillation, and fibrillation is related to viral hemagglutination activity. Because hemagglutinin (fibrillation) is type-specific, clinical isolates are usually typed by the hemagglutination inhibition test (HI).

 

Classification of Adenoviruses

Classification and since the discovery and successful isolation of adenoviruses in the 1950s, more than 100 serotypes have been discovered, of which there are 52 human adenoviruses, divided into six subtypes: A, B, C, D, E and F. Group (subgroup). Gene type 2 and type 5 adenoviruses commonly used in gene therapy belong to subgroup C in serological classification, and have 95% homology in DNA sequence.

The proliferation ability of the two is very strong, the titer can usually reach 109 pfu (plaque forming unit) / ml, and the genome copy number in a single cell can reach 104 (about 10% of the total DNA of the cell). The virus particles are relatively stable and can reach 1010 ~ 1011 pfu / ml by CsCl gradient centrifugation, which meets the requirements of animal experiments.

 

Induced disease

Adenoviruses can infect the respiratory tract, gastrointestinal tract, urinary tract, bladder, eyes, liver, etc. About 1/3 of the known serotypes of human adenovirus are usually related to human diseases, but one serotype can cause different clinical diseases. Conversely, different serotypes can cause the same disease.

 

Respiratory infections

Typical symptoms of respiratory infections are cough, stuffy nose, and pharyngitis with fever, chills, headache and muscles

Pain, etc., include the following 4 different syndromes.

1. Acute febrile pharyngitis usually occurs in infants and children. It is caused by group C virus and has symptoms such as cough, nasal congestion, fever, and throat ulcers. These manifestations are difficult to distinguish from mild respiratory infections caused by other viruses.

2. Pharyngoconjunctival fever is similar to acute febrile laryngitis, but conjunctivitis often occurs at the same time. Pharyngeal conjunctival fever has an epidemic tendency, such as pool conjunctivitis, which is mostly caused by group B adenovirus types 3 and 7, which is better after healing, and generally has no sequelae.

3. Acute respiratory diseases (ARD): This syndrome is characterized by pharyngitis, fever, cough, and general malaise. It is often prevalent among recruits in the military, and is usually caused by sudden tension, fatigue, and gathering. This infection is mostly caused by adenovirus types 4 and 7, and can also be seen in type 3.

4. Pneumonia adenovirus pneumonia accounts for about 10% of childhood pneumonia, mostly caused by adenovirus types 3 and 7. The mortality of adenoviral pneumonia in young people is 8% to 10%. Pneumonia is also a type of acute respiratory disease in recruits serious performance.

 

Eye infection

Adenovirus-induced mild eye infections are a complication of respiratory infections and laryngitis. Follicular conjunctivitis can be caused by many types of adenovirus, similar to chlamydial conjunctivitis, and is self-limiting. Keratoconjunctivitis caused by adenovirus types 8, 9, and 37 is a severe infection and highly contagious. It begins with acute conjunctivitis and spreads to the auricular lymph nodes, followed by keratitis.

 

Gastroenteritis

Many adenoviruses replicate in intestinal cells and are excreted with feces, but most of the serotypes are not related to gastrointestinal diseases. Adenoviruses of type 40 and 41 can cause gastroenteritis in infants and young children (under 4 years), causing abdominal pain and diarrhea. Group C adenovirus can cause intussusception in some infants.

 

Other diseases

Adenovirus types 11 and 12 can cause acute hemorrhagic cystitis in children, and the virus appears in the urine. Type 37 can cause cervicitis in women and urethritis in men, often by sexually transmitted infections.

People with low immune function can cause occasional or severe viral infections, especially severe respiratory infections and viral hepatitis in organ transplant patients, mostly caused by adenovirus types 1, 5 and 7.

AIDS patients can be infected with a variety of serotypes of adenovirus, and heterozygous strains with intermediate antigenicity can appear, often with lethal adenovirus infection.

The main reason is that E1A protein of adenovirus can transactivate HIV transcription and accelerate HIV replication. Clinically found that 37% of AIDS patients with viral diarrhea are caused by adenovirus.

After adenovirus infection, can obtain lasting immunity to the same type

Compared with most pathogens of respiratory infections, the body’s reinfection with adenovirus can produce effective immunity. What protects is the circulating neutralizing antibodies produced in the body. Normal healthy adults also typically have polytype antibodies.

About 40% to 60% of people aged 6 to 15 years have neutralizing antibodies of type 1, 2 and 5, but very few antibodies of type 3, 4 and 7. Mother’s antibodies can protect babies from severe adenovirus respiratory infections.

 

Life cycle

The adenovirus life cycle can be divided into two distinct phases that cannot be separated. The first stage involves adenoviral particle attachment and entry into host cells, release of the genome into the host cell nucleus, and selective transcription and translation of early genes. At this stage, cells are ready for viral genome replication and adenovirus late gene expression and finally release mature infectious particles, the second stage. The first phase will be completed in 6-8 hours, and the second phase is faster, only 4-6 hours.

 

Adhesion and entry into cells

The process of adenovirus infecting cells begins with the adenovirus ciliary ganglion region adhering to specific receptors on the cell surface. Because human adenovirus mainly shares a receptor with Coxsackie B virus, this receptor is called coxsackie / adenovirus receptor (CAR).

Next, the tripeptide RGD on the surface of the penton of the viral ciliary base binds to αvβ3 and αvβ5 integrin on the cell surface, and internalizes the adenovirus into the cell and enters the lysosome through endocytosis.

In the acidic environment of the lysosome, the conformation of the adenovirus capsid will change, and it will be released from the lysosome to escape the digestion of the lysosome. Finally, the adenovirus particles are translocated to the nucleus, and the viral DNA is released into the nucleus through the nuclear pore.

 

Compared with liposome transfection, adenoviral genome entry into the nucleus is a very efficient process, which can generally reach 40%. Although the efficiency of the former entering the cytoplasm is comparable to the latter, the efficiency of DNA entering the nucleus is only 1 / 1000.

 

Transcription and replication

Once the viral genome enters the nucleus, a series of complex and orderly progressive amplification and transcription processes are performed.

 

Generally, the viral DNA begins to copy as a dividing line, and the adenovirus genes are roughly divided into early (E1 – 4) and late transcription units (L1 – 5) according to the sequence of transcription time.

 

Various adenovirus genes can be further divided into smaller transcription units. For example, the E1 region can be further divided into E1A and E1B. Each transcription unit has at least one unique promoter. After the adenoviral genome enters the nucleus, the cell transcription factor first binds to the enhancer upstream of the E1A region and expresses the E1A protein.

 

The role of this protein is to regulate cell metabolism and make viral DNA more easily replicated in cells. E1A protein can also activate the promoters of other early genes (E1B, E2A, E2B, E3 and E4).

 

Among them, E2B drives three other early gene transcription unit terminal protein precursors (pTP, precursor terminal protein), which are related to viral replication. Expression of single-stranded DNA binding proteins (ssDBP) and DNA polymerase (DNA pol, DNA polymerase).

 

The expression products of these three genes are tightly combined into a complex with at least three cellular proteins. Interactions initiate replication of the viral genome.

 

In general, DNA replication is initiated by RNA, but in adenoviruses it is called protein-priming.

 

As mentioned earlier, each single strand of the adenovirus double-stranded DNA is bound by a pTP protein at the 5′ end, and pTP forms a phosphodiester bond between its Ser-OH and the dCMP 5′ phosphate at the 5 ′ end of the DNA. Adenovirus DNA replication first uses dCMP with 5 ‘end bound to pTP as a primer and 3’ terminal inverted repeat (ITR) as a template for strand displacement synthesis and replacement.

 

Adenovirus

The single-stranded molecule can be self-annealed and cyclized to form a pan-handle-like circular molecule, which then uses the same mechanism to synthesize offspring double-stranded DNA molecules.

Viral genome replication usually begins hours after infection, while transcription and translation of early genes are turned off, and late genes begin to express. The transcription of most of the late genes is regulated by a common Major Late Promoter (MLP). In fact, the activity of MLP is closely related to the replication of the viral genome.

Studies have shown that once the adenoviral genome begins to replicate, the activity of MLP will be significantly enhanced, which we will elaborate in another article.

Late genes primarily encode structural proteins of the adenovirus. The structural proteins of the virus accumulate in the nucleus to form a viral capsid.

The genome of the virus is packaged to form infectious virus particles, and finally the host cell is lysed and released to complete the life cycle of the adenovirus. Adenovirus has obvious species specificity.

Human wild-type adenovirus type 5 (wtAd5) can express early genes after infecting other non-human cells (such as murine cells), and the genome can also be replicated to a certain degree and can form. Some immature virus particles cannot form mature virus particles and cannot infect other cells.

 

What is the Diagnosis (Identification method) of Adenovirus?

There are following ways od adeno virus diagnosis:

Morphological examination

Intestinal adenoviruses that are difficult to culture are crudely extracted from stool samples and observed by electron microscope or immunoelectron microscopy.

 

Virus isolation and identification

1. Isolated culture specimens should be collected from the infected site as soon as possible. Collect patient throat, eye secretions, feces, urine, etc., treat with antibiotics overnight, centrifuge the supernatant to inoculate sensitive cells (293, Hep-2 or HeLa cells, etc.). After incubation at 37 ° C, typical CPE, that is, cells Rounding, agglomeration, and stringing are the most prominent manifestations of many diseased cells clustered together in the shape of grapes.

2. Virus identification uses fluorescently labeled anti-hexanone antibodies and isolated cultured cells to identify adenoviruses. Hemoagglutination inhibition (HI) test or neutralization test (NT) can be used to detect genus and group-specific antigens. And identify the serotype of the virus.

Adenoviruses can be quickly identified using Shell vial technology. Virus specimens were treated with antibiotics and centrifugation. The supernatant was inoculated into a shell vial culture flask with cells, incubated for 1 to 2 days, and the antigenic epitope was detected with a specific hexon monoclonal antibody. Viral antigen can also be detected by direct staining of nasal mucosal epithelial cells in patients.

Use DNA hybridization or endonuclease digestion to identify isolated and cultured viral DNA. PCR can be used for the diagnosis of adenovirus infection. Primer design is based on the sequence of adenovirus hexon, VAI and VAII coding regions, which can detect all serotypes.

It is highly sensitive and can detect potential adenoviruses in some patients. Adenovirus 41 BgIII-D fragment was used as a probe to diagnose adenovirus diarrhea. The detection rate was up to 80%.

 

Serology

Common serological methods include IF, CF, EIA, HI, and NT tests. Patients are tested in duplicate during the acute and recovery phases. If the serum antibody titer during the recovery phase increases by four times or more than the acute phase, it is diagnostic . Rapid detection of serum can be used ELISA method or latex agglutination test.

 

Antigen detection

It is often used to directly detect adenovirus infections in the respiratory and gastrointestinal tracts. It is faster and more sensitive. Immunofluorescence (especially for respiratory specimens, throat swabs, and biopsy specimens) and enzyme immunoassay (especially for stool specimens) are common methods. Compared with cell culture, the sensitivity of adenoviruses detected by immunofluorescence is increased by 40% – 60%, other methods for direct determination of antigens include immunochromatography and latex agglutination. Studies have confirmed that compared with cell culture detection methods, the sensitivity of the immunochromatographic kit can reach 90%.

 

How can we prevent Adenovirus Infection?

Adenovirus Prevention principle: Adenovirus inactivated formaldehyde vaccines have been used for prevention in some populations, and may be replaced by live attenuated vaccines of human diploid cell culture in the future.

However, because adenoviruses have carcinogenic effects on animals, people have doubts about the role and safety of whole virus vaccines.

In addition, strengthening the disinfection of water in swimming pools and baths can minimize the risk of water-borne conjunctivitis outbreaks. Strictly aseptic operations should be performed during eye examinations.

The equipment used should be fully sterilized and epidemic conjunctivitis can be controlled happened. There are still no effective drugs for the treatment of adenovirus infections.

 

Genetic research

Main functions of adenovirus gene products

E1 region gene expression product

It can be further divided into E1A and E1B. E1A is mainly composed of two components, namely 289R (or 13S) and 243R (or 12S). The main function of these E1A proteins is to regulate cell metabolism and make cells more susceptible to virus replication. E1B19K is homologous to the expression product of the cell Bcl-2 gene, and can prevent apoptosis or necrosis by inactivating and removing members of the Bax family. The E1B55K gene product can down-regulate the transcription level of the p53 gene. Of course, this regulatory function is not absolute. Other adenoviral genes (such as E4 orf6) are also involved in this process. In addition, the E1B55K gene product is also involved in viral replication, transcription of viral late mRNA, and viral RNA transport.

 

E2 region gene expression product

Can be divided into E2A and E2B. Among them, E2A is DNA binding protein (DBP, DNA binding protein). E2B has two main products, namely the terminal protein precursor (pTP) and viral DNA polymerase (pol).

The three proteins interact with at least three intracellular factors to initiate adenoviral DNA replication and the transcription and translation of the virus’ late genes.

 

E3 region gene expression product

The main function is to destroy the host’s immune defense mechanism, and has nothing to do with the replication of the viral genome. One of the products of the E3 gene, 11.6 Kd, is called adenovirus death protein (ADP) because it can lyse cells and release virus particles at the late stage of virus infection.

The gp 19K protein can bind to the heavy chain of MHC class I molecules on the endoplasmic reticulum, prevent its transport to the cell surface, and delay the expression of MHC I.

RID α & β and 14.7Kd can inhibit TNF-induced apoptosis, promote Fas degradation, and down-regulate TNF receptor levels.

 

E4 region gene expression product

The gene product of E4 region is usually called orf 1 – 6/7, which is mainly related to the metabolism of viral messenger RNA. It also promotes viral DNA replication and shuts down host protein synthesis.

Studies have found that some E4 products can bind to DNA-activated protein kinases and prevent viral DNA from tandem. Since the kinase can activate the p53 gene, it is believed that some E4 region gene products can inhibit apoptosis.

Many E1B and E4 gene products are involved in antagonizing E1A protein function. For example, E4 orf4 inhibits the activation of the E2F promoter by E1A. E4 orf3VA RNA makes some untranslated RNA transcribed by adenoviruses related to adenovirus resistance to host cell immunity.

 

Communication characteristics

Wide range of hosts Low human pathogenicity

Adenovirus vector systems can be widely used for the expression of human and non-human proteins. Adenoviruses can infect a range of mammalian cells, so they can be used to express recombinant proteins in most mammalian cells and tissues.

It is important to point out that adenoviruses are epithelial, and most human tumors are derived from epithelial cells.

In addition, the replication genes and pathogenic genes of the adenovirus have been quite clear, and have been circulating in the population (70-80% of adults have neutralizing antibodies for adenovirus).

Human infection with wild-type adenovirus produces only mild self-limiting symptoms, and ribavirin treatment is effective.

Infect and express genes in proliferating and non-proliferating cells

Retroviruses can only infect proliferative cells, so DNA transfection cannot be performed in non-proliferating cells, but the cells must be kept in continuous culture.

Adenoviruses can infect almost all cell types, except for some lymphoma cells that are resistant to adenovirus infection.

Adenovirus is the best system for studying the gene expression of primary non-proliferating cells. It can directly compare the results obtained in transformed cells with those in primary cells.

 

Can effectively propagate high titer

The adenovirus system can produce 1010 to 1011VP / ml, and can reach 1013VP / ml after concentration, which makes it very suitable for gene therapy.

 

Homologous to human genes

Adenovirus vector systems generally use human viruses as vectors and human cells as hosts, so they provide an ideal environment for accurate post-translational processing and proper folding of human proteins. Most human proteins are expressed at high levels and are fully functional.

 

Does not integrate into the chromosome

Retroviruses can randomly integrate into host chromosomes, causing gene inactivation or activating oncogenes. Adenoviruses, except egg cells, are not integrated into the chromosome in almost all known cells and therefore do not interfere with other host genes. Integrating a single copy of the virus into an egg cell is a better system for generating transgenic animals with specific characteristics.

Ability to expand in suspension

293 cells can adapt to suspension culture, this adjustment can make the virus expand a lot. A large number of facts have proved that suspended 293 cells can express recombinant proteins in 1-20L bioreactors.

Ability to express multiple genes simultaneously

This is the first expression system that can be used to design multiple genes in the same cell line or tissue. The simplest method is to insert a dual expression cassette containing two genes into an adenovirus transfer vector, or to co-transfect the target cell line with different recombinant viruses to express each protein. The determination of the MOI ratio of different recombinant viruses can accurately estimate the relative co-expression of each recombinant protein.

Because of these advantages, adenoviruses are extremely widely used in various fields such as in vitro gene transduction, in vivo vaccination, and gene therapy.

 

How can we Prevent Adenovirus Infection?

Prevention method: Adenovirus infections are mainly epidemic in winter and spring and are prone to outbreaks in kindergartens, schools and barracks recruits. In general, adenovirus is mainly transmitted through respiratory tract droplets and eye secretions through the respiratory tract or contact. Intestinal infections are mainly transmitted through the digestive tract.

Its preventive measures are similar to the prevention of other respiratory and digestive tract infectious diseases.

It is mainly to wash hands and disinfect regularly to avoid contact with patients and their respiratory droplets.

Drink plenty of water, eat more vegetables and fruits, pay attention to exercise, ventilate indoors to keep the indoor environment clean.

Try to avoid crowded public places during winter and spring seasons, wear masks when going out to avoid contact with patients to prevent infection.

As soon as the symptoms of acute fever, sore throat and conjunctivitis occur, you should go to the hospital as early as possible for isolation and early treatment.

Report collective outbreaks of more than 5 people to the epidemic prevention department in your area in a timely manner, and take effective prevention and control measures in a timely manner to avoid the spread of the disease.

In the adenovirus epidemic season, children with upper respiratory tract infections in childcare institutions should go home and rest to avoid spreading the epidemic.

After getting sick, try to see a doctor in a nearby hospital to avoid infusion in the observation room of a large hospital where patients are concentrated, to prevent cross infection. The symptoms of severe cough and dyspnea are mostly serious cases, and they should be hospitalized in time to avoid delay.

 

 

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NB The write-up is based on the most prevalent media information and is not a medical advice. Consult your Authorized Cardiologist for any Medical Treatment.

 

 

 

 

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