Virology Flashcards
1
Q
True or False: Positive RNA can be translated directed to protein.
A
True - generally translated to negative strands and then made into many positive strand
2
Q
What is the best way to view viruses?
A
Electron microscope - 20nM
-300nM
3
Q
True or false: Viruses do not need an extracellular phase to be considered a virus.
A
False - particles must pass from cell to cell, throughout body, or between individuals
4
Q
What are 5 characteristics for classifying viruses
A
- Particle type - icosahedral, filamentous, enveloped, naked
- Tissue tropism
- Disease etiology
- Serology - cross reaction
- Genome type
5
Q
Capsid
A
Protein shell of a virus - can be either naked or enveloped
6
Q
Plaque Assey
A
Tissue culture for quantitating infectious virus - apply serial dilutions of virus and drug or neutralizing antibody
7
Q
Focus Assay
A
Viruses promoting cell growth rather than death
8
Q
Virus replication is characterized by an initial _______ and then rapid growth and _____.
A
- Gully
2. Burst
9
Q
What are the properties of Picornavirus?
A
Icosahedral \+ssRNA no lipid envelope no tegument Mostly pH stable
10
Q
Enterovirus, rhinovirus, hepatovirus, parechovirus and kobuvirus are all types of what virus?
A
Picornavirus - most common agent of common cold
11
Q
Enterovirus has what type of transmission?
A
Fecal/oral - via water supply - capsid is resistant to mild sewage treatment
12
Q
What is characteristic of paralytic poliomyelitis
A
Asymmetric flaccid paralysis
13
Q
What does polio virus isolated in the oropharynx look like?
A
Asympotomatic - viremia is what looks like slight flu
14
Q
Where can picornavirus (polio) be isolated from?
A
Throat, stool, or CSF
15
Q
What is the attributed as the cause of the polio epidemic?
A
Indoor plumbing: children no longer encounter the virus at a young age when they have maternal antibodies - worse disease
16
Q
How does picornavirus/polio virus ensure its ssRNA gets translated quickly?
A
- Its + sense
2. It has a 5’ end that grabs ribosomes
17
Q
How many proteins does picorna/polio virus encode?
A
One - it uses proteases to cleave different products
18
Q
What are three targets for therapy for picornavirus
A
- Protease inhibitors
- RNA-dependent RNA Polymerase
- Block entry
19
Q
Where does picornavirus replicate?
A
Cytoplasm
20
Q
What is a major challenge for -RNA viruses?
A
Encode enzymes to translate it to +sense
21
Q
What are characteristics of adenovirus?
A
- dsDNA
- Icosahedral capsid w/ penton spikes
- fecal/oral transmission
- Frequent unapparent
respiratory infection - Many serotypes
22
Q
What is something that picornavirus and adenovirus have in common?
A
They both use the coxsackie/adenovirus receptor to mediate entry
23
Q
Where does adenovirus replicate?
A
Nucleus
24
Q
What are characteristics of an acute infection?
A
- Rapid onset
- Active virus + immune response ￫ pathogen cleared, memory
- Immunopathology from overactive immune response
25
What are characteristics of an acute/chronic infection?
1. Acute onset + immune response ￫ Virus not cleared
2. Initial response is subdued to prevent immunopathology
3. Immune response of host is set to higher activation state overall - higher cytokines
26
Latent
A viral life cycle characterized by minimal--if any--expression of a subset of viral genes and absence or lytic replication and infectious virion production
27
What are two advantages of latent virus infection?
1. Stealth
| 2. Cell maintains viral genome - difficult to target
28
What are two disadvantages of latency?
1. Spread is limited
| 2. Death of latently infected cell is dead end for virus
29
How do viruses overcome the challenges of latency?
1. Reactivation
| 2. Establish latency in long-lived cells - memory T and B, hematopoietic stem cells
30
What is a prototype virus for chronic persistant infection?
Hepatitis C
31
T cell exhaustion
Persistant infection leads to downregulation of T cells ￫ Loss of antiviral functions and death of T cells
32
Latent infections are quiescent, but result in a higher level of __________. Is this good or bad?
Circulating cytokines - this can be good since you clear infection quicker, but can also serve as a promotor for those predisposed to autoimmune infections
33
How is disease generated with latent infection?
Reactivation - the switch to the lytic life cycle
34
Tissue damage from persistant infections is associated with what?
1. Mostly from inflammation and excessive immune response
2. Very low levels from damage associated with replication
3. ROS and cytokines can cause DNA damage ￫ cancer, diabetes
35
How do you target chronic persistant infection?
1. Anti-viral therapy
| 2. Immunosuppression - careful balance
36
What functions do IFNs serve in the immune response?
1. Activate macrophages
2. Activate Th1
3. Activate B cells ￫ IgG
4. Activate memory and killer T cells
37
What limits interferons as therapeutics?
They induce inflammation, cause the symptoms associated with being sick
38
What is the "Type 1 interferon response"
Virus sensed by PRRs/TLRs triggers synthesis and secretion of IFN α/β which induce the "antiviral state" in nearby cells
39
How do nearby cells sense/implement the antiviral state?
IFN α/β bind receptors which lead to translation of PKR and OAS via Jak/Stat
40
PKR
Protein Kinase R - Shutdowns mRNA translation
1. Binds dsRNA (becomes autophos'd/active)
2. Phos eIF-2 - normally initiates peptide synthesis
41
OAS
2'-5' Oligoadenylate Synthetase
1. Binds/activated by dsRNA
2. Catalyzes synthesis of oligoadenylate from ATP
3. AAA then activates RNAse L (endoribonuclease) - dedgrades mRNA
42
What are characteristics of the antiviral state?
Overall a decrease in protein synthesis, mRNA degradation, and inc. in NK cell
1. Increased MHC I expression
2. Inc. PKR expression
3. Inc. 2'-5' OAS
43
What are the pro-inflammatory cytokines?
TNFα: Pyrogen, induces cell death - produced by activated Mφ, CD4, and NK cells
IL-1β: Pyrogen, produced by activated Mφ
IL-6
44
When a cell first senses a virus, what 2 classes of molecules are upregulated?
1. Type 1 IFNs
| 2. NK activating ligands
45
What are 3 components of the innate immune response?
1. Cytokines
2. NK Response
3. Complement
46
What are 3 components of the adaptive immune response?
1. Cytokines: IFN-γ + IL-12 ￫ Th1 and IL-2 ￫ T cell proliferation
2. T cell via Class 1 and CD8
3. B cell response (antibodies)
47
Apoptotic Response
1. From within - selfless
| 2. From outside - Fas or by TNFα on CD8 or NK cells
48
What genes regulate apoptosis?
1. p53
| 2. caspase 9 and 3 mediate cascade ￫ activated by leaky mito losing cytochrome c
49
What are 5 mechanisms to circumvent the Type 1 IFN Response/Induction of the Antiviral State?
1. Encode proteins to prevent IFN binding
2. Direct cellular phosphatase to dephos eIF-2α
3. Decoy mRNA to sequester PKR and prevent activation
4. Sequester dsRNA to prevent PKR activation
5. eIF-2α decoys to bind activated PKR
50
What are 3 mechanism to circumvent innate immune defenses?
1. Sequester IL-1β w/ viral binding protein
2. Encode homologs of complement control proteins → prevent MAC formation
3. Encapsidate viral/host complement control proteins in their membranes to evade lysis
51
What are 6 mechanisms to avoid Class 1 MHC Presentation?
1. Degrade TAP transporter
2. Block TAP transporter - no peptide in ER
3. Degrade MHC1 molecules
4. Divert MHC 1 to lysosomes
5. Retain MHC 1 in ER/Golgi
6. Downreg transcription of MHC components
52
What are 3 mechanisms to evade NK cell killing?
1. Encode decoy MHC 1 like molecules - more inhibition
2. Prevent NK cell receptor ligands from getting to cell surface
3. Remove HLA-A and HLA-B from cell surface- only HLA C - not as effective
53
Viruses can also induce the downregulation of ______ and ____.
1. Adhesion molecule ICAM-1
| 2. Costim B7 (CD80/CD86)
54
Viruses also encode which inhibitors of apoptosis?
1. p53 inhbitors
2. Bcl-2 homologs
3. Inhibitors of caspases
4. Soluble TNF receptors - prevent TNF triggered apop
55
What other characteristics can make viruses difficult to kill?
1. Latency
2. Epitope mutation
3. Infection of "privileged sites"
4. Intracellular sequestration
5. Infection via immune receptors
56
How does Merkel cell polyoma virus cause cancer?
A common deletion renders virus unable to complete replication cycle; persistant infection
More common in immunosuppressed
57
How does papillomavirus cause cancer?
E5: stimulates constitutive GFR signaling
E6 & E7: neutralize "brakes" p53 and Rb - target them for degradation
Uncouples cell division from regulatory control
58
What are the characteristics of papillomavirus?
dsDNA circular genome
1/3 infect GI, sexual transm.
Infects epithelium - causes cells in sratum spinosum to replicate
59
Rb protein
Blocks cells from going to G1 ￫ S
60
E6 (HPV)
Blocks p53 and activates telomerase
61
Development of carcinoma is associated with ___________ of the DNA into the host chromosome. ___ and ___ are always retained in cancer cells.
1. Integration
2. E6
3. E7
62
What are some of the problems with the HPV vaccine?
1. Need good mucosal immunity
2. Must recognize multiple strains
3. Can't include oncogenes
4. Political
63
Hybrid capture assay (HPV)
1. Isolate DNA
2. Introduce target RNA strands
3. Use antibodies to amplify and ID
64
Gardasil
Tetravalent vaccine with recombinant L1 VPLs
65
What etiologies can be caused by retroviruses?
1. No ill effects
2. Tumors - capture host gene
3. Wasting disease/neurological disorders
4. Immune deficiencies
66
What is the general structure and composition of retroviruses?
1. Enveloped (env) embedded in membrane, matrix under lipid bilayer
2. Capsid (groups specific antigens - gag genes)
3. 2 copies of +ssRNA genome - only "diploid" virus
67
Gag gene includes which proteins?
1. Matrix MA
2. Capsid CA
3. Nucleocapsid NC
4. Protease PR
68
What does the Pol gene encode?
Reverse transcriptase and integrase (IN)
69
What does the Env gene encode?
1. Surface SU (gp120)
| 2. Transmembrane TM (gp41)
70
What generates tissue tropism in retroviruses?
Whether the TF expressed in a given cell type are capable of binding the U3 region on the LTR
71
What generates tissue tropism in retroviruses?
The TF expressed capable of binding the U3 region on the LTR
72
How do non-transforming retroviruses cause cancer?
1. Downstream LTR is activated upon integration - up-regulates cell signals to cause growth
2. Enhancer insertion - recruit RNA Pol to wrong spot
73
When do retroviruses cleave their gag, and gag-pol proteins? What happens if this doesn't occur?
1. After budding
| 2. Non-infectious viral particles
74
The decline in deaths due to AIDS coincided with the development of what?
Protease inhibitors and the development of HAART
| -2 RT and 1 PI
75
How is HIV transmitted?
1. Inoculation in blood - transfusion, needle sticks
2. Sexual
3. Perinatal - Elective C section, don't breast feed if HIV+
76
How is HIV not transmitted?
Close personal contact - household members and healthcare workers NOT exposed to blood
77
What are the accessory proteins of HIV?
Vif, Vpr, Vpu, Nef, Tat, and Rev
78
Tat
Transactivator of transpiration - absolutely required for transcription
79
Rev
Regulator of virion expression - promotes transport of unspliced RNA from nucleus to cytoplasm
80
Vpu
Promotes virion release from cell by inhibiting host protein tetherin - blocks release of enveloped viruses from cell
81
What is the receptor on immune cells that HIV binds? Is this sufficient for entry?
CD4 - conveys immune cell tropism
| No. Need a co-receptor - confers further tropism
82
M-tropic HIV use what co-receptor?
CCR5 - natural ligands are RANTES, MIP-1α, MIP-1β
83
What type of HIV is the source of person to person transmission?
M tropic
84
Resistance to M-tropic HIV is caused by what?
32bp-deletion in CCR5 gene - non-functional co-receptor
Heterozygotes: get infected, progress disease more slowly
Homozygous: highly resistant to infection - don't express co-receptor
85
What is the mechanism of HIV fusion?
1. Binds CD4 - exposed co-receptor binding site
2. gp41 fusion domains exposed and bind to PM
3. Co-receptor binds - "snapback" ￫ membranes fuse
86
How do HIV infect T cells?
Infection at mucosal surfaces - DC cells carry HIV to lymph nodes where T cells reside and are infected
87
In early infection with HIV is replication high or low?
High in lymph nodes - immune system is keeping viremia low
88
What are 3 main disease mechanisms of HIV?
1. Directly killing CD4
2. Immune system kills infected cells
3. Immunosuppression due to loss of CD4 T cell help
4. Dysfunctional Mφ
89
What are characteristics of the acute infection phase of HIV?
1. High viremia
| 2. Initial drop in CD4
90
What are characteristics of the symptomatic phase of HIV?
1. CD4 depleted <200/μL
2. High viral load
3. Opportunistic infections
91
What are 5 classes of anti-HIV drugs?
1. RT inhibitor (NRTI/NNRTI)
2. Protease inhibitor
3. Fusion inhibitor
4. Entry Inhibitor
5. Integrase inhibitor
92
In what locations is HIV found in order of increasing half life?
1. Circulation: 6 hrs
2. CD4: 1 day
3. Mφ: 1-2 years
4. Memory T cells: 5 years
93
What are symptoms of acute retroviral syndrome?
Fever, myalgia, weight loss, rash, malaise, oral ulcers - seen 2-6 weeks after infection
94
What are the pros and cons of the antigen (RNA) tests for HIV?
Detects infection after 10-14 days - HIV viral load/RNA
| Expensive - pooled RNA testing
95
Combination test HIV antibody/antigen
Cheaper, quicker than ELISA, slower than HIV RNA test
96
What is the chance of an untreated mother transmitting HIV to her baby? Treated?
25%
| 0-1% (HAART, AZT during labor, formula feeding)
97
What determines the likelihood of HIV transmission?
Viral load
98
What is the definition of a healthcare exposure to HIV?
1. Percutaneous injury - needle stick
2. Contact of mucous membrane or non-intact skin with
Blood, tissue, CSF, amniotic fluids, etc
99
Post exposure prophylaxis
2-3 drugs ASAP following exposure (<72 hours)
| -expensive for
100
Pre-exposure prophylaxis (PrEP)
2 anti-HIV drugs, prophylaxis for at risk individuals - adherence correlates to prevention
101
Influenza virus is part of which family?
Orhtomyxovirus
102
What are the influenza genus differences based on?
Matrix and nucleoprotein antigens - subtypes base on HA and NA
103
What is the difference between antigenic drift and antigenic shift?
Drift: minor changes in HA or NA or both
Shift: occurs in HA or NA a result of gene reassortment between human and animal strains
104
What is the pathogenesis of Influenza A virus?
Acute Respiratory Disease
- Viral replication and CD8 response destroy epithelium
- Interferon response ➝ Flu Symptoms
105
What are complications with Influenza?
1. Primary viral pneumonia
2. Secondary bacterial pneumonia
3. Myositis/cardiac involvement
4. Neurologic syndromes: GBS, Reye's, encephalopathy
106
How is influenza diagnosed?
Clinical signs/epidemiology
Lab: Rapid antigen capture - detect NP in 15 min
RT-PCR
107
What family does Rhinovirus belong to?
Picornavirus
108
What are characteristics of Rhinovirus?
1. +ssRNA
2. IgA most important in limiting re-infection
3. >150 serotypes
4. Common cold
5. Like growing at 33 degrees
6. Transmission via resp. secretions
109
Are they any effective methods of controlling/preventing rhinovirus?
No. Protease inhibitors are experimental
110
What are characteristics of Coronavirus?
1. +ssRNA
2. Cause common cold - URT
3. No vaccine (SARS/MERS)
111
What are characteristics of the Paramyxoviridae family?
1. -ssRNA
2. Proteins:
F: Fusion protein
HN: Hemagglutinin - attachment & Neuraminidase - release (Not in RSV)
NS1 and NS2: nonstructural proteins that modulate immune response?
112
Where do paramyxoviruses replicate?
Cytoplasm
113
What steps are targets for treatment of paramyxoviruses?
Attachment/fusion
RNA replication
Transcription
114
What are the 5 Paramyxoviruses for this class?
1. Human Parainfluenza Virus
2. Respiratory Syncytial Virus
3. Human Metapneumovirus
4. Hendra and Nipah Virus
5. Measles
115
What are the symptoms of human parainfluenzavirus (HPIV)?
1. Acute upper and lower respiratory illness-often infants, elderly, IC
2. Croup, bronchiolitis and pneumonia
116
Is there a vaccine for HPIV?
No, currently in pediatric phase I trials
117
What are the symptoms of Respiratory Syncytial Virus?
Localized infection of respiratory tract- no viremia
Pneumonia - syncytia
Bronchiolitis - host immune response Th2 (Hist/PGD2) in infants - wheezing
118
What is the #1 cause of fatal acute respiratory tract infection in infants and young children?
RSV - narrow airways are obstructed via syncytia forming giant cells
Maternal Ig doesn't protect
Natural infection doesn't protect
119
Is there a vaccine for RSV?
No
120
How do you treat a premature/IC infant with RSV?
1. Aerosolized ribavirin
| 2. Passive immunization w/ anti-RSV Ig and Ig against fusion protein
121
How do you treat a premature/IC infant with RSV?
1. Aerosolized ribavirin
| 2. Passive immunization w/ anti-RSV Ig and Ig against fusion protein
122
How do you treat hMPV?
Supportive mostly
| Immunoglobulins (IVIG) and humanized monoclonals for fusion protein
123
Hendra and Nipah viruses
Emerging resp. viruses w/ high mortality rate in SE Asia and Africa (from bats)
124
What are symptoms of Adenovirus infection?
1. Pharyngitis, conjunctivitis, GI, Hemorrhagic cystitis
| 2. Viremia (Resp infection)
125
In the respiratory tract, Adenovirus can cause destructive productive infection, _____________________, and latent infection in tonsils or adenoid tissue.
Persistant infection with virus shedding
126
What populations is adenovirus common in?
Infants, young children, military recruits
127
Is there a vaccine for adenovirus?
Yes - for military recruits
128
Why is the MMR vaccine (live-attenuated) so effective?
1. Natural infection protects against re-infection - single antigenic type
2. Virus has systemic replication phase prior to establishing infection in target organ
3. Humans are only known host
129
What are the symptoms of measles?
Maculopapular rash w/ cough, conjunctivitis, photophobia, Koplik spots
130
What are complications associated with measles?
Otitis media, croup, pheumonia, blindness, enchephalitis (immune systems been exposed worse rxn)
131
What does atypical measles look like?
More intense rash distally, possible vesicles, petechiae, purpura
132
Measles is _____ infectious among susceptible populations.
Extremely
133
What are the symptoms of Mumps?
1. Asymptomatic (~75%)
2. Parotitis - b/l w/ fever
3. Swelling of other glands
4. CNS involvement (mild meningitis, rare encephalitis)
134
What is the initial life cycle for Measles and Mumps?
1. Inoculation of respiratory tract
2. Local replication
3. Viremia
4. Systemic infection
5. Targets tissue
135
What are mumps outbreaks associated with currently?
College campuses - are boosters needed?
136
What are the symptoms associated with Rubella?
1. Mild rash - children
2. Severe rash, arthritis, arthralgia - adults
3. Congenital defects in neonates < 20 wks
- can be asymptomatic
137
What family does Rubella belong to?
Togavirus
138
Congenital Rubella Syndrome
Heart defects, deafness, IU growth retardation, failure to thrive, mortality w/i first year, mental retardation
139
What are the characteristics of Parvovirus?
1. ssDNA
2. Icosahedral, non-enveloped
3. Replicates in nucleus
4. Dependent on host DNA rep fx
140
What cells does Parvovirus B19 infect?
Actively replicating (S-phase) RBC progenitors in bone marrow (viremia) via blood group P antigen
141
What symptoms are associated with Parvovirus?
1. Rash, bright red cheeks, arthralgia (circulating immune complexes)- Erythema infectiosum/Fifth Disease
2. Slight drop in hemoglobin (normal host)
3. Life-threatening aplastic crisis (chronic hemolytic anemia host)
142
How long is the incubation period for Parvovirus B19? When do characteristic symptoms appear?
1. 7 days
| 2. Day 20
143
What happens if parvovirus gets transmitted to fetus?
Still birth, generalized edema, anemia, congestive heart failure -- Associated with death, not abnormalities
144
What is the most common cause of severe diarrheal illness requiring hospitalization in infants and young children?
Rotavirus
145
What are characteristics of Rotavirus?
11 dsRNA segments - 12 genes
No envelope, 3 layer capsid
Multiple antigenic groups (Group A causes human disease)
146
What are the antigens of Rotavirus?
VP4 neutralization antigen (P serotype-28)
VP7 neutralization antigen (G serotype-19)
VP6 subgroup antigen (A-E)
NSP4: acts as enterotoxin
147
NSP4 (Rotavirus)
Enterotoxin
1. Raises intracellular Ca → disrupts tight junctions
2. Secreted into lumen, bind receptor → inc. Intracellular Ca
3. Stimulate enteric nerves/act on goblet cells inc. Ca → efflux of Cl
148
Is there a vaccine for Rotavirus?
Yes. Two.
RotaTeq: Live attenuated 3x orally at 2, 4, and 6 months
Rotarix: Live attenuated 2x orally at 2 and 4 months
149
What are considerations for Rotavirus?
1. Intussusecption
2. Reversion to virulent strain
3. Viremia
4. Shedding of virus by vaccine recipients
5. Immunosuppressed children
150
What family is Norovirus apart of?
Calici family
151
What symptoms are associated with Norovirus?
1. Acute gastroenteritis - winter dt new strain
2. Short-lived ~ 3 days D/V/N, cramps, headache, malaise, fever
3. Most serious infection in young children/elderly
152
What are characteristics of Norovirus?
1. +ssRNA - Picornalike
2. Protease (Pro)
3. Polymerase (Pol)
4. Structural proteins
153
What is the most common cause of foodborne illness in the US?
Norovirus
154
Why is Norovirus so easily transmitted?
1. Low infectious dose
2. Prolonged asymptomatic shedding ~2 wks
3. Stable in environment
155
What viruses that are apart of the Enterovirus group do we need to know for this class?
1. Poliovirus
2. Enterovirus
3. Coxsackie A Virus
4. Echovirus
156
What determines tissue tropism for enterovirus infection?
Receptor bound
157
How are enteroviruses transmitted and what is their life cycle like?
1. Fecal/oral or respiratory
2. Replicate in GI/oropharynx
3. Viremia
4. Target tissues
158
What viruses cause hand, food and mouth disease?
Coxsackie A and Enterovirus 71
159
What viruses cause muscle infections i.e. myocarditis and pericarditis?
Echo, Coxsackie A, B
160
What viruses cause encephalitis and paralytic disease?
Polio and Coxsackie
161
What viruses cause meningitis?
Echo, Polio and Coxsacie
162
Everyone of the enteroviruses can cause which disease?
1. Respiratory tract infections
2. Undifferentiated fever
3. Paralytic disease
4. Encephalitis, meningitis
163
Most viral meningitis in the summer is caused by what virus?
Enterovirus
Children; fever, irritability, poor eating, hard to awaken
Adults: Headache, F/C, N/V, stiff neck, sensitivity to light
164
What are symptoms of hand, foot and mouth disease?
Mild fever, sore throat, fatigue, loss of appetite, vesicular lesions on hand, feet, mouth
165
What factors contribute to emergence of new virus disease?
1. Inc. population density
2. Increased travel
3. Changes in social behavior
4. Changes in environment
5. Changes in industry/technology
6. Changes in microorganisms
166
What are characteristics of herpesvirus?
Evenloped, iscosahedral, linear dsDNA
| Not labile in environment
167
What viruses are α herpesviruses?
HSV1 and 2
| Varicella zoster virus
168
What are some characteristics of α herpesviruses?
Short replication cycle
Broad host range
Latency in sensory ganglia
169
What viruses are β herpesviruses?
Cytomegalovirus (CMV)
| Roseolovirus (HHV-6)
170
What are some characteristics of β herpesviruses?
Longer replication cycle
Latency in glands
Host range limited
171
What viruses are γ herpesviruses?
EBV and Kaposi's Sarcoma
172
What are some characteristics of γ herpesviruses?
Slow replication
Limited host range
Latency
173
What makes a virus a herpes virus?
1. Encode TK, PK, DNA Pol
2. Synthesis of DNA and capsid - nucleus
3. Lytic replication & latency
174
What is the general life cycle of a herpesvirus?
1. Binding/fusion
2. DNA → Nucleus
3. vhs degrades host mRNA
4. VP16 goes to nucleus to start transcription
5. Viral DNA circularizes
6. α→ β →γ gene transcription via host
7. Packaging/budding
