Ivan Trus

Efficacy of an attenuated European subtype 1 porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs upon challenge with the East European subtype 3 PRRSV strain Lena

The efficacy of a commercial attenuated European subtype 1 PRRSV vaccine was evaluated upon challenge with the East European subtype 3 PRRSV strain Lena (83.3% nucleotide identity). Two vaccination experiments were carried out. Four- and seven-week-old pigs were vaccinated with the modified-live vaccine. Upon vaccination, virus specific IPMA antibodies were detected in all vaccinated animals with titers ranging from 10(2.8) to 10(4.6). No virus neutralizing (VN) antibodies were detected after vaccination. Eight (exp. 1) or six (exp. 2) weeks after vaccination, pigs were challenged with 10(6) (exp. 1) resp. 10(5) (exp. 2) TCID50 of the European subtype 3 PRRSV Lena. Upon challenge, non-vaccinated animals showed fever during 5.1 (exp. 1) or 7.7 (exp. 2) days. In vaccinated pigs, the duration of fever was reduced by 1.8 (exp. 1) or 3.5 (exp. 2) days. The modified-live virus vaccine reduced the mean duration of nasal shedding and viremia. In non-vaccinated pigs, virus shedding lasted 5.8 days (exp. 1), resp. 8.3 days (exp. 2). This period was reduced to 3.6 (exp. 1), resp. 3.0 (exp. 2) days in vaccinated animals. Viremia was observed during a shorter period in vaccinated (exp. 1: 7.4 days, exp. 2: 4.8 days) than in non-vaccinated groups (exp. 1: 11.8 days, exp. 2: 12.3 days). Starting from 5 days post challenge, virus titers in nasal secretions and sera were significantly lower in vaccinated animals (P<0.05). Virus-neutralizing antibodies were detected at low titers (≤ 16) after 7 days post challenge in vaccinated animals and 28 days post challenge in control animals. In conclusion, it can be stated that vaccination of pigs with an attenuated European subtype 1 vaccine provides a partial protection against a subsequent exposure to the highly pathogenic East European subtype 3 PRRSV strain Lena.

Heterologous prime-boost vaccination with H3N2 influenza viruses of swine favors cross-clade antibody responses and protection

The emergence of multiple novel lineages of H1 and H3 influenza A viruses in swine has confounded control by inactivated vaccines. Because of substantial genetic and geographic heterogeneity among circulating swine influenza viruses, one vaccine strain per subtype cannot be efficacious against all of the current lineages. We have performed vaccination-challenge studies in pigs to examine whether priming and booster vaccinations with antigenically distinct H3N2 swine influenza viruses could broaden antibody responses and protection. We prepared monovalent whole inactivated, adjuvanted vaccines based on a European and a North American H3N2 swine influenza virus, which showed 81.5% aa homology in the HA1 region of the hemagglutinin and 83.4% in the neuraminidase. Our data show that (i) Priming with European and boosting with North American H3N2 swine influenza virus induces antibodies and protection against both vaccine strains, unlike prime-boost vaccination with a single virus or a single administration of bivalent vaccine. (ii) The heterologous prime-boost vaccination enhances hemagglutination inhibiting, virus neutralizing and neuraminidase inhibiting antibody responses against H3N2 viruses that are antigenically distinct from both vaccine strains. Antibody titers to the most divergent viruses were higher than after two administrations of bivalent vaccine. (iii) However, it does not induce antibodies to the conserved hemagglutinin stalk or to other hemagglutinin subtypes. We conclude that heterologous prime-boost vaccination might broaden protection to H3N2 swine influenza viruses and reduce the total amount of vaccine needed. This strategy holds potential for vaccination against influenza viruses from both humans and swine and for a better control of (reverse) zoonotic transmission of influenza viruses.Influenza: More efficient, more protective vaccinationsBoosting an influenza vaccine with a different strain offers twice the protection for half the volume of vaccine. Pigs and humans need two doses of killed influenza vaccine—a “priming” dose, with a “booster” given approximately 4 weeks after—in order to provide immunity. Kristien Van Reeth, of Belgium’s Ghent University, and collaborators demonstrated that differing the strain of the primer and booster elicits antibodies that protect against both strains, negating the need for two doses of both strains. The swine vaccinated in Van Reeth’s study also had increased immunity against strains that the animals were not vaccinated against but bore similarities to the viruses used. This cross-protection corresponds with conclusions from similar research into other animals and could eliminate the need for unnecessary vaccinations whilst increasing vaccine efficacy in swine and presumably in humans.

Presence of DNA extracellular traps but not MUC5AC and MUC5B mucin in mucoid plugs/casts of infectious laryngotracheitis virus (ILTV) infected tracheas of chickens

Although it has been speculated that the tracheal obstructions and asphyxiation during acute infectious laryngotracheitis (ILT) are due to mucoid plugs/casts formed by mucus hypersecretion, there are no reports demonstrating this. Hence, in the present study, we first examined if the main respiratory mucins, MUC5AC and MUC5B, are expressed in the mucosae of larynx, trachea and bronchi of mock-inoculated and ILTV infected chickens. Second, the tracheas with plugs/casts were stained for mucins (MUC5AC and MUC5B) and nuclear material (traps). MUC5AC and MUC5B were produced by the mucosae of larynx, trachea and bronchi of mock-inoculated chickens. Interestingly, MUC5AC and MUC5B were exclusively present in the dorsal tracheal region of the cranial and middle part of trachea of mock-inoculated chickens. In ILTV infected chickens, the tracheal lumen diameter was almost 40% reduced and was associated with a strongly increased tracheal mucosal thickness. MUC5AC and MUC5B were scarcely observed in larynx, trachea and bronchi, and in tracheal plugs/casts of ILTV infected birds. Surprisingly, DNA fibrous structures were observed in connection with nuclei of 10.0±7.3% cells, present in tracheal plugs/casts. Upon inoculation of isolated blood heterophils with ILTV, DNA fibrous structures were observed in 2.0±0.1% nuclei of ILTV inoculated blood heterophils at 24hours post inoculation (hpi). In conclusion, the tracheal obstructions and suffocation of ILTV infected chickens are due to a strong thickening of the mucosa (inflammation) resulting in a reduced tracheal lumen diameter and the presence of mucoid plugs/casts containing stretched long DNA-fibrous structures (traps) but not MUC5AC and MUC5B mucins.

Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes

Porcine circovirus type 2 (PCV2) is associated with various diseases which are designated as PCV2-associated diseases (PCVADs). Their severity varies among breeds. In the diseased pigs, virus is present in monocytes, without replication or full degradation. PCV2 entry and viral outcome in primary porcine monocytes and the role of monocytes in PCV2 genetic susceptibility have not been studied. Here, virus uptake and trafficking were analyzed and compared among purebreds Piétrain, Landrace and Large White and hybrid Piétrain × Topigs20. Viral capsids were rapidly internalized into monocytes, followed by a slow disintegration to a residual level. PCV2 uptake was decreased by chlorpromazine, cytochalasin D and dynasore. The internalized capsids followed the endosomal trafficking pathway, ending up in lysosomes. PCV2 genome was nicked by lysosomal DNase II in vitro, but persisted in monocytes in vivo. Monocytes from purebred Piétrain and the hybrid showed a higher level of PCV2 uptake and disintegration, compared to those from Landrace and Large White. In conclusion, PCV2 entry occurs via clathrin-mediated endocytosis. After entry, viral capsids are partially disintegrated, while viral genomes largely escape from the pathway to avoid degradation. The degree of PCV2 uptake and disintegration differ among pig breeds.

Preferential use of Siglec-1 or Siglec-10 by type 1 and type 2 PRRSV strains to infect PK15 S1–CD163 and PK15 S10–CD163 cells

Cellular entry mediators define whether the cell is permissive to PRRSV infection. Porcine sialoadhesin (pSn, Siglec-1) and CD163 are main entry mediators facilitating infection of porcine macrophages by PRRSV. Recently, Siglec-10 was demonstrated to be an alternative receptor for PRRSV. To examine if virulence and pathogenicity of PRRSV strains could be correlated with the use of different Siglecs, a PK15 cell line recombinantly expressing Siglec-1 and CD163 (PK15S1–CD163) and a PK15 cell line recombinantly expressing Siglec-10 and CD163 (PK15S10–CD163) were used to compare the virus replication of 7 genotype 1 subtype 1 strains (G1s1), 2 genotype 1 subtype 3 (G1s3) strains and 5 genotype 2 (G2) strains. Some strains (08VA (G1s1), 13V117 (G1s1), 17V035 (G1s1), VR2332 (G2)) were poor virus producers (<104 TCID50/mL), while other strains (07V063 (G1s1), 13V091 (G1s1), Su1-Bel (G1s3), MN-184 (G2), Korea17 (G2) and SDSU-73 (G2)) easily grew up to ≥106 TCID50/mL. PK15S10–CD163 cells exhibited a higher efficiency in virus production per infected cell than the PK15S1–CD163 cells. The G1s1 strains LV and 07V063 infected more cells in the PK15S1–CD163, whereas the 94V360 and 08VA strains preferred PK15S10–CD163. The highly virulent G1s3 strains Lena and Su1-Bel showed a strong preference for PK15S1–CD163. The G2 strains MN-184, SDSU-73, Korea17 had a much higher infection rate in PK15S10–CD163, while the reference strain VR2332 and the NADC30 strain had a slight preference for PK15S1–CD163. Differences in receptor use may influence the outcome of a PRRSV infection in pigs and explain in part the virulence/pathogenicity of PRRSV strains.

Genetic and pathogenic characterization of a Russian subtype 2 PRRSV-1 isolate

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory problems. Data about the virulence and pathogenicity of subtype 2 PRRSV-1 strains are limited. The main purposes of this investigation were to characterize the full genome sequence of the subtype 2 PRRSV-1 WestSib13 strain and to compare the pathogenicity with that of the subtype 1 PRRSV-1 Lelystad strain. Comparison of the whole genome sequence of the WestSib13 strain with that of PRRSV-1 prototype strains revealed a 76.2% (subtype 1 Lelystad virus) and 79.0% (subtype 3 Lena virus) identity, respectively The virulence and pathogenicity of the European subtype 2 PRRSV strain WestSib13 and the European subtype 1 PRRSV strain Lelystad were compared in 3-week-old piglets upon inoculation of 105.4 TCID50 of virus. Non-infected animals (control group) as well as animals infected with the Lelystad strain were clinically healthy until 14days post challenge. In contrast, animals infected with the WestSib13 strain demonstrated dyspnea starting at 3days post-inoculation (dpi). All piglets in this group died between 5 and 8 dpi. During that period, fever was not observed in WestSib13-infected animals. Viremia was detected in animals from both infected groups starting from 2 dpi. Viral loads in serum and lungs upon euthanasia were significantly higher (3 log10) in the WestSib13-infected than in the LV-infected animals. Taken together, this study provides the full genome sequence and the unusual virological and clinical outcome (high level viremia without fever) of the novel WestSib13 strain.

MCMV exploits the spleen as a transfer hub for systemic dissemination upon oronasal inoculation

Murine cytomegalovirus (MCMV) infection in mice is a commonly used animal model for studying human cytomegalovirus (HCMV) infections. In our previous studies, a mouse model based on an oronasal MCMV infection was set up for mimicking a natural infection, and the spleen was hypothesized to regulate viremia and virus dissemination to distal organs such as submandibular glands. Here, the role of the spleen during an MCMV infection was investigated by the comparison of intact and splenectomized Balb/c mice. Both highly passaged MCMV Smith and low passaged MCMV HaNa1 were used. Various samples were collected at 7, 14, and 21 days post inoculation (dpi) for analyses by virus isolation/titration, co-cultivation and qPCR. The results showed that for both virus strains, 1) cell-associated virus in PBMC (determined by co-cultivation) was detected in intact mice but not in splenectomized mice; 2) the mean viral DNA load in PBMC of splenectomized mice was 4.4-(HaNa1)/2.7-(Smith) fold lower at the peak viremia (7dpi) in contrast to that of intact mice; and 3) infectious virus in the submandibular glands was detected later in splenectomized mice (14dpi) than in intact mice (7dpi). Moreover, the average virus titers in submandibular glands of splenectomized mice were 10-(HaNa1)/7.9-(Smith) fold lower at 14dpi and 1.7-(HaNa1)/2.1-(Smith) fold lower at 21dpi compared with that of intact mice. Upon inoculation with MCMV Smith, infectious virus was found in the kidneys and liver of intact mice, but not in splenectomized mice. Taken together, all these data clearly demonstrate that virus dissemination to distant organs is reduced in splenectomized mice, further confirming the importance of the spleen as a viremia booming site for a natural MCMV infection.