INFLUENZA A VIRUS IN SWINE:OVERVIEW OF DISEASE & DIAGNOSTICS

Boehringer-Ingelheim Summer Health Seminar

Phil GaugerIowa State University Veterinary Diagnostic Laboratory

August 21, 2015pcgauger@iastate.edu

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

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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

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neum

onia

Cas

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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%

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30%

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50%

2014

2013

2012

2011

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2014 2013 2012 2011 2010

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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

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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

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1 Cl

uste

r

H3 IAV Cluster Distribution

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

0%

10%

20%

30%

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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!