Sequences: The different methods of passage

[mixin]

I see the passage history noted on sequences often but I don't know what they all mean. The MDCK is one I know; I assume when I see "C" that it's some sort of a cell line? E1 and E2 are passaged through eggs? I've also seen an X/E2. Does "Original" mean it had no passage and the sample was viewed directly through a microscope? What is "p1"?

I found this site that lists many cell lines but I did't find the others I was looking for. It has good pictures.

http://www.microscopyu.com/galleries/fluorescence/cells.html

Madin-Darby Canine Kidney Epithelial Cells (MDCK Line)
S. H. Madin and N. B. Darby initiated the MDCK line in 1958 by from the kidney tissue of an adult female cocker spaniel. The cells exhibit typical epithelial morphology and stain positive for keratin. MDCK cells demonstrate susceptibility to a number of viruses, including infectious canine hepatitis, coxsackievirus B5, reoviruses 2 and 3, vesicular stomatitis (Indiana strain), adenoviruses 4 and 5, vesicular exanthema of swine, and vaccinia. The cells, which are negative for the enzyme reverse transcriptase, are known to be resistant to poliovirus 2 and coxsackieviruses B3 and B4. The MDCK line is a popular tool for studies focusing on the processing of beta-amyloid precursor protein and its proteolytic products.

[AlaskaDenise]

Re: Sequences: The different methods of passage

The glossary here may be of assistance.

Also, there are many MDCK lines....the referenced site shows a few here:


Catalogue No. Cell Line Name Keywords
90102523 DoCI1 (S+L-) Canine kidney, Moloney Sarcoma Virus infected
84121903 MDCK Canine Cocker Spaniel kidney
85011435 MDCK Canine Cocker Spaniel kidney
02050101 MDCK-Protein Free Canine Cocker Spaniel kidney
05071502 MDCK-SIAT1 Canine Cocker Spaniel Kidney Sialic Acid Over Expression
00062106 MDCK I Canine Cocker Spaniel kidney
00062107 MDCK II Canine Cocker Spaniel Kidney


.

[JJackson]

Re: Sequences: The different methods of passage

As I recently read this on MCDK-SIAT1 I will provide the link here for reference.
MDCK is alpha 2,3 rich and 2,6 poor which applies selection pressure during passaging. They engineer a additional gene into the MDCK genome which doubled the number of a2,6 receptors. Also MDCK gives less reliable neuraminidase inhibitor test results. If you do read the paper I thought they had not considered a third option of bias in neuraminidase testing which I asked Vincent Racaniello about our Q & A can be found in the comments section here.

[mixin]

Re: Sequences: The different methods of passage

Thanks AD for that good link; I didn't realize there were that many cell lines.

JJackson, the reason I asked about passage was the recent study linking D225G to egg passaging (if I understood it correctly). I had assumed MDCK was probably better.

Do you know what "original" means?

I had read that Racaniello blog but not the comments; I wish I would have.. I could have saved myself a lot of frustration about my inability to fully understand this:

Clearly there are significant differences between infection and budding that are not fully understood.

It appears I'm in good company.

[JJackson]

Re: Sequences: The different methods of passage

Mixin I do not know what they mean in most cases. Here is link to a lab supply company's 'Whole Cell Lysates for Western Blotting' list (about 250 cell lines). I note that they only supply Std. MDCK but I know that MDCK-SIAT now comes in, at least, versions 1 to 4 - so their list is partial and others suppliers must offer other ranges.
We need someone who has some relevant lab experience to chip in. All culture mediums, their substrates, nutrients and environmental variables are only going to be approximations to infection in the host species and will therefore skew the results. Even if you used human epithelial cell in culture you would still not be able to mimic host immune response so may select for strain that would have been out performed in vivo.

The quote you used form ProfVRR is from the last comment but while it does point to the current lack of knowledge on exact mechanism the earlier part does at least remove the paradox. The paradox being that if you are presented with a snap-shot of a virion adjacent to a host cell wall there would not appear to be any way to tell between these two scenarios
A] The cell was uninfected and the virion was about to bind and infect
B] The cell was infected and the virion was just released by the cell
In scenario A the virus wants its HA to bind to a sialic acid and enter the cell but in B it wants its NA to cleave off the residues and prevent binding; the two are tugging in opposite directions. If we add Oseltamivir to this picture it should help with infection but hinder release. As it seems to be effective it would appear that the net effect, over both scenarios, is to the host's benefit. If you allow for the NA being embedded in the endosome prior to budding it is then possible to envisage a mechanism - while in the endosome- which prevents fusion with the endosome wall but allows NA cleavage of sialic acid residues in the wall so when budding occurs the endosome wall is now the cell wall adjacent to the virion and is already denuded of residues so the virus can drift free. The paradox is gone because our original snap-shot were not identical closer examination would have shown that the cell walls were not identical. Now if you are a graduate student wondering what to do for your doctorate all you need to do is find out how the environment in the endosome differs from the cell wall and show it is residue free at the time of budding.

[gsgs]

Re: Sequences: The different methods of passage

I assume they grow the virus in one sample first, then they divide
it and give it to others, freeze it, make multiple tests.

Then, later, they may want to make more and grow it again etc.
Or they must grow them regularly to ensure that they don't die.

For some old viruses, I saw a list with some of them being
replicated more than hundred times

[mixin]

Re: Sequences: The different methods of passage

Here's a list of the different methods and combinations I've seen so far:

C, C1, C2, 3, 4 and all types of combinations of those
MDCK, MDCKX, MDCK1-4, MDCKp4,
M1/C1, MX/C2,
X/C1, X/E2, X2/C1
SIAT, 1-4
R1/C1, E2, P0, P1, CK2, RIIX/C1, LLCMK2/MDCK1
Harvest-Rh1
IR

I'll include the collection methods I've seen in case they use certain passages for each type of sample:

nasal swab
throat swab
bronchial wash
nasalpharnygeal swab
lung autopsy
clinical specimen

[gsgs]

Re: Sequences: The different methods of passage

below is my list from genbank records, 2009 H1N1 only
there is a need to standardize and uniformize this, so it becomes
databasable,computer-readable,analyzable. And not everyone
introduces her own wording and the number of different entries
is smaller. Also the passage data is in different record fields,
sometimes in /note or in /lab_host or in Passage_history,
sometimes combined with other data, separated by ; or not




genbank passages of 2009 H1N1 HA
----------------------------------


Canis lupus familiaris MDCK cells,61
LLCMK2 MDCK1 passage(s),3
MDCK,1
MDCK 1 passage(s),94
MDCK 2 passage(s),1
MDCK P1,1
MDCK p1 passage(s),4
MDCK passage(s),9
MDCK1 passage(s),36
MDCK2 passage(s),94
MDCK3 passage(s),9
MDCK4 passage(s),2
swine kidney 2 MDCK 2 passage(s),2
swine kidney 2 MDCK 3 passage(s),1

P0 passage(s),102
P1 passage(s),1
p1 passage(s),80
P3 passage(s),2

RhMK 1 passage(s),4
RhMK1 passage(s),5
RhMK2 passage(s),1
harvest-Rh1 passage(s),20
one passage(s),2

E1 passage(s),52
E3 passage(s),1
embryonated chicken eggs 1 passage(s),1
embryonated chicken eggs passage 1CE-4dpi,5
embryonated chicken eggs passage 1CE-6dpi,2


MDCK:318
P:185
Rh:30
E:61=10.3%

---------------------------------------

ORIGINAL,15
ORIGINAL-NP WASH,1
0,12
Original,2
original,20
original specimen,1

E1,12
E2,67
E2/E1,2
E3,2
E4,1
C1/E2,2
X/E2,2

C1,168
C1-2,1
C1-3,1
C1-IR,3
C1-NP WASH,1
C1/C1,20
C2,23
C2/C1,3
CACO1/C1,1
CX,1
CYNO1M1/C1,2
X/C1,8
X/C2,1
X1/C1,1
X2/C1,1
c1,14
c2,1

M1,4
M1/C1,34
M1/C2,5
M1M1/C1,1
M2/C1,7
M2/C2,2
MDCK pimary isolate,1
MDCK1,6
MX/C1,13

R1/C1,3
RII1/C1,3
RIIX/C1,2
X,3
passage 1,1


O:51
E:88=18.6%
C:250
M:73
rest:12

----------------------------------------

egg:149 out of 1068 with given passage history = 14.0%

passage history: E1+1",1
passage history: M1",2
passage history: MDCK1",32
passage history: MDCK1+1",5
passage history: MDCK2",1
passage history: MDCK2+2",2
passage history: MDCK2; resistance to Oseltamivir",1
passaged in MDCK cells",35
passaged in rhesus monkey kidney cells",1
passaged twice in MDCK cells",2

E:1
other:81

---------------------------------------

2 passages in MDCK cells",9
A/Guangdong/1/2009(H1N1)v; originally submitted as,1
Contains a mixed population of nucleotide A and G,,1
MDCK passage",2
embryonated chicken eggs passage 1CE-3dpi",1
lineage: swl; passage history: MDCK1",1
lineage: swl; passage history: MDCK1+1",10
lineage: swl; passage history: MDCK1; resistance to,1
lineage: swl; passage history: MDCK2+1",5
lineage: swl; passage history: MDCK2; resistance to,1
lineage: swl; passage history: MDCK3+1",1
lineage: swl; passage history: MDCK4+1",1
lineage: swl; passaged in MDCK cells",3
lineage: swl; passaged in rhesus monkey kidney,1
passage MDCK1",1
passage egg 1",1
passaged in MDCK cells",1

egg:2
rest:36


================================================== =====



total:
-------

MDCK:318
P:185
Rh:30
E:61=10.3%

O:51
E:88=18.6%
C:250
M:73
rest:12

E:1
other:81

egg:2
rest:36

----

egg=152= 12.8% of passage-specified samples = 6.1% of all samples
rest=1036

total samples = 2475

[gsgs]

Re: Sequences: The different methods of passage

http://www.ncbi.nlm.nih.gov/genomes/FLU/help.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC226993/

more samples were SIV(swine influenza virus)-positive with ECE(embryonated chicken eggs)
than with tissue culture(MDCK)
suggesting that ECE remains the system of choice for isolation of SIV