Fig. 1
A schematic representation of the experimental timeline. (a) Scheme of virus passages; (b) Scheme of the introduction of contact piglets to experimentally inoculated animals. In each passage/contact group, two piglets were used. p – passage; HI – haemagglutination inhibition; rRT-PCR – real time reverse transcriptase PCR; inocul. – inoculation; i.n. – intranasally; r.t. – rectal temperature; n.s. – nasal swabs; N – passage number; EI – experimentally inoculated; CE – contact-exposed
Fig. 2
Rectal temperature of experimentally inoculated (EI) and contact-exposed (CE) piglets in individual passages. (a) EI gilts; (b) EI barrows; (c) CE gilts; (d) CE barrows; dpi – days post inoculation; dpc – days post contact; I – first group; II – second group
Fig. 3
Pathological changes in the lungs of animals from selected passages. (a) experimentally inoculated (EI) gilt at passage (p) 10, lung lesion score (LLS) = 2; (b) EI gilt at p12, LLS = 3; (c) EI gilt at p24, LLS = 8; (d) EI barrow at p24, LLS = 9; (e) contact-exposed (CE) group I gilt at p24, LLS = 11; (f) CE group I barrow at p24, LLS = 6
Fig. 4
rRT-PCR results. (a) results for all tested tissue samples from EI piglets in each passage; (b) results for all tested tissue samples from CE piglets. p – passage; CE – contact-exposed; b – barrow; g – gilt; I – first group; II – second group
Fig. 5
rRT-PCR results for nasal swabs from the EI and CE piglets. (a) results from animals at passage 20; (b) results from animals at passage 24. p – passage; EI – experimentally inoculated; CE – contact-exposed; b – barrow; g – gilt; I – first group; II – second group; dpi – day post inoculation; dpc – day post contact
Fig. 6
rRT-PCR results for each tissue type from EI and CE piglets. NM – nasal mucosa; T – trachea; RCrL – right cranial lobe; RML – right middle lobe; RCaL – right caudal lobe; LCrL – left cranial lobe; LML – left middle lobe; LCaL – left caudal lobe; AL – accessory lobe; strong positive Ct < 30; weak positive Ct 30–35; negative Ct > 35
Table 1
Amino acid changes in the proteins of virus strains obtained from selected passages
| Gene | a.a. position | Inoculum | vEIp9 | vEIp16 | vEIp20 | vCEp20 | vEIp24 | vCEp24 |
|---|---|---|---|---|---|---|---|---|
| 144 | R | Q | Q/* | * | § | * | § | |
| PB2 | 477 | R | G | G | G | § | G | § |
| 554 | I | L | L | L | § | L | § | |
| 701 | D | N | N | N | § | N | § | |
| PB1 | 654 | S | * | * | G | § | G | § |
| 370 | L | * | * | */I | § | * | § | |
| PA | 465 | I | */T | * | * | § | * | § |
| 613 | E | * | */K | */K | § | */K | K | |
| 159 | S | * | */R | * | § | * | § | |
| 180 | K | E | E | E | § | E/G | § | |
| 189 | E | * | * | * | § | */D | § | |
| 203 | D/N | N | N | N | § | N | § | |
| HA | 204 | E | D | D | D | § | D | § |
| 239 | G | * | */E | E | § | D | § | |
| 286 | D | * | */E | * | § | * | § | |
| 411 | G | S | S | S | § | S | § | |
| NP | 63 | I | M | M | M | § | M | § |
| 132 | R | K | K | K | § | K | § | |
| 30 | I | * | * | L | § | L | § | |
| 34 | V | * | * | */I | I | */I | § | |
| 95 | G | S | S | S | § | S | § | |
| NA | 127 | L | M | M | M | § | M | § |
| 131 | A | T | T | T | § | T | § | |
| 135 | T | * | * | * | § | * | S | |
| 364 | N | S | S | S | § | S | § | |
| M1 | 248 | M | I | I | I | § | I | § |
| M2 | 19 | C | Y | Y | Y | § | Y | § |
| NS | 56 | M | */I | I | I | § | I | § |
| 226 | V | I | I | I | § | I | § |
[i] a.a. – amino acid; vEI – virus isolate from experimental inoculated piglets; vEI – virus isolate from experimentally inoculated piglets; vCE – virus isolate from contact-exposed piglets; p – passage; PB2 – basic protein 2; PB – basic protein 1; PA – acid protein; HA – haemagglutinin; NP – nucleoprotein; NA – neuraminidase; M – matrix protein; NS – non-structural protein; A – alanine; R– arginine; N – asparagine; D– aspartate; C – cysteine; E – glutamate; Q – glutamine; G – glycine; I – isoleucine; L – leucine; K – lysine; M – methionine; S – serine; T – threonine; Y – tyrosine; V – valine; * – the same a.a. residue as inoculum; § – the same a.a. residue as vEI; italic text – a.a. residues in inoculum; underlining – subsequent substitution