Article Type : Research Article
Authors : Rajcani J, Banati F, Szenthe K, Gyurjan I, Stipkovits L and Szathmary S
Keywords : Porcine respiratory and reproductive system virus (PRRSV); Usual interstitial pneumonia (UIP); PRRSV antigen detection
Toget.Together 28 piglets (aged over 2 months) were infected with 105 TCID50 of porcine respiratory and reproductive syndrome virus (PRRSV) into both nasal nostrils using an inoculum of 2x150 microliters (together 300 µl). Another 9 piglets served as uninfected controls. On days 11 and 18 post-infection (p.i.), either 12 and/or 16 infected animals were sacrificed to take the tissue samples from tonsillar area, each lung lobe as well as the spleen and liver. At both intervals, blood was removed to isolate white blood cells and to obtain serum for specific antibody detecting ELISA assay. At histological examination, the typical picture of usual interstitial pneumonia (UIP) was seen in the lungs of 23 out of 28 infected animals (82 %). In each such case, in the lungs the thickened inter-alveolar septa revealed widespread mononuclear (mainly lymphocyte) infiltration occasionally reaching an extensive intensity. By immunohistochemically staining for N-protein this was found in the ciliary epithelium cells lining the bronchial tree by nearly all piglets who developed UIP (in 21 out of 23 animals, i.e. in 91 %). In contrast, the squamous epithelium at the pharyngeal and/or tonsillar areas of same piglets was less frequently positive (in 13 out of 23, 57 %).
The porcine respiratory and reproduction syndrome
virus (PRRSV) has been classified into family Arteriviridae (order
Nidovirales), along with the equine arteriitis virus and/or the lactate
dehydrogenase elevating virus of mice [1-3]. The virions form small, enveloped
particles (50-65 nm in diameter) harboring a relatively long (approximately 15
kb in size) single strand RNA genome [4]. The viral RNA (vRNA) is a
positive-sense molecule with terminal cap at 5´- end and a poly-A repeat at
3´-end [5-7]. In the course of virus replication, the vRNA is copied as whole,
being synthesized via a full length negative-strand intermediate. In the course
of vRNA replication, a complete (genomic) minus strand is generated, which
serves as template for the synthesis of the new vRNA. Also for viral mRNA
synthesis, first a negative sense RNA sequence is formed, from which the set of
positive sense nested sub genomic (sg) RNAs is being transcribed. Thus, minus
sense subgenomic (sg) RNAs are used as the template for the synthesis of the
functional positive sense sg mRNAs 23/26. The vRNA has of 2 long open reading
frames (ORF1a and ORF1b), which comprise about 75% of the total genome
sequence. This portion of the genome specifies 14 non-structural proteins (nsp)
formed by cleavage of the 2 corresponding translated polyproteins. Of special
importance are the non-structural proteins 9 (nsp9) and 12 (nsp12),
representing the vRNA replicase, termed also RNA-dependent RNA polymerase
(RdRp). The rest of the genome encodes 7 structural proteins, out of which 5
are glycoproteins (designated GP2a/Gp2, GP2b/E, GP3, GP4 and GP5) in addition
to the M (membrane) protein and the N nucleoprotein4. Both strands are
complementary to each other and at their conterminal 3´-end and are equipped
with a common leader sequence at their 5´- ends27/28. The viral genome is
equipped with several (but at least two) conserved transcription regulatory
sequences (TRS), located in the front of ORF1a and ORF2a which encode the
structural protein GP2a and the envelope glycopreotein (Gp2b/E). Two different
strains were isolated in the US (VR2332) and in Europe (Lelystadt) relevealing
serologic as well as genomic differences [8-13]. Experimental infection with
the PRRSV isolates can be lethal in newborn and/or 3-week-old piglets. A key
event is the involvement of porcine alveolar macrophages, which represent an
important target for infection allowing virus spread [14,15]. To date, at least
two macrophage surface molecules are known as entry mediators: the siglec sialoadhesin
and CD163, a scavenger receptor10. The PRRSV-induced pneumonia is characterized
by thickening of inter-alveolar septa due to infiltration with macrophages and
by the presence of occasional inflammation and cell debris within alveolar
cavity. The pneumocytes of type II which are lining the alveolar cavity may
also be PRRSV positive along with the hyperplasia of per bronchial lymphatic
tissue24/25. 3 The severity of lung lesions may vary from relatively mild to
more extensive. The viral genotypes can differ in their pathogenicity, namely
the Type 2 North American PRRSV induces more severe respiratory disease than
type 1 European virus [16]. In this paper we describe the correlation of lung
lesions as seen by standard histological examination in comparison with
immunohistochemically detection of viral N-protein along with the results of
serological tests for N-protein antibodies.
Virus. The North-American strain of PRRSV was cultured
using the MARC-145 cell line (African green monkey kidney cell line derivative
of MA-104, ATCC-CRL-12231) in EMEM medium at 37°C in the presence of 5% CO2;
the virus end point (TCID50 titer) was tested in 96-well plates as described.
(2014)31/32 and Ramakrishnan (2016)21/22, respectively. Animals. Pigs (together
28 infected animals) were inoculated into both nostrils with 105 TCID50 of
above mentioned PRRSV strain being administered in a volume of 300?l tissue
culture supernatant. The nine (9) negative control animals were inoculated in a
similar way but with a virus free culture medium; they were kept under
carefully checked conditions of strong isolation to avoid any possible contact
with the virus-inoculated piglets. Specimen sampling and histological
examination. Blood samples were taken on days 0,2,4,6,8,10,15,17 post-infection
for serological examinations. At given intervals (on days 11 and 18)
post-infection (p.i.), the animals were succumbed and selected tissue samples
(coming from each lung lobe, from both tonsils including adjacent pharyngeal
area, from spleen and liver) were immediately immersed into cold 10% neutral
formalin, fixed for 24 hr and embedded into paraffin. Sections were cut in an
amount to stick to at least 3 parallel slides. Those destined for the standard
histological examination were stained by hematoxylin and erythrosine (HE),
while the rest was handled by immunohistochemically staining. Briefly, the
slides were rinsed in phosphate buffer saline (PBS) and then dehydrated in a
series of corresponding reagents as described. The mix of commercial
monospecific mouse ascites derived antibody against the N-protein of PRRSV had
been purchased from 4rtilab and contained both, the SDOW-17 as well as the
SR30-A reagents. Both anti-N antibody solutions were freshly mixed before use
in an equal (1:1) ratio. In the second layer, an alkaline phosphatase labelled
anti-mouse IgG antibody was applied. An additional parallel slide which had
been included, was treated with the second antibody only (it served as staining
control for detection of any putative non-specific background staining). 4
Saliva (oral fluid) collection. The oral fluid was pooled from each animal
separately using the Civets sues oral fluid rope (IDEXX) ELISA titer
measurements. The obtained saliva samples were examined for the presence of
class IgA antibody specific against PRRSV using the Oral Fluids (IDEXX PRRS OF)
kit as recommended by the manufacturer. In case of blood samples the positive
control monospecific serum to class IgG antibody against the PRRSV was tested
using the INgezim PRRS 2.0 ELISA kit (purchased from Eurofins) strictly
following the recommendations of the manufacturer [17].
As documented in the interalveolar septi in normal lung tissue are very thin in order to ensure the diffusion of oxygen into blood capillaries, where the erythrocytes circulate. Occasionally, a few mononuclear cells (mainly lymphocytes) might be seen in the peribronchial connective tissue. Surprisingly, in 4 out of 9 non-infected (control) piglets, a slight focal thickening of interalveolar septi was noted along with accumulation of a mild interstitial infiltrate consisting of mononuclear cells, mainly lymphocytes (Figure 1).
Figure 1: Histological picture of lung tissue in uninfected (control) piglets.
· In the left (piglet no.
5). The normal lung structure at low power view shows the thin interalveolar
septa devoid of infiltrate; the peribronchial (and/or perivascular) connective
tissue reveals a minimal infiltrate of very few mononuclear lymphocytes (magn
x100).
· In the right (piglet no.
2). Unlike to Fig. 1A, this area lung tissue area shows thickened interalveolar
septa due mild accumulation of mononucellular lymphocytes. Similar mild (M)
non-specific (NS) interstitial infiltrate (II) was found in the lungs of 5 out
of 9 uninfected controls.
As expected, staining for PRRSV in any control lung tissue sample was negative including areas revealing the above mentioned mild interstitial infiltrate (Figure 2).
Figure 2: Histological findings in the lungs of PRRSV infected piglets.
· In the left above (piglet
no. 16). An area of the lung tissue in the infected animal showing mild focal
thickening of the interalveolar septa and dilatation of small vessels (magn
x100).
· In the right above
(piglet no. 16). In contrast to the area shown in the left, this one reveals
typical UIP with severe thickening of interalveolar septa and an abundant
mononuclear interstitial infiltrate along with dilatation and hyperemia of the
small blood vessels.
· In the left below (piglet
no. 25). The lung tissue of an animal who developed typical UIP showing a
widespread mononuclear infiltration of the interalveolar septa and
peribronchial connective tissue (magn. x100).
In the right below, the same piglet (no. 25). The mononuclear infiltrate in the peribronchial area consists mainly of lymphocytes; the thickening of interalveolar septa is prominent (magn. x 240).
As expected, in the majority of infected animals (23 out of 28, 82%) the lung tissue revealed the typical picture of usual interstitial pneumonia (UIP). In these, the interalveolar septi were thickened along with the presence of a rich mononuclear cell (lymphocyte) infiltrate. In addition, in UIP the capillaries of the alveolar wall were widened along with occasional focal bleeding into the alveoli in result to an injury of endothelial cells. High power view of altered areas confirmed that the interstitial infiltrate consisted mainly of lymphocytes. Occasionally (in example by piglet no. 40) the infiltrate in question was so extensive that the original lung structure became altered or completely different so that it could not be recognized either (Figure 3).
Figure 3: Severe interstitial
pneumonia due to PRRSV infection.
Staining with anti-N antibody showed the presence of PRRSV antigen predominantly in the columnar ciliary epithelium lining the bronchial tree. Details demonstrated the presence of viral antigen in the cytoplasm of acinus cells building the small mucous glands, situated below the lamina muscular is of the bronchial wall. The alveolar lining was rarely positive for the N-protein. Occasionally, the type II alveoli lining cells could be found harboring the N-protein (Figure 4).
Figure 4: Staining for N-antigen in the respiratory tract and spleen of PRRV infected piglets.
· In the left above (piglet
no. 34). The lung tissue shows overwhelming staining for N-protein, namely the
bronchial epithelium, the parabronchial mucous glands and the flat lining of
the aveolar wall (magn. 80x).
· In the right above
(piglet no. 12). An intrapulmonar bronchus enlarged: the N-protein can be seen
in the cytoplasm of ciliary epithelium cells lining the bronchial tree; a few
negative goblet cells can be seen as well (magn. 120x).
· In the left below (piglet
no. 45). The N-protein is present in the cytoplasm of cells lining the alveolar
wall and in several mononuclear phagocytes invading the interalveolar septi
(magn. 400x).
· In the right below (piglet no. 44): the white pulp of the spleen shows lymphatic follicles formed mainly by lymphocytes which are positive for N-protein (magn. x120) (Figure 5).
Figure 5: N-protein in pharyngeal epithelium and salivary gland of PRRSV infected piglets
· In the left (piglet no.
26). The N-protein in tonsillar area can be found mainly within the cytoplasm
of proliferating squamous epithelium cells at both suprabasal and/or
intermedial layers; a few basal epithelium cells are positive as well (magn.
220x)
· In the right (pig no. 13). The N-protein can be seen in the acini of submandibular salivary gland (magn. 220x).
The N-protein could be also found in the cytoplasm of
alveolar macrophages moving from intraalveolar space across the interalveolar
septa to the local 5 lymphatic capillaries in order to reach the sinuses of
regional lymph nodes. Nevertheless, in some piglets the local peribronchal
lymph nodes did not hamper the virus spread, so that via the infected virus
carrier cells viremia has developed (see below) involving remote organs such as
spleen and/or liver. In the spleen, the reticular cells of regional sinuses
could be found positive along with the lymphocytes forming the lymphatic
follicles. In the liver, the N-protein could be detected in the cuboid
epithelium cells forming the intrahepatal biliary ducts. Outside of lung
tissue, the N-protein of PRRSV was found especially in the nonhornified
squamous epithelium of the pharyngeal area including that over tonsils. Here
the virus antigen occupied the deeper layers of stratified epithelium, namely the
multiplying parabasal cells as well as those in the medium layer. The virus was
also found in salivary glands, namely in the submandibular gland, where the
N-antigen was harbored in the cytoplasm of acinus cells. Noteworthy, the
presence of viral N-antigen in the acinar cells of salivary glands was
relatively rare in comparison to the tonsillar and/or pharyngeal squamous
epithelium, which still were relatively more frequently positive. However, the
real incidence of N-protein in the former was difficult to assess, since the
sections of salivary glands could not be regularly found. By testing the
occurence of the virus in the blood, viremia was found to peak on day 6. While
by day 10 p.i. already no virus could be detected. As mentioned in the section
Materials and Methods, development of the specific class IgG anti-N antibody
response was followed by ELISA. As documented on (Figure 6B), unlike to virus
presence, no N-specific serum antibodies occurred before day 11 p.i., but on
day 18 p.i. they were detected in high levels. The low amount of serum
antibodies on day 11 might be related due to their binding to virus particles.
Contemporarily, first traces of the anti-N protein class IgA antibodies in the
saliva also appeared from day 11. As the levels of blood lymphocytes concerns,
these started to decrease from day 4 post-infection, returning back to the
original level on day 10. The kinetics of the polymorphonuclear leukocytes
(i.e. blood neutrophils) in the blood revealed a similar outcome (data not shown)
[18-20].
As a member of the family Arteriviridae, the porcine respiratory and reproductive syndrome virus (PRRSV) belongs to the order Nidovirales together with the Coronaviridae and Roniviridae families, 26/27. PRRSV was originally divided into European type 1 and North American type 2 genotypes. Later on, the East European PRRSV isolates have been found to be of the European genotype, but forming different subtypes [6]. A novel virus, namely the 6 Belarusian strain Lena, has been recently characterized as a highly pathogenic East European subtype 3, which differs from European subtype 1 Lelystad and North American US5 strains at genetic as well as antigenic levels [11]. A novel PRRSV strain isolated on a French pig farm has been recently identified to represent a modified recombinant consisting from two genotype l live vaccine strains, namely the VP-045BIS and DV ones22/23. For example, the kinetics of antibody responses directed against nonstructural virus coded proteins (nsp) can be analysed in pigs experimentally exposed to the virus23/24. In such case, high antibody reactivities especially against nsp1, nsp2, and nsp7 were noted. Among the latter, nsp7 recombinant protein based ELISA showed good sensitivity and specificity most suitable for diagnostic development especially for identification and differentiation of type 1 and type 2 PRRSV. Several nonstructural proteins (such as nsp1, nsp2, nsp5, nsp7 nsp9, nsp10 and nsp11) have been implicated in the induction of IFN-? and also in the development of the cell-mediated immune response [17]. On other hand, the induction of neutralizing antibodies (NAs) may be delayed and/or their levels may remain low, which is not only the problem of early diagnostic, but is also of importance regarding effective virus elimination. NAs may protect against disease if present in sufficient quantities before infection, but they do not seem to be essential for clearing virus in blood during the course of the infection. PRRSV is able to modulate innate responses, probably through the regulation of IFN-? and IL-10 responses [14].
Figure 6: Detection of vRNA in the serum of infected pigs in comparison with the specific antibody response. Viremia has been detected on days 6 and 8 post-infection, while the anti-N protein class IgG antibody began to rise from day 11 reaching high levels on day 15 and/or 18 post-infection. The local class IgA antibody formatioin in the saliva showed a kinetics similar to that found in serum. The blood lymphocyte number started to decrease from day 4 and remained low until day 10, later on returning to the early post-infection level on day 14. The blood neutrophils revealed a similar kinetics (data not shown).
Table 1: Histological lesions and the presence of N-antigen in infected piglets.
Animal |
PRRSV |
Results |
Uninfected |
None |
No N-antigen was seen neither in bronchial
epithelium (0/9) nor in tonsils (0/9). |
Infected |
Yes |
Histological picture of UIP*
has developed in the lungs of 23 out of 28 infected piglets; the N-antigen was seen in the bronchial
epithelium of 21 infected animals, while their alveolar lining was positive
in 5 cases only. Outside of lungs, the N-protein was detected in the tonsilar
epithelium of 13 out of 28 infected piglets; in contrast, the spleen was positive in 3 piglets and the salivary gland in 1 animal only. |