Subject: MHV - Summary

From: <[email-masked]>
Date: Tue, 16 Nov 1993 20:12:17 CST

Sorry for the delay in my summary.  I got a bit busy with an
experiment.  Meanwhile, here's what I discovered.

A Medline did discover a few interesting papers which describes a PCR
test for MHV.

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"Detection of rodent coronaviruses in tissues and cell cultures by
using polymerase chain reaction" by: Homberger-FR; Smith-AL;
Barthold-SW in J-Clin-Microbiol.  1991 Dec; 29(12):

Abstract:  A polymerase chain reaction (PCR) method was developed for
the detection of rodent coronaviruses in biological material by using
reverse transcriptase and two primers which flanked an M gene
sequence of 375 bp.  PCR detected all of 11 different strains of mouse
hepatitis virus (MHV) as well as rat sialodacryoadenitis virus but not
bovine coronavirus or human coronavirus strains OC43 and 229E.  The
M gene sequences of bovine coronavirus and human coronavirus OC43
are homologous to that of MHV, but minor differences exist in the
primer regions, preventing annealing of the primers.  For detecting
MHV-Y in tissue samples, PCR was faster than and at least as sensitive
as either of the two bioassays (infant mouse bioassay and mouse
antibody production test).
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"Sequence analysis and molecular detection of mouse hepatitis virus
using the polymerase chain reaction."  by: Kunita-S; Terada-E; Goto-K;
Kagiyama-N in Lab-Anim-Sci.  1992 Dec; 42(6): 593-8

Abstract:  Sequence analysis of the nucleocapsid protein genes of five
strains of mouse hepatitis virus (MHV) disclosed that the 3' region of
the nucleocapsid protein gene contains highly conserved sequences
unique to MHV.  We designed a pair of primer to amplify cDNA from
such sequences of MHV by using the polymerase chain reaction (PCR).
Six isolates of wild-type MHV, as well as prototype viruses, were
amplified successfully and detected in ethidium bromide-stained agarose
gels.  The sequence identity of PCR products was readily verified by
confirming target size and a Mf1I site within the target.  The
sensitivity of our PCR assay was estimated to be sufficient to detect
a single cell infected with MHV in biologic materials than current
methods such as virus isolation, the infant mouse bioassay, and the
mouse antibody production test.
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In addition to the Medline, I did receive this interesting response to
my posting:

Reply-To: "barrys2327" <[email-masked]>
Message-Id: <[email-masked]>
To: [email-masked]
Date: Tue, 02 Nov 93 18:42:29 EST
Subject: RE: MHV Testing

Kathy,

A scientist in our laboratory has recently developed a set of reagents
for detection of murine viral antibodies.  One of the reagent sets is
designed for the detection of murine antibodies to MHV.  The format of
the detection system is a 96 well microelisa plate.  The test takes less
than three hours to run and can test up to 92 samples.  I believe the
price will be approximately $370, working out to $4.00 per test.  It is
necessary to have a microelisa plate reader and a multichannel pipettor
to perform the assay.  The reagents are going to go on sale in early
December.

Feel free to email me with any additional question you have.  You can
obtain ordering information at 800.523.7620 (voice).

Barry Schmetter
Diagnostic Product Research Laboratory
Organon Teknika / Biotechnology Research Institute
[email-masked]
*************************

Since we do not have a microelise reader, I did not do a follow-up to
this interesting suggestion.

Finally, I did contact a Stephanie Weisbroth of ANMED Biosafe, 7642
Standish Place, Rockville, MD  20855 - phone (301.762.0366).  Who did
offer to do a single agent test (only for MHV) for only $325 per
tumor/cell line.  She even suggested a cheaper method: we grow our
tumors ourselves in an isolated area for 4-5 weeks and collect the
serum.  Dilute the serum 1:10 in PBS and mail it to her on ice and they
will run an antibody production test for only $7 per serum sample.

I think this is the way to go for the initial screening of our tumor
lines.  If they test negative... it may then be worth it to invest in the
proper primers and run a PCR once every 3-6 months as we propagate
this tumor in the athymic nudes.

Thanks to all 3 of you who responded and again, sorry for the long
delay.

-Kathy