Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) causes huge economic losses to the European pig industry. PRRSV-1 is divided into 3 subtypes and exhibits considerable antigenic heterogeneity. Due to its high mutation rate, PRRSV-1 is constantly evolving, and highly virulent, particularly subtype 3 strains, are continually emerging. The mechanism(s) underlying PRRSV-1 virulence have not been fully elucidated. In vivo studies have implicated replication kinetics, cell tropism and an enhanced pro-inflammatory cytokine response as potential contributing factors. However, few strains have been directly compared and differences in in vivo study design have hindered comparison, thus limiting our understanding of PRRSV-1 virulence. To address this knowledge gap, we sought to develop a reverse genetics and ex vivo model system, to attempt to identify correlates of PRRSV-1 virulence and attenuation in vitro. Herein we describe the use of primary porcine bone marrow-derived macrophages (BMDM) to investigate the growth kinetics and induced cytokine profiles of the highly virulent SU1-Bel strain, the low virulence 215-06 strain and the attenuated Olot/91 strain. We show that infection of BMDM with virulent PRRSV-1 strains induced higher expression of IL-6 and IL-8 and lower expression of TNF-? when compared with the attenuated strain. In addition, BMDM infected with SU1-Bel secreted significantly more IFN-? than those infected with PRRSV-1 strains of lower virulence. Interestingly, despite inducing less IFN-? than SU1-Bel, Olot/91 induced much higher levels of expression of several interferon-stimulated genes (ISGs), suggesting that Olot/91 may be less able to counteract type I IFN signaling which may contribute to its attenuated phenotype.

Marek's disease virus (MDV) is an important oncogenic alpha-herpesvirus that induces Marek's disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant-GX0101Deltapp38-and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas-namely, 4F9, 31G7, 34F2, and 35G9-were demonstrated to secrete specific antibodies against MDV-1's pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses-especially for large DNA viruses such as herpesviruses.

Eight infectious bursal disease virus (IBDV) genogroups have been identified based on the sequence of the capsid hypervariable region (HVR) (A1 to A8). Given reported vaccine failures, there is a need to evaluate the ability of vaccines to neutralize the different genogroups. To address this, we used a reverse genetics system and the chicken B-cell line DT40 to rescue a panel of chimeric IBDVs and perform neutralization assays. Chimeric viruses had the backbone of a lab-adapted strain (PBG98) and the HVRs from diverse field strains as follows: classical F52-70 (A1), U.S. variant Del-E (A2), Chinese variant SHG19 (A2), very virulent UK661 (A3), M04/09 distinct (A4), Italian ITA-04 (A6), and Australian variant Vic-01/94 (A8). Rescued viruses showed no substitutions at amino acid positions 253, 284, or 330, previously found to be associated with cell-culture adaptation. Sera from chickens inoculated with wild-type (wt) (F52-70) or vaccine (228E) A1 strains had the highest mean virus neutralization (VN) titers against the A1 virus (log2 15.4 and 12.7) and the lowest against A2 viruses (log2 7.4 to 7.9; P?=?0.0001 to 0.0274), consistent with A1 viruses being most antigenically distant from A2 strains, which correlated with the extent of differences in the predicted HVR structure. VN titers against the other genogroups ranged from log2 9.3 to 13.3, and A1 strains were likely more closely antigenically related to genogroups A3 and A4 than A6 and A8. Our data are consistent with field observations and validate the new method, which can be used to screen future vaccine candidates for breadth of neutralizing antibodies and evaluate the antigenic relatedness of different genogroups.