The biology of the “new” porcine coronavirus infections in gnotobiotic swine

Steven Krakowka, DVM, PhD, Professor (emeritus)&

Jianrong Li, DVM, PhD,Associate Professor

Department of Veterinary BiosciencesCollege of Veterinary Medicine

The Ohio State UniversityColumbus, OH 43210 (USA)

krakowka.1@osu.edu or krakowkasummit@yahoo.com

Gnotobiotic vs. GermfreeGnotobiotic (GN) animal: A laboratory animal species which is devoid of all

“known” microfauna and microflora.

Germfree (GF) animal: A laboratory animal species in which the microfauna

and microflora are defined or specifically known.

Advantage(s) vs. conventional or even spf animals: In practical terms, the

contributions of microbial interactions to disease development are removed

as are any effects of prior immune activation (innate* or acquired). Thus,

resultant clinical disease/ lesions are directly related to the effects of the

pathogen(s) under study.

*Innate protective responses are essentially not operable in gnotobiotic pigs

prior to challenge infection(s). In GN piglets, all markers of activation are min-

imally (if at all) expressed, lymphoid tissues are inactive, macrophages and/or

dendritic cells, T-, B-, NK- lymphocytes and plasma cells are essentially not

present in lamina propria of mucosal and respiratory surfaces. Piglets are

hypogammaglobulinemic at birth. If IgM is detectible, this suggests in utero

infection.

Why are piglets so amendable to study under gnotobiotic conditions?

Immune competent prior to birth and so the immune responses areinduced. Maternal antibodies are not transferred across the placenta.

Piglets are “self-sufficient”, litter sizes provide for “same age” controls; the principals can be infected/vaccinated or treated early in life.

Gnotobiotic swine: The immediate “goal” is to get a litter of term (142-4 days of gestation) piglets into isolation units alive and living

under sterile conditions.

Derivation: A Caesarian section is performed upon sedated sows under epiduralanesthesia (A). The uterus is removed “intact” (B), entered into an air-tight “dunk tank” (C) and then into a transfer unit (D). Transfer units are then hookedto pentub units (E) that can hold up to six piglets. (average litter size = 11-15).

(A) (B) (C) (D)

(D)(E)

(D) (D)

Swine husbandry and sampling:

Piglets are fed SimalacR or PermalatR

(synthetic milk replacements) 3X/day;

The liquid diet is used to 35 days age.

Heat lamps for first two weeks of age.

Piglets are observed 3X/day for illness

(anorexia, diarrhea). Temperatures are

never elevated in infectious diseases.

Blood samples (jugular venipuncture,

swabs, skin tests, biopsies etc.) are

taken as needed.

Agents studied at OSU with germfree methods:

Bacterial species (Gram negatives): H. pylori (human): 1984-1999; H. cerdo, H. heilmannii (swine): 2004-2008.

Viruses: Rotaviruses (human), 1983-1986.

Porcine circoviruses (PCV1, PCV2), 1998-2011.

Porcine torque teno virus (TTVs), 2006-2010.

Norovirus (humans, pigs), 2011-present.

Coronaviruses (pigs), 2013-present.

1. Coronaviruses (TGE, PEDV and PdCV) high mortality

2. Porcine rotaviruses ???

3. Porcine noroviruses ???

immediate virus suspects clinical presentation?

piglets with diarrhea?

porcine rotavirus

porcinenorovirus porcine

coronaviruses

Family: Coronaviridae

Genus: Alphacoronavirus (bats): TGE, PRCV, PEDV Genuses: Betacoronavirus, Gammacoronavirus

Genus: Deltacoronavirus (birds): PdCV

S (spike) protein

M (membrane) protein

E (envelope) protein

S (spike) helical

nucleocapsid

31,000 NThelical ssRNA

80-160 nm diametersurface epithelial

cell tropism

endoplasmic reticulum

(+) RNA strand

ribosomes

(-) RNA strand

viral proteins transcribed, by subgenomic (-) RNAs

viral polymerase

N proteins and viral RNAsself-assemble to nucleocapsids

virions “bud” off the Golgi & released

Coronaviruses:

1. Attachment and membrane fusion

2. + RNA codes for the viral polymerase.

4. - RNA strand codes for viral + RNA;

5. Subgenomic RNAs code for other viral proteins.

6. N protein binds to + RNA to make the viral nucleocapsid.

7. N protein binds to M protein in ER and then “buds” into ER to get the lipid envelope with S protein and HA.

8. Virions transported to cell surface by Golgi and are released.

Part 1: Porcine epidemic diarrhea virus (PEDV) infection in GN piglets.

dehydration (PID 3) lethargy (PID 3)

diarrhea (PID 3)

19-day oldpiglets werechallenged orally with

104 TCID50 PEDV.

Clinical findings in PEDV-infected piglets:

Days after infection: -1 and 0 PID 1 PID 2 PID 3

154 158 154 158 154 158 154 158

- - 3 3 3 3 3 3

- - + - + - - -

- - +/- +/- +/- + + +

- - - - + + + +

- - - - - - - - 7.0 9.0 7.0 9.0 7.0 8.5 <7.0 7.5

- - - - - < * < <

Clinical Signs in PEDV- infected GN piglets:

Diarrhea

Vomiting

Dehydration

Lethargy

Anorexia

Body wt (lbs)

Fever

* Body temperatures well below normal.

PEDV infection, PID 3

PEDV infection, PID 4

Bulk collection of intestinal contents after collection

of small segments for viral qPCR,

virus re-isolation and histopathology.

Cross sections of duodenal mucosal thickness after challenge with PEDV:

The signature lesion of PEDV (like TGE) is: “villous atrophy”

PEDV-naive PEDV-infected, PID 3

PEDV, duodenum, PID 3 PEDV, jejunum, PID 3

PEDV, ileum, PID 3 PEDV, colon, PID 3

Pathogenesis of “villus atrophy”

1.Epithelial cell necrosis and sloughing; the remainingepithelia “flatten” out to cover the defects (A).

2. Smooth muscle fibers in the “exposed” denuded villicontract (smooth muscle) resulting in shortened villi (A).

3. Coronavirus-mediated fusion (due to the major viral envelope glycoprotein) occurs between cells (B) and alsobetween adjacent villi (C).

4. The net effect is 99% reduction of absorption surfaceand resultant mal-absorptive diarrhea (D).

B

C

D

A

smooth muscle fibers

2014-158, PEDV- infected, PID 3.

2014-162, PdCV- infected, PID 3.

Anti-PEDV IgG binds to PEDV viral antigens in the cytoplasm of mucosal enterocytes infected with PEDV (left panel); anti-PEDV IgG does not react with PdCV antigens in

mucosal epithelial enterocytes of a PdCV-infected GN piglet (right panel).

1. Primary antibody: Post-immunization terminal serum from a GN

piglet vaccinated twice IM with inactivated PEDV in alum adjuvant.

2. Secondary antibody: Biotinylated goat anti-porcine IgG; strepta-

vidin-HRP; substrate, H202.

immunohistochemistry

Part 2: Porcine deltacoronavirus (PdCV)

infection in gnotobiotic swine

24 hr 48 hr 72 hr

PdCV origin: Todd Price, DVM,

origin fecal sample 1, received

at OSU on February 21, 2014.

PdCV virions recovered from the intestinal tract of a PdCV-infected gnotobiotic piglet, 48 hr PI.

84 nmdiameter

150 nm diameter

PdCV structure by TEM:

infectious virion

Noninfectious virion (?)

vomiting

profuse watery diarrhea

fatal dehydration

Clinical features of PdCV disease in GN swine:

1. Afebrile; no odor.

2. The diarrhea appears to me to be“painless”.

3. As long as there is some hydration,piglets continue to consume all of themilk diet they can hold.

4. Within 24 hrs, they are “sticky”.

PdCV cell tropism and viral cytopathic effects (CPE) in vivo

Goblet cells appear to berelatively “resistant” to infection

by both PEDV & PdCV.

Gastric pit epithelial cells are also infected by PdCV.

Aside from prominent villus atrophy, there is extensive intestinal villus epithelial

cell syncytia and cell necrosis featuring pyknosis, karyorhexis and karyolysis.

IN PIGS, THE ESOPHAGEAL LINING DESCENDS INTO THE CORPUS OF THE STOMACH AS THE NONGLANDULAR

GASTRIC CARDIA OR PARS ESOPHAGEA. porcine gastric anatomy

gastric cardia

gastricfundus

gastricantrum

nonglandular cardia

esophagus duodenum

pyloric bulb

lesser curvature of the stomach

greater curvature of the stomach

fundic diverticulum

gastric cardia

gastric fundus

gastric antrum

24hr PI 48hr PI 72hr PI

PdCV-associated gastric lesions:

gastric epithelial celldegeneration and necrosis

PdCV-associatedcell fusion

(syncytial giant cells)

48hr HX colon

duodenum

jejunum

ileum

colon

24hr PI 48hr PI 72hr PI

24 hrs 48 hrs

cell swelling, cytoplasmic vacuolation, syncytia

loss of brush border and cell swelling

return of brush border(microvilli) and returnto normal cell size

72 hrs

PdCV: Duodenum (Todd strain 1)

Curiously, although duodenal contents contain high amounts of viral RNAs, PdCV viral cytopathic effects are mild, transient and within normal limits by 72 hrs. In contrast, PEDV lesions are still severe in the duodenum at 72 hrs.

PdCV: Jejunum(Todd strain 1), 24 hrs (left) and 72 hrs (right)

PdCV: Ileum(Todd strain 1), 24 hrs (left) and 72 hrs (right)

Is there any evidence that OSU PdCV (Todd) strain 1 replicates in the respiratory tract? After all, tracheal and bronchial epithelium are

derived from embryonic endoderm, just like the gastrointestinal tract.

GN piglet: uninfected control formalin-inflated

lung, 23 days of age

PdCV, 24 hr PIbronchi and lungsPdCV, Ohio CVM1

Are the PdCV-positive materials in some bronchi by IHC evidence of infection of bronchial epithelium or is this inhaled PdCV vomitus?

PdCV, 24 hr PIbronchi and lungsPdCV, Ohio CVM1

PdCV, Ohio CVM1

48 hr PI

PdCV, Ohio CVM1

48 hr PI

C

PEDV-infected, stained withanti-PEDV antibody

PdCV-infected, stained with anti-PEDV antibody

Uninfected control, stained with anti-PEDV antibody

PEDV-infected, stained withanti-PdCV antibody

PdCV-infected, stained with anti-PdCV antibody

Uninfected control, stained with anti-PdCV antibody

Immunohistochemistry stains on CV-infected gnotobiotic piglet duodenum:Lack of serologic cross-reactivity between PEDV and PdCV.

Anti-PEDV reacts only with PEDV; anti-PdCV reacts only with PdCV; neither react with uninfected control tissue.

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other tissues

duo jej ileum col total total feces blood spl liver kid lung sm int colon

For PdCV, mild proteases (trypsin) are needed to expose the active binding siteon the virion S protein for binding to host enterocytes. However, prolonged

protease exposure inactivates PdCV virions.

Given this “bizarre” factoid, where would one find infectious virus and where would one find the most viral RNAs? Why???

anatomic oddities of the pig immune system:

1. Pigs differ from other species in anatomical organization of the hemato-poietic system and lymphocyte re-circulation patterns.

2. Epithelial-chorial placentation means that there is no maternal transfer of Ig to fetuses in utero. All passive immune protection is acquired from thecolostrum, first 24 hrs of life.

Spare the children,give the vaccine!(an old polio vaccine slogan)

What aboutvaccinations?

Pigs lack both a common thoracic duct and afferent lymphatics. As a result, lymphocytes leave the blood through high endothelial venuoles (HEVs), filters through lymph nodes and exits back into the venous side of the tract. Thus, “central” immune responses are anatomically separated from local surface- and mucosal-draining lymph nodes.

pig

Thus local mucosal immune responsesare largely self-contained to the surface

and draining lymph nodes.

What are the implications of this???

highendothelialvenuoles

Antigen-sampling and processing structures, distinct from mucosallymphoid follicles, line the gi tract of pigs. Unlike mucosal follicles

(left), these are encapsulated and possess efferent lymphatics.In the lower bowel are called lymphofollicular complexes.

Gastric mucosal follicles (inflammation): no

lymphatics, not encapsulatedGastric lymphofollicular

complexes

Gastric lymphofollicular complexesare autonomous immunologic

organs with T & B cells, APCs andefferent lymphatics. They are capable of making an immune

response, independent of systemicstructures (nodes, spleen, etc).

Colostrum and immune protection in neonatal pigs

• Piglets are immunocompetant during the last trimester in utero; piglets with precolostral antibody titers have been infected in utero.

• Piglets are “physiologically immature” at birth and are born without Igs (agammaglobulinemic) and without ANY in utero-origin maternal IgG or IgA immunoglobulins.

• “First” colostrum is rich in IgG, IgM and the spectrum of antibody specificities reflects that of the sow. These Igs are absorbed into the piglet, first 24-48 hrs of life.

• Colostrum contains leukocytes; maternal-origin secreted lymphocytes, macrophages and even PMNs are also absorbed and these localize primarily in piglet intestinal lamina propria.

• After intestinal closure, lacteal secretions/milk contains primarily IgA, whose primary purpose is to neutralize environmental antigens and down-regulate aggressive immune responses to these antigens.

1. We KNOW that they are secreted from the sow mammary gland and are T-lymphocytes (the cytotoxic or CD8 phenotype), activated macrophages and PMNs.

2. We also know that they are NOT homologous to the piglets’ MHC type(s) and that they lack IL-2 receptors (eg. they DO NOT proliferate to IL-2 growth signals from the piglet).

3. Absorbed maternal leukocytes MAY provide the piglet with short-lived or “temporary” cytotoxic T cell immune protection in gut. Maternal cells MAY be the source of interferon alpha, a major inducer of innate (immediate) immune responses.

A little known “factoid”: Porcine colostrum contains many leukocytes; maternal-origin leukocytes are absorbed intact into the gut of neonates and are oriented

towards the luminal surface!

Immunity on mucosal surfaces is “separate” from immunity in central lymphoid organs (lymph nodes, spleen, etc.)

Given the unavoidable anatomical facts and knowing that the porcine CVsproductively infect gastric and small intestinal enterocytes, induces transient(at least three days) viremia and the CVs do not cross-react with each other

(or TGE for that matter) how should we approach the concept of vaccinations for PEDV and SdCV?

What are the options?

1. Natural infection, recovery of sows and “adequate” colostral protection for subsequent litters?

This is not working; farms experience repeated outbreaks.

2. Parenteral (IM) vaccination(s) with killed products? This won’t work with high environmental viral challenge.

3. Oral vaccinations with killed products?

The real “role” of IgA is to suppress the immune response to enteric antigens. Experience

with TGE says this won’t work.

4. A combination of oral and IM booster vaccinations?

This has a good chance of inducing effective (but short-lived) mucosal immunity.

5. Modified-live vaccine?

This should work as persistence of antigen in the pigs and environment will provide positive

booster effects.

PdCV: Conclusions (so far)

1. PdCV is a novel enteric pathogenic epitheliotropic coronavirus infection of swine.

2. PdCV infects mature ciliated absorptive cells, immature columnar and cuboidal cells but not goblet cells in the gastric and intestinal pits.

3. Epithelial lesions progress from hydropic degeneration, coagulation necro-sis and cell lysis with syncytium formation of infected epithelial cells.

4. As a result, villi contract and fuse together producing a characteristic lesion of the enteric coronaviruses known as “villous atrophy”.

5. Epithelial enterocyte loss is evident by 24 hrs after infection in the three major divisions of the stomach (cardia, fundus and antrum), the small intestine(duodenum, jejunum and ileum). The cecum and colon (spiral, transverse, descending) are not productively infected but contain high levels of CV RNAs.

6. Epithelial enterocyte regeneration and resultant villus elongation is evident in the duodenum by or before 72 hrs PI.

7. PdCV may replicate undergo self-limited replication (24 hrs) in ciliated cellsof the respiratory tract.

Feature PED coronavirus SdCV (delta) coronavirus

origin(s) Alphacoronavirus (bats) Deltacoronavirus (birds)

diarrhea, dehydration yes (China) yes (Hong Kong)

mortality >90% 40-50%

tropism, viral CPE enterocytes, lysis, GCs enterocytes, lysis, GCs

villous atrophy yes, duodenum, jejunum, ileum yes, jejunum, ileum

infectious virus ileum, colon duodenum

viral RNAs all segments all segments

viremia ??? yes

gastric lesions +/- yes

respiratory tract no possible

Comparing the new porcine coronaviruses:

Conclusions, even though they may be pre-mature!

PdCV infection is a “real” disease and will complicate control of PEDV as there is no serologic cross-reactivity between these two coronaviruses.

There are other viruses that cause viral diarrhea (rotaviruses, noroviruses); at the moment, these are not significant causes of mortalities in infected herds.

A respiratory component may be involved with PdCV; we are only “a mutationor two away” from a MERS-like disease; may have human health implications.

Vaccination(s) are our best option for control of the coronavirus infections. If akilled product is used, a combined parenteral and oral formulation will likelyperform best in the field.

Ultimately, modified live products will be the best preventative approach.

The avian origin of PdCV may implicate birds in transmission/spread of PdCV.

“New” disease-causing viral agents will continue appear in the industry.

The dedication, expertise and life-long

friendships provided by the personnel

of The Gnotobiotic Life Laboratory

(Judy Dubena) and the Infectious

Disease Laboratory (Susan S. Ringler)

are gratefully acknowledged.

As well the graduate students and staff

of Dr. Jianrong Li’s Molecular Virology Laboatories (Yaomai Ma, Fanfei Lou,

Sheya Li, Erin DiCaprio and others are

recognized for their efforts and drive in

developing the porcine coronavrus

program here at The Ohio State

University.

Financial support for portions of this

work was provided by the NPC, Des Moines, IA.