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INFLUENZA A VIRUS IN SWINE:OVERVIEW OF DISEASE & DIAGNOSTICS
Boehringer-Ingelheim Summer Health Seminar
Phil GaugerIowa State University Veterinary Diagnostic Laboratory
August 21, [email protected]
Iowa State University-VDL• Ames, Iowa
• Full Service & Fully Accredited Laboratory
• Total ≈ 125 People – 22 Faculty– 103 Technical Staff
• Veterinary Diagnostic Medicine: 8 Total Sections1. Pathology2. Virology and Molecular Diagnostics3. Bacteriology4. Serology/Immunology5. Toxicology & Nutrition6. PhAST (Clinical Pharmacology)7. Epidemiology8. IT
ISU VDL Revenue by Species
2009 2010 2011 2012 2013 2014 20150%
20%
40%
60%
80%
100%
Porcine Bovine Poultry Canine/FelineEquine Sm. Ruminant Other
Year
Perc
ent o
f Tot
al R
even
ue
Porcine: 78%
Where the Pigs Are Located in US
Swine byways:≈ 500,000 pigs/week imported to IA to be grown
IAV-S: The Virus• Influenza A virus in swine
– Orthomyxoviridae family– Genome:
• Negative-sense • Single-stranded• Segmented• Enveloped• RNA virus
• Influenza B and C viruses– Human pathogens; C viruses occasionally in pigs
IAV-S: The Genome• Segmented RNA virus
– Eight segments; 10-11 proteins• 1: Polymerase basic 2 (PB2)• 2: Polymerase basic 1 (PB1)• 3: Polymerase acidic (PA)• 4: Hemagglutinin (HA)• 5: Nucleoprotein (NP)• 6: Neuraminidase (NA)• 7: Matrix (M or M1/M2)• 8: Non-structural (NS1/2)
Influenza in Swine: History• H1N1 IAV isolated in swine in 1930
– 1918 Spanish flu spillover• Humans into swine
– One IAV subtype in swine for 80 years• 1918 to 1998, genetically stable in swine
• H3N2 IAV– 1998 triple reassortant virus
• Human to swine introduction• Genes with swine, human and avian influenza
– Increasing genetic diversity since 1998
ISU-VDL Frequency of Respiratory Disease Diagnosis
Pathologists diagnosis of IAV respiratory disease
PRRSVIA
V-S
S. suis
P. multo
cida
M. hyo
pneumoniae
H. para
suis
A. suis
Bacter
ial
Idiopathic
B. bro
nchise
ptica
APPPCV
A. pyo
genes Vira
l
Salmonell
aPRCV
Parasit
ic0%
10%
20%
30%
40%
50%45.1%
23.9%
6.6% 6.3% 5.7%2.5% 2.2% 2.1% 1.3% 1.2% 1.2% 0.9% 0.7% 0.3% 0.1% 0.0% 0.0%
2014-2015
Pathogen
Perc
ent o
f Tot
al P
neum
onia
Cas
es
Influenza
ISU-VDL: 2014 - 2015
Detection of IAV-SSeasonal influence but detected throughout the year
Influenza A Virus: Challenges
• Influenza Challenges in Swine– Respiratory disease
• Endemic infections, co-infections– Production & economic losses
– $10.31 per market pig– 64% due to lost production
» AVG, FE, Top hogs marketed– 36% due to veterinary related expense
– Ubiquitous virus, transmission– Virus genetic diversity
• Increasing antigenic drift and shift– Zoonotic potential– Cross-protection/prevention
Why is IAV Changing?• Increased mixing of pigs
– Increased mixing of viruses with different genetics• Large pool of viruses for exchanging genetic material• Antigenic shift
– Influenza has a segmented genome• 8 segments that contain the code for proteins• Antigenic shift
– Influenza is an RNA virus• Common for replication errors to occur in their genome• Antigenic drift
IAV: Evolution
Antigenic drift
Selective point mutations in the
genome
• Mild genetic variations• Impact cross-reactivity
• Slow genetic changes• Difficult to recognize over time
• RNA virus• Polymerase errors during replication
• May evade populations immunity• New antigenic variant
IAV: Evolution
Antigenic shift
• Large genetic changes• Impact cross-reactivity
• Two viruses must infect the cell• Simultaneous replication• Exchange segments of
genome
• Major antigenic changes• Much different IAV
• H5N1 highly pathogenic AIV• H5N2 and H5N8
• These are reassortants• With endemic LPAI
Reassortment of entire gene segments
IAV Transmission and Disease• Virus transmission
– Direct contact: nasal secretions, aerosolized virus• Most common form of transmission• However, detected in air, outside infected barns, 1 mile from infected farm
• Virus infection– Epithelium of the upper and lower respiratory tract
• Nasal mucosa, trachea, lungs (bronchi and bronchioles)• Virus isolation
– Shedding virus begins 1-3 days post inoculation (dpi)– Shedding may last 4-5 days, occasionally 7 dpi– Virus is confined to the respiratory tract
• No productive infection in blood, tissues, fetuses• Abortions are diagnosed from samples collected from the dam
Influenza A Virus: Gross Lesions
• Cranioventral or lobular • Red to purple
consolidation– Locally extensive,
• slight to moderately firm– Resilient, non-collapsing,
+/- edema– Mixed infections are
common: may appear different or more severe
IAV: Cranioventral consolidation
Normal Lung
IAV-S: Gross Lesions• Influenza A virus
– Suckling & nursery pigs• May observe subtle lesions
• Influenza A virus―“Checker-board”― Lobular pattern
IAV: Microscopic Lesions• Necrotizing bronchiolitis
– Causes coughing• Destroys epithelium
– Causes inflammation• Around the airway
Look for these descriptions in the histopath
Diagnosing IAV• Important to know the cause of sick pigs
– Need a diagnosis for treatment and prevention• Antemortem or Postmortem
– Presumptive diagnosis• Coughing, ‘barking’ pigs, slow, off feed• Test for IAV in oral fluid samples, nasal swabs• Not always easy to determine a diagnosis
– Etiologic diagnosis• Submit tissue samples• Clinical signs• Test for IAV in lung tissue• Histopathology demonstrates a lesion in lung
IAV: Diagnostics• Antemortem Diagnostics: direct detection of virus
– Oral fluids• Samples collected from a large population
– Increased chance of detection• Presumptive diagnosis with detection
– Correlate clinical signs with detection of IAV
– Nasal swabs• Collect sufficient numbers to increase detection• 10% prevalence, collect 40-50 swabs
– Pool swabs up to 5 for PCR • Neonatal/nursing piglet suspect influenza
– Collect from febrile pigs
Oral Fluid Samples: Number of Tests
2010 2011 2012 2013 20140
20k
40k
60k
80k
100k
2,083
14,265
35,281
60,383
94,199
Fiscal Year
Num
ber o
f Tes
ts
IAV: Diagnostics• Postmortem Diagnostics
– IAV-S diagnosis is based on detection and lesions• Necrotizing bronchiolitis hallmark lesion
– PCR or immunohistochemistry for detection of virus• PCR more sensitive
– Evaluate Ct levels– Suggests semi-quantitative levels of virus
• IHC detection within a lesion– Indicates the presence of influenza– Where influenza replicates– If IHC negative
» Detection may be too late » Lower sensitivity compared to PCR
Tissue Collection• Agents on your differential list
– Know where they like to replicate/reside• Sample from the lesions• If there are no detectable gross lesions
– Collect multiple sections– Microscopic lesions are not necessarily evenly distributed– Focus collection on the organ system(s) to which the
clinical signs are referable • Coughing pigs – need lungs
• Submit fresh and formalin-fixed samples– Should be appropriate size – Bigger is not always better
Tissue Collection
• Be clean• Reduces erroneous results
• “Size Matters”
• Keep fresh tissues chilled (double bag)
Fresh: golf ball size
Fixed: thin like a flash drive
Tissue Collection• Fixed tissues:
– Formalin • 10:1 ratio of fixative to tissue for optimal fixation • As quickly as possible• Within 15 - 30 minutes of death
– Slice tissues into thin sections (0.5 – 1.0 cm thick)– Understand where the agents on your differential
list reside and sample accordingly– Do not allow fixed material to freeze
• Dilute 10% formalin with equal volumes of 70% alcohol– Winter months
If no gross lesions: Location for collection of 5 slices of
lung for histopath
1
2
34
5
Include airway cross sections
Include affected and adjacent unaffected tissue
Histopathology
IAV Diagnostics: When to Sample
IAV: Diagnostics• Antemortem Diagnostics: indirect detection
– Serology• Nucleoprotein antibody ELISA
– Detects antibody to influenza across species– Natural exposure antibody, vaccine antibody, maternal antibody– Does not suggest cross-protection
• Hemagglutination inhibition antibody– Functional assay
» Requires panel of anti-sera and virus of interest» Antisera panel updates are necessary
» Keep pace with changing viruses in circulation– Cross HI assays are helpful for predicting cross-protection
» Titer 40 or higher
Influenza Virus PCR & Subtyping• Influenza virus detection by PCR
– Screening PCR assays• Is influenza present in the sample?
– Lung samples– Oral fluids– Nasal swabs
– Subtyping PCR• Detects if H1, H3 / N1, N2
– Two separate PCR reactions– May be less sensitive than screening assays
• Interpretation: different combinations of subtypes
Influenza Virus PCR & SubtypingResult Category Result
No Result Untypeable Unknown/NegativeH1 Incomplete Partial SubtypeH3 Incomplete Partial SubtypeN1 Incomplete Partial SubtypeN2 Incomplete Partial Subtype
H1/H3 Incomplete Partial subtype/Mixed InfectionN1/N2 Incomplete Partial subtype/Mixed InfectionH1N1 Single Single subtypeH1N2 Single Single subtypeH3N1 Single Single subtypeH3N2 Single Single subtype
H1/H3N1 Multiple Mixed infection/PartialH1N1/N2 Multiple Mixed infection/PartialH1/H3N2 Multiple Mixed infection/PartialH3N1/N2 Multiple Mixed infection/Partial
H1/H3N1/N2 Multiple Mixed infection
Resample/Retest
Mixed Infections
Mixed Infections
IAV Sequencing Success: HA Gene
Specimen ScreeningPCR Ct
Success (direct) Success with VI Success (direct&VI)
% % %
Lung <25 68.83% 21.43% 90.26%25-29.9 0.00% 19.23% 19.23%30-38 0.00% 0.00% 0.00%Total 46.70% 16.74% 63.44%
Nasal Swab <25 56.67% 26.67% 83.33%
25-29.9 37.50% 18.75% 56.25%30-38 0.00% 0.00% 0.00%Total 41.07% 19.64% 60.71%
Oral Fluid <25 55.74% 16.39% 72.13%
25-29.9 14.71% 11.76% 26.47%30-38 0.00% 0.00% 0.00%
Total 29.10% 10.45% 39.55%JQ Zhang; ISU VDL
IAV Virus Isolation SuccessSpecimen Screening PCR Ct VI Success
%Lung <25 89.63%
25-29.9 75.17%30-34.9 25.84%35-<40 3.43%Total 55.92%
Nasal Swab <25 88.52%25-29.9 61.76%30-34.9 28.81%35-<40 0.00%Total 60.40%
Oral Fluid <25 45.56%25-29.9 17.92%30-34.9 5.16%35-<40 0.00%
Total 17.42%
JQ Zhang; ISU VDL
Influenza A Virus: Clinical Presentation• Acute, fulminating IAV-S
– Classical presentation• Potentially less frequent• Rapid transmission, fever, anorexia, coughing• Dyspnea, mortality, rapid recovery
– Occurs after genetic or antigenic variant IAV introduction– Little to no cross-protective immunity in the group
• Straightforward diagnosis: lesions, diagnostic tests• Rarely confounded with secondary pathogens• Induction of high levels of immunity post-infection• All ages may experience this presentation
Influenza A Virus: Clinical Presentation• Age-associated IAV-S
– Waning maternal antibody from dam• Most common at 10-12 weeks of age but variable• Exposure to endemic IAV in the herd
– Passive immunity decreases over time, pigs are susceptible– IAV infected pigs in the group expose the others
• Protracted timeline, slow progression in the group– Slowly moves through a group, 2-3 weeks
• Clinically variable in presentation– Less dramatic or lower severity– Mild to moderate coughing
• Complicated by concurrent infections– Bacteria
Influenza A Virus: Clinical Presentation• Piglet influenza: nursing to newly weaned pigs
– Perceived as a subtle but persistent clinical expression• Mitigated clinical signs• Scattered/sporadic coughing• 10% of litters affected; endemic infection
– Associated with variable passive protection• Maternal antibody is absent or provides poor/partial cross protection
– Clinical signs/severity may increase with stress• Weaning, movement, transport to the nursery, co-mingling• Passive protection continues to wane, increasing prevalence
– May be difficult to diagnose in this population
IAV-S: Viral Shedding• Grow/Finish swine
– Virus is shed in large quantities– 24-48 hours to 5-8 days post-infection
• Piglets/Neonates/Nursing Pigs– Virus is shed in low quantities– Detection may be poor even with PCR
• Mitigated shedding due to partial immune protection– Look for febrile piglets, sample entire litter
• The coughing piglet may not be the best/optimum sample• Virus shedding may be minimal at that time
• Breeding swine– IAV shedding is minimal
• Unless new introduction of a variant IAV
IAV: Viral Ecology• Three subtypes co-circulate in US swine
– H1N1, H1N2 and H3N2 subtypes most common• Occasional H3N1 subtype• Occasional human-like viruses
• H1 and H3 IAV– Seven antigenic clusters co-circulate
• H1 viruses: Six clusters that includes pandemic H1N1• H3 viruses: Cluster IV with subclusters
– Clusters I, II, III are rarely if ever detected
IAV Subtype Detection
Mixed H3N1 H3N2 H1N2 H1N1
0%
10%
20%
30%
40%
50%
2014
2013
2012
2011
2010
2014 2013 2012 2011 2010
Year
Perc
ent o
f tot
al IA
V su
btyp
es
H1 IAV: Ecology• H1 viruses
– Most diverse subtype in circulation– α, β, γ, δ-1, δ-2 (human seasonal), Pandemic H1N1– γ and δ-1 viruses are most common
• Largest clusters, commonly detected– The α, β and δ-2 are minor clusters in circulation– Rare to find the entire pandemic H1N1 virus
• Pandemic matrix gene is commonly detected in IAV-S– Clusters are important for vaccine selection
H3 IAV: Ecology• H3 viruses
– Characterized by recent genetic diversity• Post-pandemic H1N1 introduction
– Subclusters evolve from the H3 Cluster IV• Six different clades• A, B, C, D, E, F (Kitikoon, 2013; Anderson 2014)
• Clade A most common
H1 IAV Cluster Distribution
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
0%
10%
20%
30%
40%
50%
60%
70%
α-clusterβ-cluster
γ-clusterγ-2-cluster
pandemicδ1-cluster
δ2-cluster
α-cluster β-cluster γ-cluster γ-2-clusterpandemic δ1-cluster δ2-cluster
Year
Perc
ent H
1 Cl
uste
r
H3 IAV Cluster Distribution
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Unclustered
II
IV
Unclustered I IIIII IV Human-like
Why are IAV clusters important?
Clusters included in US influenza vaccines for swine
Influenza Sequencing &Vaccines: Application
• H1 and H3 cluster comparison– Utilize vaccines with same clusters
• Based on sequencing• Genetically compare vaccine and field IAV HA
– Evaluate the amino acid similarity• Prefer >95% homology• Amino acids may affect antigenic cross-reactivity
– H3 viruses receptor binding site: 6 amino acids– H1 viruses are more complicated
Hemagglutination Inhibition
• Hemagglutination inhibition test or cross-HI– Evaluates potential vaccine protection against virus
• Based on HI titer– HI assay requirements
• Need antiserum from vaccinated pigsOR
• Individual vaccine antigen antiseraAND
• Field virus isolated from the farm – Interpretation
• HI titer > 40 suggests efficacy
IAV-S: Future
• Novel vaccine platforms are needed– LAIV and intranasal
• Improved cross-protection between vaccines– Components that ensure cross-protection
• Parameters to determine cross-protection– Beyond the genome or combination– Antigenic cartography
• Serological tests determine mucosal immunity• Immune correlates of protection
Acknowledgements
• Jianqiang Zhang & Karen Harmon• Amy Vincent• ISU VDL• ISU-VDL Pathologists and Staff
Questions?
THANK YOU!