213 ASSESSMENT OF THE SUSCEPTIBILITY OF PORCINE PRE-IMPLANTATION EMBRYOS TO PSEUDORABIES VIRUS (PRV) AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) USING SUBZONAL MICROINJECTION

It is known that porcine pre-implantation embryos before the morula stage are refractory to infection with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) (Bolin et al. 1981 Am. J. Vet. Res. 42, 1711-1712; Prieto et al. 1996 Theriogenology 46, 687-693, respectively). The effects of PRV and PRRSV on embryonic cells of morulae and blastocysts are unknown. Therefore, the objectives of the present study were to (1) assess the effects of PRV and PRRSV exposure on further embryo development, and (2) determine whether PRV and PRRSV are able to replicate in embryonic cells. Zona pellucida (ZP)-intact morulae (6 days post-insemination, 6 dpi) and early blastocysts (7 dpi) were microinjected subzonally with approximately 3 nL of 109 TCID50/mL PRV (strain 89v87, second passage in swine testicle cells) or 108.6 TCID50/mL PRRSV (Lelystad virus strain, 13th passage in swine alveolar macrophages). Control embryos were microinjected under the same circumstances with phosphate-buffered saline (PBS). Hatched blastocysts (8 dpi) were exposed for 1 h at 39�C to 105 TCID50/mL of PRV or PRRSV of the same strains used for injecting earlier embryonic stages. Control hatched blastocysts were incubated with PBS. Each group of morulae and blastocysts consisted of approximately 20 embryos. Embryonic development was assessed every 12 h. At 48 h post injection, the percentage of infected embryos and the percentage of viral antigen positive cells per embryo were determined by immunofluorescence. Subzonal microinjection of ZP-intact morulae and blastocysts with PRV inhibited in vitro development in comparison to the controls. Moreover, under direct immunofluorescence, PRV antigen-positive cells were detected in association with the embryos. Exposure of hatched blastocysts to PRV inhibited further embryo development; the majority (16/20) of the embryos degenerated 24 h after incubation. Perivitelline microinjection of ZP-intact morulae and blastocysts with PRRSV and incubation of hatched blastocysts with PRRSV did not inhibit in vitro development in comparison to the controls. No PRRSV antigen positive cells were detected in association with the embryos. Based on these results, it can be deduced that embryonic cells of morulae and blastocysts are susceptible to PRV infection but refractory to PRRSV infection. Another argument substantiating insusceptibility of embryos to certain viral pathogens is the demonstration of the lack of virus receptors at a given embryonic cell stage. Therefore, the expression of sialoadhesin, the receptor that mediates the internalization of PRRSV in cells, was investigated in hatched blastocysts (n = 10). By indirect immunofluorescence using monoclonal antibody 41D3 directed against porcine sialoadhesin, no positive signals were detected. The result of this experiment strengthens the statement that embryonic stages up to the hatched blastocyst stage are refractory to PRRSV infection.

197SUSCEPTIBILITY OF PORCINE MORULAE AND BLASTOCYST STAGE EMBRYOS TO PSEUDORABIES VIRUS AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

Porcine preimplantation embryos are refractory to infection with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) during the 2–4 to 16-cell stage as described by Bolin et al. (1981 Am. J. Vet. Res. 42: 1711–1712) and Prieto et al. (1996 Theriogenology 46: 687–693), respectively. Research on the effects of PRV and PRRSV on embryonic cells of morulae, blastocysts and hatched blastocysts is limited. Therefore, the objectives of the present study were (i) to assess the effects of PRV and PRRSV exposure on further embryonic development, and (ii) to determine whether PRV and PRRSV are able to replicate in embryonic cells of porcine morulae and blastocysts. In vivo produced ZP-intact and ZP-free morulae (6 days post-insemination), early blastocysts (7 days post-insemination), and hatched blastocysts (8 days post-insemination) derived from 22 superovulated sows were exposed to 105 TCID50 PRV (strain 89v87, second passage in swine testicle cells) or to 105 TCID50 PRRSV (Lelystad virus strain, 13th passage in swine alveolar macrophages) for 1h at 39°C. Control embryos were incubated under the same circumstances without viruses. Each group of morulae and blastocysts consisted of approximately 20 embryos. Embryonic development was assessed every 12h and differences in rates of development were analyzed using Chi-square analysis or Fisher’s exact test. At 48h post-incubation, embryos were collected and examined for viral antigen by indirect immunofluorescence. Further embryo development of ZP-intact and ZP-free morulae and blastocysts was not affected by exposure to PRV or PRRSV compared to controls (P<0.05). Moreover, using indirect immunofluorescence, no PRV or PRRSV antigen-positive cells were detected. Exposure of hatched blastocysts to PRV inhibited further embryo development as 100% (n=5) of the embryos degenerated 24h after viral exposure. This was significantly different (P<0.05) from the controls and the PRRSV-incubated hatched blastocysts that did not experience any negative influence on embryo development. Based on these results it can be concluded that embryonic cells are not susceptible to a PRRSV infection up to the hatched blastocyst stage. Embryonic cells of morulae and blastocysts are refractory to PRV, but the virus has a detrimental effect on further embryo development of hatched blastocysts. More experiments are necessary to confirm these results and to investigate whether, or at which preimplantation stage, embryos are susceptible to a PRRSV infection.

Genetic diversity and phylogenetic analysis of glycoprotein 5 of European-type porcine reproductive and respiratory virus strains in Spain

The gene encoding glycoprotein 5 (ORF5) of 21 porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Spain and two European-type vaccines currently available in that country were analysed using RT-PCR and sequencing. Sequences were then compared with other European-type sequences available through GenBank. Results showed percentages of similarity to Lelystad virus (LV), which, in most cases, were below 90 %. In contrast, two strains were very similar (>99 %) to a PRRSV variant from the Czech Republic. Evolutionary trees showed three types of strains: one grouped old Spanish sequences; a second grouped isolates from this study together with two Czech variant strains; and the third comprised other GenBank sequences. Regarding the predicted protein sequences, some isolates from this study showed a low degree of similarity to LV (below 50 %) and most of the strains examined had additional N-linked glycosylation sites compared to LV. These results provide evidence of the existence of variant PRRSV strains in Spain with characteristics that may be advantageous for immune evasion.

Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses

We determined 22 partial porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences, representing pathogenic field strains mainly from Poland and Lithuania, and two currently available European-type live PRRSV vaccines. Also, the complete ORF7 of two Lithuanian and two Polish strains was sequenced. We found that Polish, and in particular Lithuanian, PRRSV sequences were exceptionally different from the European prototype, the Lelystad virus, and in addition showed a very high national diversity. The most diverse present-day European-type PRRSV sequences were from Poland (2000) and Lithuania (2000), and exhibited only 72·2% nucleotide identity in the investigated ORF5 sequence. While all sequences determined in the present study were clearly of European type, inclusion of the new Lithuanian sequences in the genealogy resulted in a common ancestor for the European type virus significantly closer to the American-type PRRSV than previously seen. In addition, the length of the ORF7 of the Lithuanian strains was 378 nucleotides, and thus intermediate between the sizes of the prototypical EU-type (387 nucleotides) and US-type (372 nucleotides) ORF7 lengths. These findings for the Lithuanian PRRSV sequences provide support for the hypothesis that the EU and US genotypes of PRRSV evolved from a common ancestor. Also, this is the first report of ORF7 protein size polymorphism in field isolates of EU-type PRRSV.

Porcine Reproductive and Respiratory Syndrome Virus Comparison: Divergent Evolution on Two Continents

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3′-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5′-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5′ leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.