We are dealing with an unknown/yet to be identified virus suspected to belong to chicken coronavirus [infectious bronchitis virus]. There was a history of high mortality and evidence of kidney swelling in a flock of chickens where an outbreak occurred. Inoculation of 10 day old embryonated chicken eggs with tissue homogenate samples derived from the affected kidney (10% w/v) causes 50-60% embryo death within 5days pi [passage 2] and later 70-100% within 3days pi [p3 and above]. Severe congestions were among others features observed on the dead embryo. Suprisingly, at 10 e1 dilution, there were zero mortality but embryo were slightly dwarf with evidence of curling of the toe.
There was evidence of virus growth in cell culture [using kidney cells] with CPE characterized by cell rounding, syncitial cells formation, and cells detachment within 4-5day pi [at p1] and within 2-3 days pi [as the passages increases i.e p2 above]. All effort to amplify avian coronavirus using different sets of universally designed primers [to detect the conserved n-gene or even the variable S1-gene sequences] yielded either unspecific band or no detectable band at all. Samples were tested negative by Haemagglutination, negative for Newcastle disease virus using PCR, negative for mycoplasma gallisepticum using PCR and negative for Avian influenza virus using PCR. We thought of performing negative staining and direct RNA sequencing , however, the tedious nature of sucrose gradient, effect of ultracentrifuge speed on the virus envelope protein, lack of specific/random primers, cost of next-gene sequence and chances of having more of host associated mRNA in nextgene sequencing data especially if the sample is not purified are challenges to us. Could there be some clue or advise/protocol to use; could some expert render technical assistance here? Note: mutation occur greatly in
Hi, you are welcome to send me the sample for analysis. We have few methods to deal with such samples (http://www.ncbi.nlm.nih.gov/pubmed/?term=pyrc+k)
Hi, you are welcome to send me the sample for analysis. We have few methods to deal with such samples (http://www.ncbi.nlm.nih.gov/pubmed/?term=pyrc+k)
Do you have electron microscope to detect that your virus is a corona virus?
Do you have any pathogenicity test in your assay?
The pathogenicity test is the main criterion for the identification of virus suspected of being the aetiological agents of a unknown disease.
We work with viruses of native plants, most of which are new. Next gen sequencing (Illumina) of RNA after removal of ribosomal RNA, assembly and sorting contigs by size results in virus sequences at the top of the list (av length of human mRNAs only ~800+ bases). Then need to look for common motifs etc in these sequences - we have found many actual and potential new viruses this way - of course after this evidence is needed to support their functionality (eg expressed protein, transfer to non- infected plants etc).
There is an interesting article from the CDC that supports use of culture and electron microscopy to identify novel pathogenic virusues;
1: Goldsmith CS, Ksiazek TG, Rollin PE, Comer JA, Nicholson WL, Peret TC, Erdman DD, Bellini WJ, Harcourt BH, Rota PA, Bhatnagar J, Bowen MD, Erickson BR, McMullan LK, Nichol ST, Shieh WJ, Paddock CD, Zaki SR. Cell culture and electron
microscopy for identifying viruses in diseases of unknown cause. Emerg Infect Dis. 2013 Jun;19(6):886-91.
The other alternative, of course is non-specific amplification of RNase, DNase treated culture supernatants followed by next generation sequencing.
I agree with Dr. Frost about the use of culture, electron microscopy and non-specific amplification + sequencing. Using at least the two last options could give a reasonable approach for identification.
Have you tried to perform conventional PCR using consensus primers targeted to RNA-dependent RNA polymerase (RdRp) region? Our team has discovered a number of novel coronaviruses from animals, please feel free to contact us if you have any other questions.
http://www.ncbi.nlm.nih.gov/pubmed/?term=Lau+SK+and+novel+virus
You might use a re sequencing array. Bauchuan Lin et al have a number of papers on this. TesArray acquired the patents and markets the array.
Hi, we have identified several novel virus from bats.In your case, first, you need to make sure of the existence of the virus under EM. And then you can get some nucleatide acid sequence by random PCR.
I'm agree with mr Yang, firstly electron microscopy of sample and then multilication of nucleic acid and blasting could be useful.
Just an idea ... could you increase the viral titre in cell culture by cold shocking your cells?
I have a virus with no identity in any of the sequence in the NCBI database. How can I characterized this virus?
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