Influenza ‘A’ Virus in Swine: Overview of Disease and Diagnosis - Dr. Phil Gauger, Iowa State University Veterinary Diagnostic Laboratory, from 2015 Summer Swine Health Seminar, August 22, 2015, Wrightsville Beach, North Carolina, USA. More presentations at http://www.swinecast.com/2015-boehringer-ingelheim-carolina-swine-health-seminar

1. INFLUENZA A VIRUS IN SWINE:
OVERVIEW OF DISEASE &
DIAGNOSTICS
Boehringer-Ingelheim Summer Health Seminar
Phil Gauger
Iowa State University Veterinary Diagnostic Laboratory
August 21, 2015
pcgauger@iastate.edu

2. Iowa State University-VDL
• Ames, Iowa
• Full Service & Fully Accredited Laboratory
• Total ≈ 125 People
– 22 Faculty
– 103 Technical Staff
• Veterinary Diagnostic Medicine: 8 Total Sections
1. Pathology
2. Virology and Molecular Diagnostics
3. Bacteriology
4. Serology/Immunology
5. Toxicology & Nutrition
6. PhAST (Clinical Pharmacology)
7. Epidemiology
8. IT

3. ISU VDL Revenue by Species
0%
20%
40%
60%
80%
100%
2009 2010 2011 2012 2013 2014 2015
PercentofTotalRevenue
Year
Porcine Bovine Poultry Canine/Feline
Equine Sm. Ruminant Other
Porcine: 78%

4. Where the Pigs Are Located in US
Swine byways:
≈ 500,000 pigs/week imported to IA to be grown

5. 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

6. 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)

7. 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

8. ISU-VDL Frequency of
Respiratory Disease Diagnosis
Pathologists diagnosis of IAV respiratory disease
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%
0%
10%
20%
30%
40%
50%
PercentofTotalPneumoniaCases
Pathogen
2014-2015
Influenza
ISU-VDL: 2014 - 2015

9. Detection of IAV-S
Seasonal influence but detected throughout the year

10. • 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. IAV-S: Gross Lesions
• Influenza A virus
– Suckling & nursery pigs
• May observe subtle lesions
• Influenza A virus
―“Checker-board”
― Lobular pattern

17. IAV: Microscopic Lesions
• Necrotizing bronchiolitis
– Causes coughing
• Destroys epithelium
– Causes inflammation
• Around the airway
Look for these descriptions
in the histopath

18. 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

19. 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

20. Oral Fluid Samples: Number of Tests
2,083
14,265
35,281
60,383
94,199
0
20k
40k
60k
80k
100k
2010 2011 2012 2013 2014
NumberofTests
Fiscal Year

21. 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

22. 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

23. 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

24. 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

25. If no gross lesions:
Location for collection of 5 slices of
lung for histopath
1
2
3
4
5
Include airway cross sections
Include affected and adjacent
unaffected tissue
Histopathology

26. IAV Diagnostics: When to Sample

27. 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

28. 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

29. Influenza Virus PCR & Subtyping
Result Category Result
No Result Untypeable Unknown/Negative
H1 Incomplete Partial Subtype
H3 Incomplete Partial Subtype
N1 Incomplete Partial Subtype
N2 Incomplete Partial Subtype
H1/H3 Incomplete Partial subtype/Mixed Infection
N1/N2 Incomplete Partial subtype/Mixed Infection
H1N1 Single Single subtype
H1N2 Single Single subtype
H3N1 Single Single subtype
H3N2 Single Single subtype
H1/H3N1 Multiple Mixed infection/Partial
H1N1/N2 Multiple Mixed infection/Partial
H1/H3N2 Multiple Mixed infection/Partial
H3N1/N2 Multiple Mixed infection/Partial
H1/H3N1/N2 Multiple Mixed infection
Resample/
Retest
Mixed
Infections
Mixed
Infections

30. IAV Sequencing Success: HA Gene
Specimen
Screening
PCR 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

31. IAV Virus Isolation Success
Specimen 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. IAV Subtype Detection
2014
2013
2012
2011
2010
0%
10%
20%
30%
40%
50%
Mixed
H3N1
H3N2
H1N2
H1N1
PercentoftotalIAVsubtypes
Year
2014 2013 2012 2011 2010

38. 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

39. 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

40. H1 IAV Cluster Distribution
α-cluster
β-cluster
γ-cluster
γ-2-…
pandemic
δ1-cluster
δ2-cluster
0%
10%
20%
30%
40%
50%
60%
70%
2003 2004 2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
PercentH1Cluster
Year
α-cluster β-cluster γ-cluster γ-2-cluster pandemic δ1-cluster δ2-cluster

41. H3 IAV Cluster Distribution
Unclustered
I
II
III
IV
Human-like
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
Unclustered I II III IV Human-like

42. Why are IAV clusters important?
Clusters included in US influenza vaccines for swine

43. 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

44. 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 pigs
OR
• Individual vaccine antigen antisera
AND
• Field virus isolated from the farm
– Interpretation
• HI titer > 40 suggests efficacy

45. 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

46. Acknowledgements
• Jianqiang Zhang & Karen Harmon
• Amy Vincent
• ISU VDL
• ISU-VDL Pathologists and Staff

47. Questions?
THANK YOU!