2/21/2013
Recent studies regarding
the aerobiology of
influenza and other
respiratory viruses
John Lednicky, PhD
Department of Environmental and Global Health
18 Feb. 2013
Aerobiology
The study of airborne microorganisms, pollen, seeds, and
spores, especially as agents of infection.
How do humans acquire (“catch”) influenza?
How are influenza viruses transmitted person to person?
Note uncovered
eyes
1
2/21/2013
CAUTION (especially to POLICY
MAKERS and EPIDEMIOLOGISTS):
There are many different influenza
viruses! They vary genetically, have
somewhat different biological
properties, and the type of illness
they cause is not identical.
What do influenza viruses look like?
There are many models….
Be wary of Generalizations and
Information Buckets!
Influenza A and B viruses
2
2/21/2013
Influenza C viruses lack a neuraminidase gene
Influenza type A viruses are divided into subtypes on the basis of two
proteins on the surface of the virus: hemagglutinin (HA) and
neuraminidase (NA).
For example, an “H7N2 virus” designates an influenza A virus subtype
that has an HA 7 protein and an NA 2 protein.
Only influenza A virus subtypes H1N1, and H3N2 are currently in
general circulation among people
There are 17 known HA subtypes and 10 known NA subtypes.
All known subtypes of influenza A viruses can infect birds, except
subtype H17N10 which has only been found in bats.
Host Range of Influenza Viruses
Genus
Genus
Influenzavirus A
Influenzavirus A
Influenzavirus B
Influenzavirus C
Influenzavirus B
Influenzavirus C
Orthomyxovirus Genera, Species, and Serotypes
Species (* indicates
Serotypes or Subtypes
Orthomyxovirus
Genera, Species, and Serotypes
type species)
Species (* indicates type H1N1, H1N2, H2N2,
Serotypes or Subtypes
species)
H3N1, H3N2, H3N8,
H5N1,
H1N1, H5N2,
H1N2, H5N3,
H2N2,
Influenza A virus*
H5N8,
H3N1, H5N9,
H3N2, H7N1,
H3N8,
H7N2,
H5N1, H7N3,
H5N2, H7N4,
H5N3,
H7N7,
Influenza A virus*
H5N8, H9N2,
H5N9, H10N7,
H7N1,
etc.
H7N2, H7N3, H7N4,
H7N7, H9N2,
H10N7,
Influenza B virus*
Victoria,
Yamagata
etc.
Influenza C virus*
Influenza B virus*
Influenza C virus*
Victoria, Yamagata
Hosts
Hosts
Human,, pig,
bird, horse,
seals,
cetaceans,
Human,
pig, bird,
other
horse,mammals
seals,
cetaceans, other
mammals
Human, seal, pig
Human, pig
Human, seal, pig
Human, pig
3
2/21/2013
Host Range of Influenza Viruses
Shifts and Drifts
Drift = natural mutations that
occur over times
Shift = two or more strains of a
virus combine to form a new
subtype with a mixture of
genes from the two.
Reassortant (“new”) virus
4
2/21/2013
Textbooks say you catch the flu by……..
How do you “catch” the flu?
•Most textbooks state it is through droplet (especially large)
infection
•Influenza virus experts say inhalation of small airborne
particles is also important
Distances travelled by
“expelled” droplets
5
2/21/2013
Highly Pathogenic H5N1
Avian Influenza Virus
59% fatality rate
6
2/21/2013
Is aerosolized
H5N1
infectious?
Root word = Heinous
High Heinicity Co-efficient = Hc
•Security clearance
•Select agent clearance
•Acquire H5N1 strains
•High-containment facility (BSL3+, USDA and CDC inspected/certified)
•PAPR
•Animal work requires ABSL3-ag or similar facility that is USDA and CDC
inspected/certified
•Vaccines? Shower out (treatment of black water)
7
2/21/2013
Virus Propagation in
Embryonating Chicken Eggs
Schematic: Nose-Only Inhalation Exposure System
System
Pressure
relief
HEPA
9
10
Flow Meter
Exhaust
Pump
Outside
glovebox
HEPA
System
Differential
Pressure
HEPA
P
Nose only
exposure tube
CH Technologies
5 port exposure
system
HEPA
UV Aerodynamic
Particle Sizer
P
UV APS
Flow Controller
3
87
3 Way
MFC
4 valve
Midget
Impingers
HEPA
Aerosol Generator
Valve
2
BANG
Nebulizer
Generator Bypass
HEPA
Network
Computer
7
Dry Dilution Air
HEPA
Flow Meter
Flow Meter
5
HEPA
Humidifier
System
Pressure
Regulator
Wet Dilution Air
1
Air
compressor
11
Sample
Pump
Check Valve
6
HEPA
Flow Meter
Lednicky JA, et al. 2010, Virol J. 7:e231
Inside
glovebox
5 port exposure system
ferret holder
Some Key
Components
BANG
Nebulizer
3-jet bioaerosol nebulizing
generator (BANG)
Lednicky JA, et al. 2010, Virol J. 7:e231
8
2/21/2013
EXPOSURE WORK OCCURS WITHIN A
CLASS III GLOVEBOX in an ABSL3 –
ENHANCED or ABSL3 – Ag
LABORATORY
Polystyrene Latex Microsphere (PSL) Exposure System
Concentration vs. Time Profile
TSI UVAPS Model 3314
1e+6
Total Counts
8e+5
6e+5
4e+5
2e+5
0
0
60
120
180
240
300
360
Time (sec)
1.0µm PSL
1.8µm PSL
3.0µm PSL
Make sure your cell
culture cells are not
contaminated!
*mycoplasma
**viruses
9
2/21/2013
Syncytium and vacuoles in MDCK cells
Reagent quality is important!
MDCK = PBS
(supplier 1)
MDCK = PBS
(supplier 2)
Non-viable filamentous
structures in gammairradiated serum (400x)
10
2/21/2013
Quantification of Infectious Virus Particles - a
Reed & Muench Calculation for Determination of TCID50
TCID50 = 50% Tissue culture infectious dose
Inputs:
a, Dilution factor
b, Highest dilution with mortality below 50%
c, % Mortality at highest dilution with mortality below 50%
d, Lowest dilution with mortality above 50%
e, % Mortality at lowest dose with mortality above 50%
Plaque Assay
Dissemination of live influenza virus strains and measurements in dual-linked
impingers indicates the viruses are delivered by the NBIES at 9 – 30%
efficiencies
D = R x C x texp.
D = Capp x Vm x texp
Rod shaped! Collection
efficiency in liquid medium
is affected (opposite to
mathematical predictions)
11
2/21/2013
Intranasal vs Aerosol: Exposure to aerosolized H5N1
leads to earlier clinical signs/death
Sign/Observation
Range of day(s) symptoms observed postinoculation with virusa
WS/05
intranasal
IRAQ/06
intranasal
Viet/04
intranasal
Viet/04
aerosol
N/Oc
N/O
Days 5 - 12
Days 3 - 5
Deathb
Soft stool/diarrhea
N/O
N/O
Days 3 - 5
Days 2 - 5
Fever
Days 2 - 7
Days 2 - 7
Days 2 – 9
Days 2 - 5
Inappetence
Days 2 - 7
Days 4 - 7
Days 3 - 12
Days 2 - 5
Labored breathingd/wheezing
Days 4 - 6
N/O
Day 5
Days 4 - 5
Lethargy
Days 3 - 7
Days 4 - 5
Days 3 - 12
Days 1 - 5
Neurologic signs
Aggression-dementia N/O
N/O
N/O
Day 4
Ataxia
N/O
N/O
Days 5 - 12
Days 4 - 5
Convulsions
N/O
N/O
Day 5
Days 4 - 5
Head-tilt
N/O
N/O
N/O
Day 4
Hind-limb paralysis
N/O
N/O
N/O
Day 4
Shaking of head (only) N/O
N/O
Days 11- 12
N/O
Shaking (whole body) or shivering
Days 5 - 6
N/O
N/O
Days 2 - 4
Sneezing
Days 5 - 6
N/O
N/O
Day 2
Weight loss
Days 1 - 7
Days 4 - 7
Days 2 - 12
Days 1 - 5
Resolution
Day 8 onwards Day 8 onwards Uncertain
N/O
Dehydration/Thin
Day 7
N/O
Days 5-12
Days 3-5
aTen-day observation period for WS/05 and IRAQ/06; twelve-day for Viet/04.
bAnimals found dead or euthanized for humanitarian reasons
cN/O; not observed.
dLabored breathing; animals exhibited open-mouth breathing with exaggerated abdominal movement.
Lednicky JA, et al. 2010, Virol J. 7:e231
Step 6
Shed intestinal lining from a ferret exposed to aerosolized VN/04
.
VN/04 titers appear higher in brain vs
lung tissues
.
12
2/21/2013
Most important findings
1. Avian influenza virus is infectious when inhaled.
2. The infectious dose of H5N1 Z-Genotype clade/subclade 1 and 2.2
viruses is apparently < 40 infectious virus particles by IN or inhalation
exposure routes.
3. Lung lesions occur only when ferrets are exposed to high doses of
viruses like H5N1 Viet 1203/2004 (2005)
4. Brain lesions occur at all doses with Z-genotype clade 1 viruses.
5. Virus titers are higher in brain than lung tissues at all doses with Zgenotype clade 1 viruses.
6. Low doses are fatal; at higher doses, animals get sick quicker but some
survive with Z-genotype clade 1 viruses.
7. Outcomes of clinical disease in ferrets mirrors those of human: mild,
medium or severe, depending on H5N1 strain/clade.
BioBubble
• Current Air Samplers don’t work well for
aerosolized viruses
• Lack of effective sampling causes major
“headaches” for aerobiology
SKC Biosampler
(sampling rate 16.5
Lpm)
Sioutas personal
cascade mpactor
Preloaded
cassette filter
13
2/21/2013
Allergy vs “Cold?
Layer of yellow pollen
on liquid-air
interphase of
biosampler collection
tube
Pine pollen
DAY 1
OFFICE ENVIRONMENT
DAY 2
DAY 3
DAY 4
DAY 5
AM
PM
OFFICE ENVIRONMENT
DAY 6
DAY 7
DAY 8
RESIDENTIAL AIR
DAY 1
AM
AM
PM
PM
Nasal Discharge, Environmental Samples, Air Sampler Samples
Cell cultures
Cell
A549
BHK-21
HeLa
LLC-MK2
LLC-MK2 + Trypsin
MDCK
MDCK + Trypsin
Mv1-Lu
Mv1-Lu + Trypsin
MRC5
NCI-292
Vero
Vero+Trypsin
33°C
̶
●
●
●
●
●
●
●
●
●
̶
●
●
Extract DNA; PCR
37°C
●
●
●
●
●
●
̶
●
̶
●
●
●
●
Bacteria
Chlamydophila pneumoniae
Haemophilus influenzae
Legionella pneumophila
Mycoplasma pneumoniae
Streptococcus pneumoniae
DNA Viruses
Bocavirus
Human Adenovirus
KI Polyomavirus
WU Polyomavirus
Extract RNA; RT-PCR
RNA Viruses
Coronaviruses 229E, HKU1, NL63, and OC43
Enteroviruses (includes Rhinoviruses)
Influenza A, B, and C viruses
Metapneumovirus A and B
Parainfluenza virus types 1, 2, 3, 4A, and 4B
Respiratory syncytial virus types A and B
Rhinovirus C
14
2/21/2013
Rhinovirus A, B, C = cause of most
respiratory infections in humans!
Rhinovirus C = recently discovered; may
cause severe infections, pneumonia, fatality
Blood, sweat, and tears
Isolate vRNA
Sequencing
U
–
–
–
–
–
–
U
U
C
A
Stem-pairing
AUG
5’..U
U
A
A
C
A
G
U
G
G
U
A
U
U
C
C
U
Some molecular features of a new Rhinovirus C
A
U
A
C
C
A
–
–
–
–
–
–
U
U
G
U
C
A
U
– C
– A
– U
G
G
ORF
U
A
U
G
G
U
U
Consensus sequence
– G
– G
G
C..3’
A
A
G
C
U
C – G
A – U
C – G
U C U
C C C A C C A A U G CA – UU A G U G C
G G A A U C U
C
| | | |
| | | |
U
| | | | | | | |
C
G
C
A
C
G
C C U U G C C
U G G U U A C G
G – C
U G U
U U U
G – C
U – A
C – G
A – U
A – U
A – U
A
G
U
CCCUCCCUUCCCCAAGUCUUU
VPg-U
5’ CLOVERLEAF and PYRIMIDINE-RICH TRACT
Optimal Energy RNA Configurations
5’ ORF start site
The IRES reveals a bait-and- switch arrangement for initiation of
translation
15
2/21/2013
3’ UTR of new
rhinovirus C
5’. .
U
A
G
U
C
C
A
A
A
A
U
A
C A
A G
C
U
A
A
U G
U-A
U-A
A-U
U-A
G
U-A
A-U
C-G
A-U
C-G
G
A
A-U
U-A
A-U
U
A
U
A
Optimal Energy RNA Configuration
3’ UTR with unbranched stem motif
before the poly-A tail. The left-most
codon (orange box) is the ORF
terminator. Other boxes (yellow)
highlight additional terminators.
New Rhinovirus C51
pHRV-C JAL-1/USA/2010 (RG)
CMV
promoter
ASC-1
Hammehead
ribozyme
5’-TTAAA… RHV-C GENOME …GATAT-3’
TT
A22
Hep Delta
ribozyme
Mlu-1
termin
-ator
Genomic cRNA = 7,155 bp
A
B
C
D
Current Influenza Situation in
Gainesville
• Influenza H1N1 and Influenza B were
circulating during the summer
• Influenza H3N2 and H1N1 circulated recently
(virus is present in aerosols)
16
2/21/2013
Adenovirus from hallway air
Negative control cells (5-day old culture)
Cells five days post-inoculation with virus
•Same virus collected in air samplers (hallways and offices)
• Same virus collected from office workers with mild URT infection
•Same virus collected on work surfaces (computers, etc).
•Residence time in air ~ 1 hr after office workers leave
•Virus detection for 3 symptomatic days
Coronavirus – Florida Nov – Dec 2012
Aspergillus fumigatus and Dolphin morbillivirus
17
2/21/2013
Coronavirus NL63
Doublemembrane
vacuole
Granular nucleoprotein
material in packet
Virus particles in
RER cisternae
with ribosomes in
place. Electron
dense granular
nucleocapside
material is visible
in some of the
virus particles.
18