SYBR Green-Based Real-Time Reverse Transcription-PCR for Typing and Subtyping of All Hemagglutinin and Neuraminidase Genes of Avian Influenza Viruses and Comparison to Standard Serological Subtyping Tests

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 101.5, 102.3, and 103.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples

Proline Metabolism and Its Functions in Development and Stress Tolerance

Proline takes an exceptional place among the proteinogenic amino acids by its specific accumulation in pollen and in response to multiple types of stress. Despite the more than 50 years of research, the biochemical pathways of proline biosynthesis and degradation still await their complete characterization in plants. Also, the molecular and physiological functions of proline metabolism in plant development and defense against stress are not yet fully understood. This chapter focuses on the current knowledge about the biochemical pathways of proline metabolism in plants, on its tissue-specific regulation and subcellular compartmentation, and on still open questions. Furthermore, we will summarize what is known about the influence of proline metabolism on plant development under optimal growth conditions and how it may support continued development despite the impact of stress. The cognate chapter “Regulation of Proline Accumulation and its molecular and physiological Functions in Stress Defense” will focus on the possible beneficial functions of proline metabolism and accumulation in the defense response against diverse stresses. With these two cohesive chapters, we aim to provide a comprehensive picture of the current knowledge and the open research questions in proline-dependent stress defense