A Comparison of LLCMK2 and RMix A549, Mv1Lu Cells for the Detection of

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A Comparison of LLC-MK2 and R-Mix (A549, Mv1Lu)
Cells for the Detection of Human Metapneumovirus
(hMPV) Sharon Setterquist, MT(ASCP) and Gregory
C. Gray, MD, MPH The Emerging Pathogens
Laboratory of the Center for Emerging Infectious
Diseases, Department of Epidemiology, College of
Public Health, University of Iowa , Iowa City, IA

• Metapneumovirus diagnostic oligos from CDC
• Gene Sequence4
  Product Length
• hMPVF2-f Primer F GAG CAA ATT GAA
  AAT CCC AGA CA 347 BP 23
• hMPVF2-r Primer- F GAA AAC TGC CGC
  ACA ACA TTT AG
• RT-PCR Reaction mix
• 74.9 µl dH20 RNAse, DNAse free (Gibco Ultra
  Pure)
• 10.0 µl 10x Stratagene (La Jolla, CA) Cloned Pfu
  DNA Polymerase 10x reaction Buffer
• 0.65 µl dNTP (25mM)
• 0.25 µl Ambion, Inc. (Austin, TX) RNase Inhibitor
  40 U/ul
• 0.80 µl MPVF2 Forward Primer (50uM)
• 0.40 µl MPVF2 Reverse Primer (50uM)
• 1.00 µl Ambion, Inc. (Austin, TX) M-MLV-Reverse
  Transcriptase (100 U/ul)

Absract Human metapneumovirus (hMPV) is a newly
described paramyxovirus frequently associated
with acute respiratory disease. The pathogen is
slow growing on the recommended LLC-MK2 cell
line, requiring two to three weeks before CPE is
observed. In an effort to find a more rapid
culture technique, we compared LLC-MK2 with the
R-Mix cell line (A549/Mv1Lu). Using three, low
passage, clinical hMPV isolates, we applied
identical inoculums, known to be minimally
detectable by RT-PCR, onto shell vials of both
medias. The vials were centrifuged for one hour,
incubated, and refed on days 3, 6, and 10. Using
a primer set known to produce a 347bp amplicon
from the hMPV F-gene, we performed a one-step
RT-PCR on the supernatants of the cultures,
comparing band intensity over time. Two of our
three hMPV isolates showed increased band
intensity at day 6 in the R-Mix cells as compared
to increased band intensity first noted on days
10 and 13 for all three isolates in LLC-MK2
cells. However, the R-Mix signals waned by day
10. Our preliminary data suggests that LLC-MK2
cells remain the cell culture of choice for hMPV,
but after more investigation, R-Mix cells may
prove useful in rapid detection. Background In
June 2001, Osterhaus1 and colleagues reported a
newly recognized human respiratory virus
(Pneumovirinae subfamily, Paramyxoviridae family)
termed the human Metapneumovirus (hMPV). hMPV is
most closely related to avian pneumovirus
serotype C. Symptoms of hMPV infections are
similar to those seen with RSV, with some
patients suffering severe respiratory disease.
Our lab has screened 150 clinical respiratory
samples by reverse transcriptase polymerase chain
reaction (RT-PCR) and found 3 positive for hMPV
(from patients aged 7, 4, and 64 years) One
published study has found 70 of infants (n30)
with severe RSV bronchiolitis to be coinfected
with hMPV2 and another study found 6.6 of
respiratory specimens screened negative for other
respiratory viruses (n337) to have evidence for
hMPV infection.3 hMPV infections have been found
to occur in both patients of all ages and may
account for a significant portion of respiratory
infections.1-4 Most recently hMPV has been
implicated in the epidemic of Severe Acute
Respiratory Syndrome or SARS. Study Objective To
determine if hMPV will grow more quickly on R-Mix
shell vials (A549 Mv1Lu, Diagnostic Hybrids,
Inc., Athens, Ohio) than LLC-MK2 shell vials
(Rhesus Monkey Kidney, Diagnostic Hybrids, Inc.,
Athens, Ohio). Methods Three low passage
clinical hMPV isolates were diluted in media so
as to produce a weak band by RT-PCR at time 0.
(Rm-03T media was used for R-Mix cells (A549
Mv1Lu, Diagnostic Hybrids, Inc., Athens, Ohio)
and CDCs hMPV growth media for LLC-MK2 cells
(Rhesus Monkey Kidney, Diagnostic Hybrids, Inc.,
Athens, Ohio), both are serum-free, containing
trypsin and penicillin/streptomycin.) Four sets
of shell vials were inoculated including one
R-Mix (A549 Mv1Lu, Diagnostic Hybrids, Inc.,
Athens, Ohio), one LLC-MK2 (Rhesus Monkey Kidney,
Diagnostic Hybrids, Inc., Athens, Ohio) and one
empty shell vial (control). All vials were
centrifuged 1 hr. at 1400 G, 24C, and incubated
at 35C. Aliquots of cell culture media were
saved on days 0, 3, 6, 10, and 13 at -70C, for
further RNA purification. Shell vial cultures
were refed on days 3, 6, and 10 with 1ml new
media. 140ul of the cell culture media was
processed using Qiagens QIAamp viral RNA
minispin protocol (QIAGEN Inc., Valencia,
CA). 11ul of the QIAGEN eluate were used in a
100ul one-step RT-PCR reaction amplifying a 347bp
product from the hMPV F-gene. Amplified product
(20 ul) was visualized by ethidium bromide
staining after electrophoresis (using a 1.2
agarose gel) with trans UV illumination. Gel
documentation was done using Bio-Rad
Laboratories, Inc. (Hercules, CA) Gel Doc with
Quantity One software.
Fig. 1 Isolate 1 shows increasing band
intensity on day 6 with R-Mix vs. day 10 with
LLC-MK2 cells.
Fig. 2 Isolate 2 is detected on days 3 and 6
by both cell lines but disappears by day 10 in
the R-Mix while band intensity increases only in
the LLC-MK2 cells.
Fig. 3 Isolate 3 shows an increase in band
intensity on day 6 with the R-Mix vs. day 13 in
the LLC-MK2 cells