Molecular and Biological Characterization of Two Very Virulent Infectious Bursal Disease Virus Isolates, Upm94/273 and Upm97/61

An atypical very virulent (w) strain (UPM94/273) and typical vv strain (UPM97/61) of infectious bursal disease virus (IBOV) isolated in Malaysia, were characterized both in vivo and at the molecular level. Comparison of the deduced amino acid sequences with other serotype 1 and 2 sequences revealed 16 amino acid residues, which were conserved only in the vvIBDV. Among the 16 unique amino acid differences, 8 were in VP1 (146 Asp, 147 Asn, 242 Glu, 390 Met, 393 Asp, 562 Pro, 687 Pro and 695 Arg), 3 were in VP2 (222 Ala, 256 lie and 294 lie), 2 were in VP3 (990 Val and 1005 Ala) and 3 were in VP4 (685 Asn, 715 Ser and 751 Asp). The importance of these unique amino acid residues is not known but they could affect the virulence of vvIBOV. The UPM94/273 also demonstrated 6 unique amino acid residues at segment A at positions Ser254, Glu270, Lys588, Ser745, Phe838 and Lys863 and 8 unique amino acid residues at segment B at positions Ala92, Ser100, Va1208, Asp253, Asp560, Asn565, Gly750 and Gly876. In addition, these amino acid substitutions have not been reported before in vvlBDV and were found only on variant, classical and/or serotype 2 strains. However, the VP5 region of both vvlBDV strains was conserved. The UPM97/6 1 demonstrated 7 unique amino acid substitutions at segment A and 4 unique amino acid substitutions at segment B. However, none of the amino acids changes have been reported elsewhere in other IBDV strains. Although the actual functions of the amino acid substitutions are not know, the unusual amino acid substitutions at segment A and/or B of both isolates may be important in virus virulence. Alignments of the nucleic acid and amino acid sequences of segment A and B followed by distance analysis allowed the generation of phylogenetic trees. Phylogenetic analysis based on segment A and B revealed that all the vvlBDV strains including U PM94/273 isolate can be clustered in a group that is distinct from classical, variant, attenuated and serotype 2 strains. However, the tree branching patterns were quite different between segment A and segment B. In addition, the vvlBDV strains showed several conserved amino acid substitutions at segment B as found in the Australian 002-73 and serotype 2 strains. These findings indicate that probably a genetic reassortment may have play an important role in the emergence of vvIBDV. Flow cytometry and real time peR assays, indicated that chickens infected with UPM97/61 induced higher percentages of apoptotic cells but lower level of viral load whereas UPM94/273 induced lower percentages of apoptotic cells but higher level of viral load, suggesting a negative correlation between viral load and apoptosis. These results indicated that U PM97/61 was more virulent than UPM94/273

Development of Real-Time Polymerase Chain Reaction Assays for the Detection and Differentiation of Infectious Bursal Disease Virus Subtypes

Two different real-time polymerase chain reaction (PCR) detection approaches based on SYBR Green I dye and Taqman probe based assays were developed for the detection and differentiation of infectious bursal disease virus (IBDV) subtypes. Both approaches were able to detect and differentiate IBDV subtypes based on the use of subtype-specific primers or subtype-specific probes where the primers were designed based on single nucleotide polymorphism (SNP) concept. After optimization of the primer combinations and PCR parameters, very virulent-specific primer, IF & IVIR, and classical-specific primer, IF & RCLA were used in the SYBR Green I real-time RT-PCR assay. Plasmid DNA carrying the VP4 gene of the references IBDV strains: very virulent strain UPM94/273 and classical strain D78 were established and used as positive controls in the real time RT-PCR. The developed assay had a dynamic detection limit which spans over 5 log10 concentration range for very virulent and spans over 7 log10 concentration range for classical strain, respectively. The correlation coefficient for amplification of very virulent and classical strain was R2 = 0.9918 and R2 = 0.9977, respectively. No amplification was found when the subtype-specific primers were used to amplify other avian RNA viruses. The performance of the SYBR Green I based assay was tested on various IBDV isolates including 10 previously characterized IBDV and 11 commercial vaccine strains. The very virulent-specific primer only detected and amplified the very virulent IBDV with threshold cycle (CT) ranged from 14.93 to 21.52 and melting temperature (Tm) between 85.6°C to 88.0°C. The classical-specific primer was only able to amplify the classical IBDV with CT value ranged from 11.99 to 20.89 and Tm between 85.6°C to 86.8°C. The diagnostic efficacy of the developed assay was also evaluated using bursal samples obtained from experimentally infected chickens. Bursal samples collected from D78 vaccine infected chickens at day 3 and 5 p.i were positive for IBDV with average CT of 23.05+1.31, Tm of 85.8+0.17°C and average CT 21.82+1.42, Tm of 86.0+0.28°C , respectively. Bursal samples collected at day 10 p.i from this group were also found positive for IBDV with average CT of 24.42+1.20 and Tm of 85.9+0.18°C. On the other hand, only bursal samples collected at day 3 and 5 p.i were found positive for yery virulent IBDV with average CT 19.39+0.72, Tm of 86.6+0.14°C and average CT 23.55+1.39, Tm of 86.5+0.19°C, respectively. In the case of samples from dual infection with different IBDV subtypes, viral RNA was detectable only on day 3 and 5 p.i. In general, majority of the bursal samples have higher very virulent virus with an average CT value ranged from 21.24+0.68 to 22.19+0.97 compared to vaccine virus with Ct value ranged from 23.88+0.74 to 25.36+1.19. The performance of the developed SYBR Green I based assay was analyzed with other standard diagnostic methods. In the uninfected control group, no obvious microscopic lesions were found in the bursa and the lesions score was less than 1.0. However, mild bursal lesions without signs of inflammation with lesions score less than 3.0 was detected from bursal tissue obtained from chickens inoculated with vaccine strain D78. Based on the lesion score, it was clear that bursal pathology developed rapidly, with complete loss of tissue architecture by day 3 p.i. when the chickens were infected with virulent IBDV. The correlation between ELISA antibody titers and real-time CT values were inversely related, where the lower titers of antibodies associated with higher level of viral RNA as found in chickens infected with very virulent strain UPM94/273. On the other hand, vaccine strain D78 induced higher detectable antibody titers than UPM94/273, which indirectly support less virus replication with late positive amplification in real-time RT-PCR. Thus, the level of viral RNA in bursal samples obtained from D78 infected chickens was lower than UPM94/273 infected chickens. A total of 37 bursal samples from IBD suspected field cases were collected and then tested on the developed assay. The developed SYBR Green I based PCR assay was able to detect 9 samples positive for very virulent, 4 positive for classical IBDV and 12 samples positive for both very virulent and vaccine strains of IBDV. Sequence analysis of the hypervariable region of the VP2 gene of the IBDV samples revealed that the residues involved in determining the virulence of VV IBDV and CL IBDV were highly conserved. For the Taqman based duplex real-time PCR assay development, a new set of primers FWDC and RVSC were designed from the conserved region of VP4 of both very virulent and classical strains. A dual-labeled fluorescent probe each specific for very virulent IBDV (ProVV) and vaccine IBDV (ProCL) were designed The performance of the developed Taqman assay was compared with other PCR methods namely conventional RT-PCR and previously developed SYBR Green I assay. The Taqman assay was found far more superior in terms of turn around time and sensitivity. With the aid of β-actin gene, the Taqman assay was also used to determine the viral load fold changes in bursal samples that were positive for both vaccine and very virulent IBDV. Majority of these samples have higher viral load fold change in very virulent than the classical strain except for three samples MB078/04, MB001/05 and MB033/05 which showed higher fold change in classical strain than very virulent strain. In conclusion, this study has successfully developed SYBR Green I based and Taqman based one-step real-time PCR assays for rapid detection and differentiation of IBDV subtypes in particular very virulent and classical IBDV strains

Effects of kefirs on glycemic, insulinemic and satiety responses

We hypothesized that three types of kefir (Lifewayy Low Fat Strawberry Kefir, ProBugs Kefir, orange flavor, and Lifewayy Low Fat Plain Kefir) would have low glycemic index (GI), high insulinemic index (II) and high satiety index (SI). Secondarily, we hypothesized that there would be no significant correlations among postprandial satiety, glucose and insulin responses. Lastly, we hypothesized that kefir, like other dairy products, would have dissociation of GI and II. To test our hypotheses, this study was divided into three phases. In Phase I, a portion of Lifewayy Low Fat Strawberry Kefir (S group) and a portion of ProBugs Kefir, orange flavor (O group) containing 50 g of available carbohydrates were tested. In Phase II, a portion of Lifewayy Low Fat Plain Kefir (P group) containing 25 g of available carbohydrates were tested. In Phase III, 240-kcals portions of all three types of kefirs were tested. In all phases a single meal, randomized crossover design was performed in which the test meals were fed to 10 healthy, male and female adults. The total glucose AUC of S group (p\u3c 0.0023), O group (p\u3c 0.0002) and P group (p\u3c 0.0002) were significantly lower compared with their respective glucose controls. A slight, but not significant inverse relationship between glycemic and satiety responses was observed with kefir beverages (r = -0.87; P = 0.13). Using a variance of component analysis, it was found that in the future, a significant relationship between the correlated effects of the treatments on GI and SI can be further tested by increasing the number of subjects to 12. Like other dairy products, kefir showed a dissociation of GI and II. Kefir can potentially be a useful food choice for patients with diabetes who are required to control their blood glucose levels

Study of near consensus complex social networks using Eigen theory

This paper extends the definition of an exact consensus complex social network to that of a near consensus complex social network. A near consensus complex social network is a social network with nontrivial topological features and steady state values of the decision certitudes of the majority of the nodes being either higher or lower than a threshold value. By using eigen theories, the relationships among the vectors representing the steady state values of the decision certitudes of the nodes, the influence weight matrix and the set of vectors representing the initial state values of the decision certitudes of the nodes that satisfies a given near consensus specification are characterized

My bitterness is deeper than the ocean : understanding internalized stigma from the perspectives of persons with schizophrenia and their family caregivers.

Background: It is estimated that 8 million of the Chinese adult population had a diagnosis of schizophrenia. Stigma associated with mental illness, which is pervasive in the Chinese cultural context, impacts both persons with schizophrenia and their family caregivers. However, a review of the literature found a dearth of research that explored internalized stigma from the perspectives of both patients and their caregivers. Methods: We integrated data from standardized scales and narratives from semi-structured interviews obtained from eight family-dyads. Interview narratives about stigma were analyzed using directed content analysis and compared with responses from Chinese versions of the Internalized Stigma of Mental Illness Scale and Affiliated Stigma Scale. Scores from the two scales and number of text fragments were compared to identify consistency of responses using the two methods. Profiles from three family-dyads were analyzed to highlight the interactive aspect of stigma in a dyadic relationship. Results: Our analyses suggested that persons with schizophrenia and their caregivers both internalized negative valuation from their social networks and reduced engagement in the community. Participants with schizophrenia expressed a sense of shame and inferiority, spoke about being a burden to their family, and expressed self-disappointment as a result of having a psychiatric diagnosis. Caregivers expressed high level of emotional distress because of mental illness in the family. Family dyads varied in the extent that internalized stigma were experienced by patients and caregivers. Conclusions: Family plays a central role in caring for persons with mental illness in China. Given the increasingly community-based nature of mental health services delivery, understanding internalized stigma as a family unit is important to guide the development of cultural-informed treatments. This pilot study provides a method that can be used to collect data that take into consideration the cultural nuances of Chinese societies