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Abstract Yellow leaf syndrome (YLS) of sugarcane is associated with sugarcane yellow leaf virus (SCYLV), a member of the family Luteoviridae . A fragment of the coat protein and readthrough domain of SCYLV wasexpressed in a bacterial expression system. The resulting protein was purified and used to immunize mice for monoclonal antibody (MAb) production. Two MAbs, 3A2E3 and 2F7H5, were selected following the screening of hybridoma cells using both plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and tissue blot immunoassay (TBIA). These MAbs can be incorporated into the TBIA assay currently used for the routine detection of SCYLV but could not be used in triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The two antibodies selected have slightly different specificities. Antibody 3A2E3 gave equivalent results to a polyclonal antiserum (raised to purified virus) in comparative testing using TBIA. The MAbs produced should provide a widely available, uniform reagent for SCYLV diagnosis with the potential to help manage YLS. [ABSTRACT FROM AUTHOR]

In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Sécurité du Médicament et des Produits de Santé where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: The worldwide use of yellow fever (YF) live attenuated vaccines came recently under close scrutiny as rare but serious adverse events have been reported. The population identified at major risk for these safety issues were extreme ages and immunocompromised subjects. Study NCT01426243 conducted by the French National Agency for AIDS research is an ongoing interventional study to evaluate the safety of the vaccine and the specific immune responses in HIV-infected patients following 17D-204 vaccination. As a preliminary study, we characterized the molecular diversity from E gene of the single 17D-204 vaccine batch used in this clinical study., Materials and Methods: Eight vials of lyophilized 17D-204 vaccine (Stamaril, Sanofi-Pasteur, Lyon, France) of the E5499 batch were reconstituted for viral quantification, cloning and sequencing of C/prM/E region., Results: The average rate of virions per vial was 8.68 ± 0.07 log₁₀ genome equivalents with a low coefficient of variation (0.81%). 246 sequences of the C/prM/E region (29-33 per vials) were generated and analyzed for the eight vials, 25 (10%) being defective and excluded from analyses. 95% of sequences had at least one nucleotide mutation. The mutations were observed on 662 variant sites distributed through all over the 1995 nucleotides sequence and were mainly non-synonymous (66%). Genome variability between vaccine vials was highly homogeneous with a nucleotide distance ranging from 0.29% to 0.41%. Average p-distances observed for each vial were also homogeneous, ranging from 0.15% to 0.31%., Conclusion: This study showed a homogenous YF virus RNA quantity in vaccine vials within a single lot and a low clonal diversity inter and intra vaccine vials. These results are consistent with a recent study showing that the main mechanism of attenuation resulted in the loss of diversity in the YF virus quasi-species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An equine West Nile virus (WNV) outbreak occurred in 2004 in the Camargue, a wetland area in the south of France where the virus was first reported in 1962 and re-emerged in 2000. WNV neutralizing antibodies were detected in resident birds and two isolates from a House Sparrow (Passer domesticus) and a Common Magpie (Pica pica) were completely sequenced. Phylogenetic analyses revealed that these isolates are closely related to strains previously found in horses in southern Europe and North Africa. More extensive investigation is required to determine whether WNV has been re-introduced or has become endemic in the Camargue.

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

Viral infections of the central nervous system (CNS) are caused by a variety of viruses, namely, herpesviruses, enteroviruses, and flaviviruses. The similar clinical signs provoked by these viruses make the diagnosis difficult. We report on the simultaneous detection of these major CNS pathogens using amplification by PCR and detection of amplified products using DNA microarray technology. Consensus primers were used for the amplification of all members of each genus. Sequences specific for the identification of each virus species were selected from the sequence alignments of each target gene and were synthesized on a high-density microarray. The amplified products were pooled, labeled, and cleaved, followed by hybridization on a single array. This method was successfully used to identify herpesviruses, namely, herpes simplex virus type 1 (HSV-1), HSV-2, and cytomegalovirus; all serotypes of human enteroviruses; and five flaviviruses (West Nile virus, dengue viruses, and Langat virus). This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis. The analytical sensitivity of the method was shown to be 500 genome equivalents ml(-1) for HSV-1, 0.3 50% tissue culture infectious doses (TCID50s) ml(-1) for the enterovirus coxsackievirus A9, and 200 TCID50s ml(-1) for West Nile virus. The clinical sensitivity of this method must now be evaluated.

Human monoclonal antibody fragments neutralizing wild-type and vaccine strains of yellow fever (YF) virus (genotypes West Africa I + II, East/Central Africa, 17D-204-WHO) were generated by repertoire cloning from YF patients. Analysis of virus escape variants identified amino acid (aa) 71 in domain II of the envelope glycoprotein (E) as the most critical residue for neutralization, with aa 153-155 in domain I contributing to the epitope. These data confirm the previous mapping of YFV neutralizing epitopes using mouse monoclonal antibodies but suggest that a conformational epitope could be formed by amino acids from domains I and II opposing each other in the dimeric form of the E protein. While the sera of the YF patients showed up to 10-fold reduced neutralizing activity against the 17D escape variants, sera from 17D vaccinees retained their neutralizing titers. Mutations in this major neutralizing epitope of YFV thus do not seem to carry the risk of immune escape in persons immunized with the YFV-17D vaccine.