Med. Mierobiol. Immunol. 162, 23--27 (1975) 9 by Springer-Verlag 1975

Permanent Canine Kidney (MDCK) Cells for Isolation and Plaque Assay of Influenza B Viruses K. Tobita Department of Microbiology, Institute of Public Health, Takanawa, Tokyo Received June 9, 1975 Abstract. A wide range of influenza B virus strains with various passage histories uniformly formed well-defined clear plaques with high efficiency in cultures of an established line of canine kidney cells (MDCK). PFU titers of the viruses assayed in MDCK exceeded the tiers assayed in ovo. With recently isolated strains such as :B/Hong Kong/5/72 and Gifu/2/73, the PFU/EIDso ratios were as high as 100 to 400. MDCK cells have been successfully employed for primary isolation of influenza B viruses from throat washings of patients by direct plaquing. Introduetion

Biological, biochemical and genetic studies of influenza B viruses have been largely hampered b y the lack of a suitable cell system which supports productive virus growth and efficient plaque formation. Although several cells have been proposed to be suited for plaque assay of influenza B viruses (Lehmann-Grube, 1963; Mizutani and Nagase, 1967; Babiker and Rott, 1968; Beare and Kcast, 1974), technical difficulties as well as poor reproducibility have limited their usefulness. Gaush and Smith (1968) described the plaque formation with several types A and 13 influenza viruses in an established line of canine kidney cells (MDCK). I n this communication, it will be shown t h a t MDCK cells are universally susceptible to strains of influenza B virus, and a wide range of the viruses form plaques with a high efficiency without prior adaptation. I t will also be shown t h a t the cells can be employed for p r i m a r y isolation of influenza B virus b y direct plaquing. Materials and Methods Viruses. Influenza B viruses included in the present study are Lee/40, Great Lake/54 (GL/54), Taiwan/62, Massachusetts/I/71 (Mass/71), Hong Kong/5/72 (HK/72) and Gifu/2/73 (Gifu/73). All strains were propagated in 1 i - d a y chick embryos and infected allantoic fluids were stored at --80 ~ C. Cell Culture. MGDK cells were kindly given to us b y Dr. R. Krug, Memorial Sloan-Kettering Cancer Center, New York, and were grown in Earle-based Eagle's MEM containing 2.2 mg/ml of NaHCOs and 10~ fetal calf serum. Fluid maintenance medium after virus inoculation was Eagle's MEM containing twice as much glucose, four times as much vitamins, 2.2 mg/ml NaHC03, and 0.20/0 bovine serum albumin. Plaquin9 Technique. MDCK cell monolayers were inoculated with 0.1 ml of virus appropriately diluted in PBS supplemented with 0.20/o bovine serum albumin (PBS-I-BA). After 30 rain at 34 ~ C, cultures received 5 ml of agar overlay medium

24

K. Tobita.

which consisted of Eagle's MEM containing twice as much glucose, four times as much vitamins, 2.2 mg/ml NaHC03, 100 [zg/ml of DEAE-dextran, 0.2o/o bovine serum albumin, and 0.6o/o Ionagar No. 2. After 3 days at 34 ~ C in a CO 2 incubator, an equal amount of second agar overlay medium containing 0.003O/o neutral red was added and the plaques were counted 6 hrs later. Titration in Fertile Eggs. Serial 10-fold dilutions of the virus were inoculated into the allontoic cavities of l l - d a y fertile hen's eggs which were incubated at 35 ~ C for 40--48 hrs. Harvested allantoic fluids were tested for virus hemagglutinin with 0.5O/o fowl red blood cells. Virus titers were expressed in 50O/o egginfecting doses (EIDs0) per ml. One-Step Growth Experiment. MDCK cell monolayers were inoculated with 10 plaque-forming units (PFU) of the virus per cell contained in 0.1 ml. After 30 min at 34 ~ C, the infected cultures received 3 ml of fluid maintenance medium and were incubated at 34 ~ C. At 2.5 hrs post-infection, unadsorbed virus was neutralized by homolog ousantiserum. Culture fluids containing scraped-off cells were harvested at 2 hrs' intervals, subjected to 3 cycles of rapid freezing and thawing, clarified by low speed centrifugation and assayed for infectivity in MDCK cells. Clinical Specimens. Clinical specimens were throat washings collected from patients during influenza epidemics, which had been shown to be positive for influenza B virus when inoculated into fertile hen's eggs via either amniotic or chorioallantoic route. Of nine such samples employed in the present study, 4 were positive on primary egg inoculation. Five were negative on first passage in ovo, but the virus was detected after an additional blind amniotic passage had been performed. Samples were directly plated on MDCK cell monolayers and plaques were allowed to develop according to the method described above.

Results

Plaque Formation by Influenza B Viruses in MDCK Cells. All influenza B viruses tested formed well-defined clear plaques in MDCK cells. There were no significant strain-to-strain differences in plaque type, although HK/72 plaques were smaller. One plaque contained 1.1--8.2 x 107 P F U of infectious virus. Representative plaques are shown in Fig. 1. E//iciency o/Plaquing. Parallel titrations of influenza B viruses showed that P F U titers in MDCK ceils exceeded EIDa0 titers (Table 1). For older strains which have a history of multiple passages in eggs, the PFU/EIDs0 ratio was below 10, but with more recently isolated strains, i.e. HK/72 and Gifu/73, this value ranged from 100--400. One Step Growth Curves. Fig. 2 shows one step growth curve of some influenza B virus strains. All viruses grew productively in MDCK cells and induced typical CPE. A single cycle of virus growth required 10 hrs. Virus yield was approximately 25 PFU/cell, but HK/72, which formed small plaques, yielded much less virus (3 PFU/cell). Demonstration o/ Influenza B Virus in Throat Washings o/ Patients. All four specimens which were positive for influenza B virus on primary amniotic or chorioallantoic inoculation formed plaques in MDCK cells. I n addition, five samples which were negative on primary inoculation in eggs but became positive

MDCK Cells as a Sensitive Host for influenza IJ

25

Fig. 1 a--e. Plaques of influenza viruses on MDCK cell monolayers. (a) B/Lee/40, (b) B/Taiwan/62, (c) B/Massachusetts/I/71, (d) B/Hong Kong/5/72, (e) B/Gifu/2/73. Plaques were observed on day 3 p.i. Mass/71

l O~

HK/'72

10'

2 ld a

-

lff

2 4

6

8 10

2 4

6

8 10

2 4

6

810

HOURS AFTER INFECTION

Fig.2. One step growth curves of influenza viruses B/Lee/40 (l,ee/40), Massachusetts/l/71 (Mass/71) and Hong Kong/5/72 (HK/72) in MDCK cells. For details see the text after one blind passage (SN-2, SN-6, NS-3, NS-4, NS-5) were directly positive when inoculated into MDCK cell monolayers (Table 2). The content of the virus in such samples was up to 1.3 • 102 P F U / m l . Plaques produced by NS-5 are shown in Fig. 3. Discussion

I n agreement with the findings of Gaush and Smith (1968), an established line of canine kidney cells, MDCK, proved to be highly sensitive for plaque assay of influenza B virus. Virtually all strains of influenza B virus replicated productively

26

K. T o b i t a Table i. Parallel t i t r a t i o n s of influenza B viruses b y various a s s a y m e t h o d s

Viruses a

Titer h

Lee/40 Great Lake/54 Taiwan/62 Massachusetts/71 Hong Kong/72 Gifu/73

HA e

EID~0

PFU

3.6 3.1 3.0 3.4 2.4 3.3

8.7 9.5 7.8 8.0 5.8 5.8

9.1 9.7 8.6 9.5 8.6 7.8

PFU/EIDa ~ ratio 2.5 1.6 63 30 400 100

a Viruses are egg-grown. b log10 u n i t s per ml. c One-half per cent chicken red cells were used. d P l a q u e - f o r m i n g units in M D C K cells.

Table 2. D e m o n s t r a t i o n i n ovo a n d in M D C K cells for influenza B virus in t h r o a t w a s h i n g s from patients Sample a

NY-6 SN-2 SN-4 SN-6 KB-4 NS-3 NS-4 NS-5 NS-6

In MDCK

I n ovo

Amniotic

Chorioallantoic

g_ b _ b -}-~: ---g-

_1_ _ --------

§ -}~: g--}+ g-

a T h r o a t w a s h i n g s were all positive for influenza B virus w h e n inoculated into fertile eggs via either amniotic or chorioallantoic route. Some of t h e m contained u n d e t e c t a b l e level of infections virus on p r i m a r y passage a n d an additional blind amniotic passage was required before the virus was d e m o n s t r a t e d . b Presence of absence of detectable virus on p r i m a r y passage.

Fig.3. P l a q u e s p r o d u c e d in M D C K cells b y a t h r o a t washing, NS-5. P l a q u e s were o b s e r v e d on d a y 3 p.i.

MDCK Cells as a Sensitive Host for Influenza B

27

in the cells, induced typical CPE, and formed well-defined, clear plaques. P F U titers of the viruses assayed in MDCK exceeded the titers as obtained in ovo, and notably, P F U titers of the more recently isolated strains were 100--400 timcs higher than EIDs0 titers. A single cycle of virus synthesis after high multiplicity of infection required 1 0 h r s in MDCK cells (Fig. 2), and the virus yield was ca. 25 PFU/cell, but Hong Kong/5/72, which formed small plaques, produced virus in much reduced a m o u n t (3 PFU/cell). Several host cell systems have been reported in thc past to bc suitable for plaque assay of influenza B viruses (Lehmann-Grube, 1963: Mizutani and Nagasc, 1967; Babiker and Rott, 1968; Beare and Keast, 1974). I tested some of these systems, including p r i m a r y chick kidney cells, p r i m a r y chick fibroblasts obtained from 5-day-old embryos, and p r i m a r y 10-day-old-chick e m b r y o fil)roblasts in combination with incorporation of trypsin in agar overlay (Tobita and Kilbournc, 1974). None was superior to MDCK cells both with respect to sensitivity and reproducibility (data not shown). Amniotic inoculation is still the most sensitive method for isolation of influenza viruses from a clinical specimen. Unique susceptibility of MDCK cells to a wide v a r i e t y of influenza B viruses with different passage histories has led to employ the cells for p r i m a r y virus isolation from t h r o a t washings of patients. All samples positive for influenza B virus on p r i m a r y in ovo inoculation were also positive in MDCK cells. Furthermore, five additional specimens which contained so little infectious virus as to be detectable only after blind passages directly formed plaques on MDCK cell monolayers (Table 2). MDCK cells appeared to be a t least as sensitive a host for isolation of influenza B virus as developing chick embryos, although precise comparison has not been made in the present study. Acknowledgements. I would like to thank Dr. Furuyama, Hygienic Laboratory of Okayama Prefecture, for supplement of clinical specimens.

Re~erenees Babiker, H. A., Rott, R.: Plaque formation by influenza viruses in monolayers of chicken kidney cells. J. gen. Virol. 3, 285--287 (1968) Beare, A. S., Keast, K. A. : Influenza virus plaque formation in different species of cell monolayers. J. gen. Virol. 22, 347--354 (1974) Gaush, C. R., Smith, T. F.: Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl. Microbiol. 16, 588--594 (1964) Lehmann-Grube, F.: A sensitive plaque assay for influenza viruses. Virology 21, 520 522 (1963) Mizutani, H., Nagase, M.: Plaque formation with influenza viruses in explants of chick embryo fibroblasts from embryos of different ages. Arch. ges. Virusforsch. 20, 278 282 (1967) Tobita, K., Kilbourne, E. D.: Genetic recombination for antigenic markers of antigenically different strains of influenza B virus. J. Virol. 18, 347 352 (1974) Dr. K. Tobita, M.D. Department of Microbiology Institute of Public Health 4-6-1, Shirokane-dai Minato-ku, Tokyo 108 Japan