Studies of the molecular genetics and epidemiology of porcine rubulavirus infection
Sammanfattning: Porcine rubulavirus (PorPV) infection emerged spontaneously in pigs in Mexico in the early 1980s. Since the report of the initial outbreak of the disease, only one full-length genome from a strain isolated in 1984 (PorPV-LPMV/1984) has been sequenced. There is therefore limited information about the genetic variation of this virus. The overall objective of this thesis was to develop molecular techniques to help in the diagnostic field and to investigate in greater detail the full genomes of several isolates, and if possible, gain insights into the persistence, molecular epidemiology and the possible reservoirs of PorPV. In addition, a characterisation of the immune response during acute and persistent infection was included. A real-time RT-PCR was developed for the detection of viral RNA from PorPV in clinical samples using TaqMan technology and primers for the P gene. This assay was highly sensitive (approximately 10 copies per reaction), specific, reproducible and a very useful tool for molecular diagnostics and for enabling studies of various aspects of PorPV throughout this thesis. RT-PCRs based on the NP and P genes were used to study the tissue distribution of the virus. Viral mRNA in the lymph nodes showed that the NP gene was consistently detected in the parotid, submaxilar, cervical and mesenteric nodes and the pancreas. Full-length genomes were sequenced from new isolates obtained from clinical cases of infected swine. The genetic comparison and phylogenetic analysis indicated that three different genetic variants of PorPV had spread in the swine population and that a new generation of circulating virus with a pronounced attenuation has begun to emerge in nature. We also report the isolation of PorPV, or a related virus, from frugivorous, insectivorous, and hematophagous bats. A partial genome sequence analysis showed a 99.97 - 100% amino acid identity to the reference strain isolated from swine. However, larger parts of the genome must be sequenced to ascertain the genetic relationship between these viruses. The study of the immune response during acute and persistent infection revealed enhanced levels of CD8+, CD4+ and CD2+ T-cells in all infected pigs at 10 days PI. CD8+ T-cell subpopulations were signiﬁcantly higher (p<0.05) at 10 and 250 days PI, and CD4+ T-lymphocytes were also signiﬁcant at 250 days PI. In summary, this work developed molecular techniques that can be used to study the pathogenesis and molecular epidemiology of PorPV. The knowledge of the presence of different virus variants in nature, associated with a wildlife reservoir of PorPV can provide greater knowledge regarding the molecular genetic changes and useful data to establish new strategies in the control of this virus in Mexico.
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