Dot-blot imunoenzimni test za dokaz protutijela za virus virusnoga proljeva goveda

Dot-blot enzyme immunoassay (DB-EIA) was utilized for the detection of bovine viral diarrhea (BVD) antibodies in infected cattle. In this assay whole particles of the NADL strain of BVD virus were used as an antigen. A total of 1250 sera from SN and ELISA tested cows were tested for BVDV antibodies. HRPO conjugated protein G was used to detect bound BVDV antibodies using TMB as the substrate. Statistical analyses did not show any significant differences in the sensitivity of DB-EIA, to the ELISA and SN tests. The assay proved to be a simple, inexpensive, reliable and rapid tool for BVD serodiagnosis.Dot-blot imunoenzimni test rabljen je za dokaz protutijela za virus virusnoga proljeva u zaraženih goveda. Kao antigen rabljen je virusni soj NADL. Ukupno je bilo pretraženo 1250 uzoraka seruma neutralizacijskim testom i imunoenzimnim testom. Protein G obilježen peroksidazom iz hrena rabljen je za dokaz specifičnih protutijela, a kao supstrat rabljen je TMB. Statističkom analizom nije ustanovljena značajna razlika u osjetljivosti dot-blot imunoenzimnoga testa, neutralizacijskoga testa i imunoenzimnoga testa. Razvijeni test je jednostavan, jeftin, pouzdan i brz te se može rabiti za serološku dijagnostiku virusnoga proljeva goveda

Characterisation of M2e Antigenicity using anti-M2 Monoclonal Antibody and anti-M2e Polyclonal Antibodies

Matrix 2 ectodomain (M2e) protein is a potential antigen for detection of influenza A virus infection in vaccinated poultry (DIVA test). However the M2e antigenicity and immune response it induces in either humans or animals are poorly understood. Seventeen M2e peptides and sixteen recombinant M2e (rM2e) proteins with amino acid (aa) changes introduced at position 10, 11, 12, 13 14, 16, 18 and 20 were compared by western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using mouse anti-M2 monoclonal antibody (mAb) 14C2, and anti-M2e peptide chicken and rabbit polyclonal antibody (pAb). The mAb 14C had the best discriminating power and indicated that all six positions contributed to the M2e antigenicity. Position 11 was the important immunodominant and affected Mab14C binding to a greatest degree. Changes in the adjacent position 14, 16 and 18 also influenced the binding, and it detected regardless of the method (WB or ELISA), or the antigen used (M2e peptide or rM2e). For chicken pAb and rabbit pAb, the immunodominant aa was position 10 and the antibody reaction was not affected by aa change at 11. The binding of rabbit pAb was also affected by changes at 14 and 16, which confirm the contribution of these positions to the M2e antigenicity. Position 10 was the only important position for the binding of chicken pAb to M2e. Overall, the study showed that the M2e antigenic sites are located between residues 10 – 18 and that aa changes at position 10, 11, 12, 14, 16 and 18 may all affect the antibody binding within the M2e protein

Detection of bovine coronavirus by RT-PCR in a field study

In the present study we used RT-PCR assay for detecting of BCoV, targeting a 730 bp fragment of the nucleocapsid (N) gene of BCoV with published primers that could amplify all BCoV strains. We evaluated presence of BCoV in diarrheic and nondiarrheic samples. 108 faecal samples from diarrheic calves and 80 faecal samples from nondiarrheic calves collected. In 13 of 108 diarrheic samples both ELISA and RT-PCR detected BCoV. In 4 of 80 samples second group (non diarrheic) BCoV was detected by RT-PCR only not capture ELISA. This report is the first detection of BCoV in Iran. The results suggest that RT-PCR is more sensitive than ELISA method to detect BCoV, especially in subclinical cases. Because these animals shed a low amount of virus in faeces we need to apply sensitive techniques, such as RT-PCR, nested PCR and real time RT-PCR

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P

Genetic diversity of Koala retrovirus (KoRV) env gene subtypes: Insights into northern and southern koala populations

Koala retrovirus (KoRV) is a recently endogenised retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the “northern” and “southern” koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV’s ongoing entry into the koala genome

Epitope mapping of avian influenza m2e protein: different species recognise various epitopes

Published: June 30, 2016A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.Noor Haliza Hasan, Esmaeil Ebrahimie, Jagoda Ignjatovic, Simson Tarigan, Anne Peaston, Farhid Hemmatzade

The map of interactions between ligands and TLR4.

A) ht-Map2191 and B) ht-FAP-P. Green dotted lines represent hydrogen bonds.</p

Fig 3 -

Ramachandran plot of A) ht-MAP2191 and B) ht-FAP-P.</p