The cytoprotective role of GM1 ganglioside in Huntington disease cells.

BACKGROUND: Huntington disease (HD) is a neurodegenerative disease where a genetic mutation leads to excessive polyglutamine (Q) repeats in the huntingtin protein. The polyglutamine repeats create toxic plaques when the protein is cleaved, leading to neuron death. The glycolipid GM1 ganglioside (GM1) has been shown to be neuroprotective in HD models, as it prevents the cleavage of the mutant huntingtin protein by phosphorylation of serine 13 and 16. Previous studies have tested GM1 in both adult-onset and juvenile-onset HD models, but this study set out to investigate whether GM1 mediated cytoprotection is influenced by the length of polyglutamine repeats. METHOD AND RESULT: This study utilized cell culture to analyze the effect of GM1 on cell viability, directly comparing the response between cells with adult-onset HD and juvenile-onset HD. HEK293 cells expressing either wild-type huntingtin (Htt) (19Q) exon 1, adult-onset HD mutant Htt exon 1 (55Q), or Juvenile HD mutant Htt exon 1 (94Q) were assessed for cell viability using the WST-1 assay. Our results suggested moderate doses of GM1 increased cell viability for all cell lines when compared to untreated cells. When comparing HEK293 55Q and 94Q cells, there was no difference in cell viability within each dose of GM1. CONCLUSION: These data suggest cellular responses to GM1 are independent of polyglutamine repeats in HD cells and provide insight on GM1's application as a therapeutic agent for HD and other diseases.

In vitro neutralization of HoBi-like viruses by antibodies in serum of cattle immunized with inactivated or modified live vaccines of bovine viral diarrhea viruses 1 and 2.

HoBi-like viruses are an emerging species of pestiviruses with genetic and antigenic similarities to bovine viral diarrhea viruses 1 and 2 (BVDV1 and BVDV2). Vaccines for HoBi-like viruses are not yet available. However, both modified live virus (MLV) and killed virus (KV) vaccines against BVDV are widely used worldwide. This study evaluated the cross reactive antibody response against HoBi-like pestiviruses in sera of cattle immunized with BVDV1 and BVDV2 vaccines. Groups "KV" and "MLV", with 25 calves each, received killed or modified live vaccines, respectively, containing both BVDV1 and BVDV2 antigens. The antibody response was evaluated by virus neutralization test. The average of geometric mean titers (GMTs) of neutralizing antibodies in serum against HoBi-like viruses in the MLV group was 12.9, whereas GMTs to BVDV1, BVDV2 and border disease virus (BDV) were 51.1, 23.5, and 12.4, respectively. In this group, neutralizing antibodies against BVDV1, BVDV2, HoBi-like viruses and BDV were detected in 100%, 94%, 68% and 68% of calves, respectively. The GMT of neutralizing antibodies in serum against BVDV1, BVDV2, HoBi-like viruses and BDV in the KV group were 24.7, 14.5, 10.4 and 11, respectively. Similarly, the percentage of animals with neutralizing antibodies against BVDV1, BVDV2, HoBi-like viruses and BDV were 84%, 56%, 34% and 44%, respectively. These results indicate that MLV or killed BVDV1 and BVDV2 vaccines induce a cross reactive antibody response comparatively weak to HoBi-like viruses, and this response would likely not suffice to confer protection.

The effects of vaccination on serum hormone concentrations and conception rates in synchronized naive beef heifers.

Crossbred beef heifers (N = 59) were vaccinated at the time of synchronization/breeding with either a commercially available bovine herpesvirus type 1 modified live virus (MLV) (one dose) or inactivated virus vaccine (one or two doses). The estrus cycle was synchronized at vaccination and heifers were artificially inseminated 8 days (one dose) or 36 days (two dose) after initial vaccination. Pregnancy rates were greater for control heifers (90%; P = 0.02) and heifers given the inactivated virus vaccine (one dose: 86%; P = 0.08; or two: 90%; P < 0.01) than those given the MLV vaccine (48%). No control heifers experienced an abnormal estrous cycle, whereas only two (two dose; 2/21) and one (one dose; 1/7) heifers in the inactive virus groups had abnormal estrous cycles and were similar to control (P > 0.10). Heifers given the MLV vaccine had a greater (P = 0.02) percentage of abnormal estrous cycles (38%; 8/21) compared with the control and inactivated groups. Of the heifers with an abnormal estrous cycle, 100% of heifers given the inactivated vaccine (one or two dose) conceived at their return estrus, whereas only 38% of heifers given the MLV vaccine conceived at their return estrus (P > 0.10). During the synchronization period, concentrations of estrogen were greater (P < 0.01) in the control and the two-dose inactivated group compared with the MLV group. After AI, progesterone concentrations were greater (P < 0.01) in control heifers compared with the inactivated and MLV groups, but were similar (P ≥ 0.18) between the inactivated and MLV groups. Therefore, naïve heifers vaccinated with the inactivated vaccine were less likely to have an abnormal estrous cycle and had significantly higher pregnancy rates compared with heifers vaccinated with the MLV vaccine. In summary, vaccination of naïve heifers with an MLV vaccine at the start of a fixed-time AI protocol had a negative effect on pregnancy success.

The cellular endosomal sorting complex required for transport pathway is not involved in avian metapneumovirus budding in a virus-like-particle expression system.

Avian metapneumovirus (AMPV) is a paramyxovirus that principally causes respiratory disease and egg production drops in turkeys and chickens. Together with its closely related human metapneumovirus (HMPV), they comprise the genus Metapneumovirus in the family Paramyxoviridae. Little is currently known about the mechanisms involved in the budding of metapneumovirus. By using AMPV as a model system, we showed that the matrix (M) protein by itself was insufficient to form virus-like-particles (VLPs). The incorporation of M into VLPs was shown to occur only when both the viral nucleoprotein (N) and the fusion (F) proteins were co-expressed. Furthermore, we provided evidence indicating that two YSKL and YAGL segments encoded within the M protein were not a functional late domain, and the endosomal sorting complex required for transport (ESCRT) machinery was not involved in metapneumovirus budding, consistent with a recent observation that human respiratory syncytial virus, closely related to HMPV, uses an ESCRT-independent budding mechanism. Taken together, these results suggest that metapneumovirus budding is independent of the ESCRT pathway and the minimal budding machinery described here will aid our future understanding of metapneumovirus assembly and egress.

The lack of an inherent membrane targeting signal is responsible for the failure of the matrix (M1) protein of influenza A virus to bud into virus-like particles.

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.

Detection and characterization of influenza A virus PA-PB2 interaction through a bimolecular fluorescence complementation assay.

The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.

Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs.

Despite the development of several recent PCR assays for the egg stages of various trichostrongyles, there have been no protocols described for preserving field samples for PCR without refrigeration. In this study, Lugol's iodine (LI), sodium azide (SA), and neutral buffered formalin (NBF) were evaluated using Haemonchus contortus eggs to determine their potential as a preservative for trichostrongyle egg samples to be processed with real-time PCR. When egg recovery, embryo development, and egg morphology were evaluated from fecal samples preserved with LI, NBF, and SA, there was equally good recovery and preservation for the first month. Preserved eggs were detectable for 1 month, but after 6 months, none could be recovered. When real-time PCR analysis was performed on eggs isolated from faeces preserved with LI and SA, there was no detectable inhibition compared to fresh, non-preserved eggs; however, NBF significantly inhibited amplification. The results from this study demonstrate that for PCR applications LI and SA are effective preservatives for H. contortus eggs, resulting in good preservation of morphology while allowing for uninhibited PCR.

Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors.

The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.

Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.

A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.