J. gen. Virol. (199), 70, 341-345. Printed in Great Britain 341 Key words: influenza C virus/interferon/persistent infection Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization By F. YAMAMOTO GOSHIMA* AND K. MAENO Laboratory of Virology, Research Institute for Disease Mechanism and Control Nagoya University School of Medicine, Showa-ku, Nagoya, Japan (Accepted 17 August 199) SUMMARY Persistent influenza C virus infection was readily initiated in Madin-Darby canine kidney (MDCK) cells at low m.o.i, and has been maintained for over 1 year. The persistently infected (p.i.) cultures were characterized by the following properties: virus infection was limited to a minority of cells, small amounts of infectious virus were produced together with low levels of interferon (IFN) and the cultures were resistant to superinfection by homologous virus and vesicular stomatitis virus, but not by influenza A and B viruses. These properties fluctuated cyclically with passage of the p.i. culture. When p.i. cultures were cured by cultivation in the presence of antiserum, the cultures lost their IFN-producing activity and became as susceptible to homologous virus as normal MDCK cell culture. The results suggest that persistent influenza C virus infection may be regulated by endogenously produced IFN. Under the condition of high m.o.i, a persistent influenza C virus infection could not be initiated in MDCK cells due to the development of cytopathic effects. Human influenza is predominantly an upper respiratory tract infection with some involvement of the lung. The optimal period for isolating influenza A virus from the upper respiratory tract is during the first 4 h of disease (Hoyle, 196), suggesting that the period of association between the virus and its host cell is relatively short and that the virus is eliminated rapidly. Outbreaks of type C influenza are usually detected retrospectively by serological means during the investigation of an epidemic caused by another type of influenza and little is known about the duration of virus shedding, mode of transmission and natural viral persistence. The survey of an outbreak of type C influenza in a children's home by Katagiri et al (193) demonstrated that the virus is shed from infected individuals for a long period (more than 9 days) and reinfection may occur soon after primary infection. Although influenza virus infections are generally cytopathic in cell cultures that support their replication, the study by Katagiri et al. (193) has led us to investigate whether the interaction of influenza C virus with cultured cells results in the establishment of a long-term infection. Madin-Darby canine kidney (MDCK) cells were grown at 35 C in Eagle's minimum essential medium (MEM) supplemented with 7~o bovine serum, 10~o tryptose phosphate broth (Difco), penicillin (100units/ml) and streptomycin (100~tg/ml) (growth medium). The C/Miyagi/77 strain of influenza virus, kindly supplied by Dr K. Tobita, Department of Virology Jichi Medical School, Japan, was grown in MDCK cell cultures maintained with MEM containing 0.1 ~ bovine serum albumin (BSA, Sigma) and acetyltrypsin (2.5 vtg/ml, Sigma) and used as stock virus. For the infectivity assay serial 10-fold dilutions of virus in MEM containing 0.1 ~o BSA (maintenance medium) were adsorbed to each of four monolayer cultures of MDCK cells in 24-well plastic plates (Falcon) for 1 h (0-1 ml per monolayer), after which 0.4 ml of maintenance medium containing acetyltrypsin (2.5 Ixg/ml) was added to each well. After a 4 day 0000-9035 199 SGM
342 Short communication IFN titre 4 4 2 <~1 4 4 3<1 <1 r~ > 3 L_ 25 Passage no. 50 75 Fig. 1. Infectivity and IFN titres in p.i. culture fluids. P.i. culture fluids were serially diluted 10-fold in maintenance medium with (open bar) or without (closed bar) acetyltrypsin and each dilution (0-1 ml) was inoculated onto each of four MDCK cell cultures in 24-well plastic plates. One h later, 0.4 ml of maintenance medium containing acetyltrypsin was added to each culture, followed by further incubation at 34 C for 4 days. The culture fluids were tested for HA activity and infectivity titres were calculated according to the method of Reed & Muench. For the IFN assay, the culture fluids were centrifuged at 54000g for 1 h to remove virus particles, the supernatants were dialysed at 4 C overnight against saline buffered with glycine-hcl (0.01 M) at ph 3.0 and the ph was shifted back to ph 7-2 by further dialysis against MEM. Confluent monolayers of MDCK cells were treated overnight with appropriate serially diluted samples and then challenged by 100 p.f.u, of VSV. The IFN titres are expressed as the reciprocal of the highest dilution at which plaque number was reduced to 50 % or less. incubation at 34 C culture fluids were tested for haemagglutination (HA) activity and infectivity titres were calculated according to the method of Reed and Muench, expressed as TCIDso. Confluent monolayers of MDCK cells (2 106) were infected with influenza C virus at an m.o.i, of 10-2 TCIDso/cell, incubated at 34 C in maintenance medium in the presence or absence of acetyltrypsin (2-5 ktg/ml) and examined for virus yields and cytopathic effects. In the presence of acetyltrypsin extracellular infectivity titres increased quickly and reached a maximum level (107"7 TCIDs0/ml) 72 h after infection, at which time a large number of cells had already detached from the plate. Uninfected cultures of cells remained unchanged. Immunofluorescence staining with rabbit antiserum against homologous influenza C virus showed that all cells were antigen-positive 4 h after infection. On the other hand, the cultures without acetyltrypsin also released progeny virus, but the maximum yield was approximately 1/10 of that in the presence of acetyltrypsin. At 96 h after infection almost all cells still remained attached to the plate and were viable, as determined by trypan blue dye exclusion, and about 20% of the cells were antigen-positive. We examined whether infection of MDCK cells by influenza C virus in the absence of acetyltrypsin led to persistent infection. A confluent monolayer of 4 x 107 MDCK cells was infected with influenza C virus at 10-2 TCIDso/cell and incubated in growth medium. At 96 h after infection, the cells were trypsinized, suspended in growth medium and subcultured at 34 C with a split ratio of 1 : 3. The cultures exhibited mild cytopathology but grew to confluence in 5 days. As shown in Fig. 1, up to the ninth passage, 105.7 to 106.3 TCIDs0/ml were detected in each culture fluid and 30 to 40% of the cells were viral antigen-positive. The virus yield,
Short communication 343 (a) Fig. 2. Presence of viral antigens in p.i. MDCK cells. Cultures grown on coverslips were fixed in acetone and examinedfor viral antigens by direct immunofluorescenceusing rabbit antiserum directed against homologousinfluenza C virus. (a) Uninfected MDCK cells; (b) p.i. MDCK cells at passage 21. Bar marker represents 50 ktm. percentage of viral antigen-positive cells and extent of c.p.e, declined with increasing passage number. At passage 1l, no c.p.e, was observed and the cells resembled normal MDCK cells morphologically and in growth rate (data not shown). Thus, development of confluent cell monolayers became possible by subculturing with a split ratio of 1:4 every 4 days, as with normal MDCK cells and the cultures have been maintained for over 1 year. As shown in Fig. 1, after passage 15 extracellular virus was detected in smaller amounts than at the end of the first nine passages and 1 to l0 ~ of cells in the cultures exhibited virus-specific fluorescence (Fig. 2). It should be noted that at passages 30 to 32 and 45 to 50, virus yields were not detectable and viral antigen-positive cells were as low as 0.1 ~ and that mild cytopathology was seen in the cultures at passages 37 to 40 and 56 to 60, which produced virus at relatively high levels (Fig. 1). When serial dilutions of persistently infected (p.i.) culture fluids for the infectivity assay were made in the absence of acetyltrypsin, the titres were detected in reduced amounts (closed bar in Fig. 1). MDCK cells (2 x 106) were infected with influenza C virus at an m.o.i, of 5 TCIDso/cell. At 24 h after infection extracellular virus titres reached a plateau and all cells exhibited virusspecific fluorescence, but c.p.e, was not observed. Following further incubation c.p.e, appeared and 0 ~ of cells in the culture came off the plate 4 days after infection. The same result was also obtained when the infected cells were incubated with growth medium. All cells died in the following 2 weeks and the establishment of persistent influenza C virus infection was unsuccessful. P.i. cultures were passaged serially four times in growth medium containing rabbit antiserum directed against influenza C virus (final dilution 1:100), followed by one additional passage in the absence of antiserum. Both antigen-positive cells in p.i. culture and extraceuular virus became undetectable (Table 1), suggesting that persistent infection is maintained by horizontal transmission of the virus released from infected cells. However, three further passages of the cells in the absence of antiserum resulted in the appearance of viral antigen in a small proportion of cells, accompanied by low levels of virus yield.
344 Short communication Table 1. Production of superinfecting homologous and heterologous viruses in persistently infected cultures Virus yield (HA units) Trypsin (-)* Trypsin (+)* Superinfecting ~ x Cells virus (m.o.i.) 24 h p.i. 4 h p.i. 24 h p.i. 4 h p.i. MDCK Original influenza C virus (1) 12 256 12 256 p.i. - 39t Original influenza C virus (1) Mock 2 16 32 16 Cured:~ Original influenza C virus (1) 16 64 32 12 p.i. - 39 Mock <2 <2 <2 <2 MDCK p.i. - 39I" Influenza B virus (0-1) Influenza B virus (0-1) MDCK VSV (0-1) p.i. - 39t MDCK VSV (0.1) Influenza A virus (1) p.i. - 27t Influenza A virus (1) Mock 24 h virus yield (p.f.u./ml) 7.5 x 107 7-2 x 107 1-6 x 10 a 2.2 x 106 1.9 x 106 3.7 x 106 1 x 104 (TCIDs0/ml) * Cultures were incubated in the presence or absence of acetyltrypsin after superinfection. t Passage number of p.i. culture. Persistently infected cells were subcultured in the presence of homologous rabbit antiserum at passages from 35 to 3, followed by further subcultivation in the absence of antiserum. Several lines of evidence have suggested the involvement of interferon (IFN) and defective interfering (DI) particles in the initiation and maintenance of p.i. (Youngner & Preble, 190). To test this possibility, the fluids were collected from confluent p.i. cultures and examined for IFN by plaque reduction assay with vesicular stomatitis virus (VSV). Virus-yielding p.i. cultures produced low levels of IFN, btat non-yielding p.i. cultures (passages from 30 to32 and from 45 to 50) did not (Fig. 1). When p.i. cultures were cured by treatment with antiserum, IFN in the culture fluid became undetectable. To assess the involvement of DI particles in the maintenance of p.i. culture, confluent monolayers of MDCK cells (2 x 106) were infected with mixtures of several dilutions of virus stock of influenza C virus (0-1 ml) and an equal volume of p.i. culture fluid at passage 35. Any mixedly infected culture produced infectious virus at the level found in the cultures infected with the corresponding virus stock alone (results not shown). This was taken to suggest that DI particles, if any, are not present in the p.i. culture fluid in amounts sufficient to interfere with the replication of stock virus. We examined the susceptibility of p.i. cultures to superinfection by homologous and heterologous viruses. Confluent monolayers of p.i. cells (2 x 106) were superinfected with virus stock of influenza C virus at an m.o.i, of I TCIDso/cell. As shown in Table 1, extracellular HA titres appeared to be similar to those in mock-superinfected p.i. culture and were significantly lower than HA titres in the supernatants of infected, normal MDCK cells. Similar results were also obtained when acetyltrypsin was added to the cultures. When p.i. cells were superinfected with VSV at an m.o.i, of 0.1 p.f.u./cell, the virus yield was approximately 1/50 of that in normal MDCK cells. In contrast, the growth rates of influenza A/WSN/34 and B/Kanagawa/73 in p.i. cells were comparable to that in normal MDCK cells. P.i. cultures cured by treatment with antiserum were examined for their susceptibility to superinfection by homologous virus and as shown in Table 1, these cultures produced the virus at higher levels than the p.i. cultures did. While the present study was in progress, Camilleri & Maassab (19) reported a persistent infection of MDCK cells by influenza C virus, which was established by cultivation of cells that survived the crisis period of to 12 months after high-multiplicity infection. We have found that a persistent influenza C virus infection is readily initiated in MDCK cells at low m.o.i. This contrasted with the infection by influenza A/WSN/34 or B/Kanagawa/73 which was cytopathic
Short communication 345 in MDCK cell culture at any m.o.i. (data not shown). Establishment of persistent infections by influenza A virus require the presence of a high proportion of DI particles in the virus inoculum (De & Nayak, 190; Frielle et al., 194). Apparently virus-yielding p.i. cultures produced low levels of IFN and were resistant to superinfection, not only by homologous virus but also by heterologous VSV, but the cultures lost these biological activities when cured by antiserum treatment. These findings suggest the involvement of endogenously produced IFN in the maintenance of persistent infection. A low level production of IFN in p.i. cultures will allow the maintenance of persistent infection, whereas high levels of IFN production might lead to recovery of the p.i. culture from persistent infection. Selection of a less cytocidal, temperature-sensitive mutant, but not the presence of DI virus or IFN, is critical in the maintenance of persistent infections by influenza A virus (De & Nayak, 190; Frielle et al. 194). Whether this has also been the case with the persistent infection reported by Camilleri & Maassab (19) is unclear, but in our system the p.i. virus does not exhibit temperature sensitivity (data not shown). Our experiments have shown that influenza viruses A/WSN/34 and B/Kanagawa/73 were less susceptible to the antiviral action of IFN than influenza C virus and VSV. Influenza A/WSN/34 is relatively resistant to the antiviral action of IFN (Krug et al., 195; Katze et al., 19). If superinfection occurred in influenza C virus-infected individuals, superinfecting influenza A and B viruses could be isolated preferentially and type C influenza would be found retrospectively only by serological means. One characteristic feature of type C influenza is prolonged virus shedding, compared with other types of influenza (Katagiri et al., 193). This implies that naturally occurring influenza C virus infection may not be so rapidly self-limiting. Reinfection with influenza C virus is common despite demonstrable antibodies in subjects (Homma, 195). Our study suggests that prolonged virus shedding might be due to the presence of low levels of IFN produced during the course of infection, which might control the spread of the infection in the respiratory tract. We thank E. Iwata and T. Tsuruguchi for their able technical assistance and Y. Kimura for his valuable discussion. This work was supported by research grants from the Ministry of Education, Science and Culture of Japan and from the Yakult Science Foundation. REFERENCES C~ILLERI, J. J. & ~, ~. F. (19). Characteristics of a persistent infection in Madin-Darby canine kidney cells with influenza C virus. Intervirology 29, 17-14. DE, B. D. & NAYAK, D. I'. (190). Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells. Journal of Virology 36, 47-59. I~aF.LLE, D. W., HUANG, D. D. & OtmG~rER, J. S. (194). Persistent infection with influenza A virus, evolution of virus mutants. Virology 13, 103-117. HOMMA, M. (195). Epidemiological characteristics of type C influenza. Journal of Cellular Biochemistry, supplement 9C, 27. HOYLE, L. (196). The influenza viruses. In Virology Monographs, vol. 4. Vienna: Springer-Verlag. KATAGIRI, S., OmZUMI, A. & HOMMA, M. (193). An outbreak of type C influenza in a children's home. Journal of Infectious Diseases 14, 51-56. KATZE, M. G., TOMITA, J., BLACK, T., KRUG, R. M., SAFER, B. & HOVANESSIAN, A. (19). Influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 6000 Mr protein kinase. Journal of Virology 62, 3710-3717. KRUG, R. M., SHAW, M., BRONL B., SHAPIRO, G. & HALLER, O, (195). Inhibition of influenza viral mrna synthesis in cells expressing the interferon-induced MX gene product. Journal of Virology 56, 201-206. YOUNGNER, J. S. & PREBLE, O. T. (190). Viral persistence: evolution of viral populations. In Comprehensive Virology, vol. 16, pp. 73-135. Edited by H. Fraenkel-Conrat & R. R. Wagner. New York & London: Plenum Press. (Received 24 April 199)