#qRTPCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

Characterization of Arabidopsis thaliana Line with T-DNA Insertion in the Inositol Polyphosphate 5-Phosphatases8 Gene - Cytology and Genetics

Inositol polyphosphate 5-phosphatases (5Tase) are the important enzymes of the phosphatidylinositol (PI) signaling pathway and have been found to play important roles in plant growth, development, and stress responses. Most of the Arabidopsis genes encoding Inositol polyphosphate 5-phosphatases have been characterised. However, promoters of genes encoding Inositol polyphosphate 5-phosphatases of plants have not been characterized so far. Here, we report the characterization of Arabidopsis thaliana SALK mutant lines having T-DNA insertion in the upstream intergenic region of the Inositol polyphosphate 5-phosphatases8 (At5Tas8) gene. The location of T-DNA insertion in the SALK line was confirmed by PCR, and plant homozygous and hemizygous for the T-DNA insertion were identified. The homozygous plants were observed for the morphological difference as well as for the root phenotype. However, no significant morphological differences were observed in the mutant and wild-type plants. The expression analysis using qRT-PCR revealed a similar level of At5Tase8 transcript in the mutant and wild-type plants suggesting T-DNA insertion lies beyond the At5Tase 8 promoter. In silico analysis of the 3000 bp sequence upstream of the translation start site covering the T-DNA insertion site has revealed the presence of potential cis-regulatory elements for heat, light, drought, salt, sugar, and hormone. Besides, most predicted cis-elements were located downstream of the T-DNA insertion site, further supporting that the promoter of At5Tase8 lies within the 2738 bp sequence upstream of the translation start site. Further study is required to delineate the At5Tase8 promoter using promoter-reporter fusion in the transgenic Arabidopsis.

Testing of retail cheese, butter, ice cream and other dairy products for highly pathogenic avian influenza in the US

The recent outbreak of highly pathogenic avian influenza (HPAI) in dairy cows has created public health concerns about the potential of consumers being exposed to live virus from commercial dairy products. Previous studies support that pasteurization effectively inactivates avian influenza in milk and an earlier retail milk survey showed viral RNA, but no live virus could be detected in the dairy products tested. Because of the variety of products and processing methods in which milk is used, additional product testing was conducted to determine if HPAI viral RNA could be detected in retail dairy samples, and for positive quantitative real-time RT-PCR (qRT-PCR) samples to be tested for presence of live virus. Revised protocols were developed to extract RNA from solid dairy products including cheese and butter. The solid dairy product was mechanically liquified with garnet and zirconium beads in a bead beater diluted 1 to 4 with BHI media. This pre-processing step was suitable in allowing efficient RNA extraction with standard methods. Trial studies were conducted with different cheese types with spiked in avian influenza virus to show that inoculation of the liquified cheese into embryonating chicken eggs was not toxic to the embryos and allowed virus replication. A total of 167 retail dairy samples, including a variety of cheeses, butter, ice cream, and fluid milk were collected as part of nationwide survey. A total of 17.4% (29/167) of the samples had detectable viral RNA by qRT-PCR targeting the matrix gene, but all samples were negative for live virus after testing with embryonating egg inoculation. The viral RNA was also evaluated by sequencing part of the hemagglutinin gene using a revised protocol optimized to deal with the fragmented viral RNA. The sequence analysis showed all viral RNA positive samples were highly similar to previously reported HPAI dairy cow isolates. Using the revised protocols, it was determined that HPAI viral RNA could be detected in a variety of dairy products, but existing pasteurizations methods effectively inactivate virus assuring consumer safety. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported by US Department of Agriculture (USDA)-Agricultural Research Service Project No. 6040-32000-081-00D and the US Food and Drug Administration InterAgency Agreement 6040-32000-081-037I. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript or is available in Genbank (sequence data)