Susceptibility of different cell lines to Avian and Swine Influenza viruses

Journal of Virological Methods 185 (2012) 82–88

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

Susceptibility of different cell lines to Avian and Swine Influenza viruses Tina Lombardo ∗ , Silvia Dotti, Sabrina Renzi, Maura Ferrari Cell Culture Centre, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Via Bianchi 9, 25124 Brescia, Italy

a b s t r a c t Article history: Received 23 December 2011 Received in revised form 1 June 2012 Accepted 12 June 2012 Available online 20 June 2012 Keywords: Avian Influenza Swine Influenza Cell culture Avian embryo Isolation Propagation

Influenza outbreaks are widespread in swine and avian populations. Disease control is jeopardized by the extreme antigenic variability of virus strains. Primary isolation of Influenza virus is performed using embryonated chicken eggs (ECE), but alternatives to ECE are badly needed. Although various cultured cells have been used for propagating Influenza A viruses, few types of cells can efficiently support virus replication. One of the most commonly cell lines used in order to isolate Influenza A virus, is represented by the Madin Darby Canine Kidney (MDCK) cell line, but cells derived from primary swine organs (kidney, testicle, lung and trachea) can also be employed. The aim of this study was the evaluation of NSK, MDCK, UMNSAH/DF1 cell lines suitability, compared to ECE for isolation and propagation of Avian and Swine virus subtypes. The results indicated both NSK and MDCK could provide an appropriate substrate for cultivating either Avian (AIV) or Swine (SIV) Influenza virus strains, especially for high pathogenicity Avian Influenza ones. Furthermore, NSK appeared more susceptible than MDCK cells for primary isolation of AIV. In contrast, UMNSAH/DF1 cell line seemed to be less permissive to support Avian virus growth. Furthermore, no SIV replication was detected except for one subtype. Additionally, the results of this study indicated that not all virus strains seemed to adapt with the same efficiency to the different cell lines. On the contrary, chicken embryos were shown to be the most suitable biological system for AIV isolation. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Influenza A viruses are enveloped, single-stranded RNA viruses, which belong to the Orthomyxoviridae family. Based on the antigenicity of their surface glycoproteins represented by hemagglutinin and neuraminidase molecules, they are classified currently into 16 hemagglutinin (H1–H16) and 9 neuraminidase (N1–N9) subtypes (Fouchier et al., 2005). Their reassortment gives origin to several subtypes with varying pathogenicity degrees. High Pathogenicity Avian Influenza (HPAI) viruses evolve from low pathogenic precursors specifying HA subtypes H5 or H7; this process is characterized by the acquisition of a polybasic HA cleavage site. Functionally, virus infection of host cells is mediated by the binding between HA and sialic acid-containing receptors (Olsen et al., 2006). The HPAI form, in contrast to Low Pathogenic (LPAI) one, can be responsible for severe outbreaks with extraordinary mortality causing severe losses to the poultry industry. In contrast, there are fewer hemagglutinin and neuraminidase types circulating in mammals, particularly in pigs, in which H1N1, H3N2 and H1N2 are the three main currently widespread subtypes (Massin et al., 2010; Olsen et al., 2006). All the three viral subtypes (H1N1, H3N2,

∗ Corresponding author. Tel.: +39 0302290248; fax: +39 0302290392. E-mail address: [email protected] (T. Lombardo). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.06.008

H1N2) have been associated with disease and there are no indications of differences in virulence among subtypes (Van Reeth, 2007). A common feature of influenza viruses from different species and subtypes, is their capacity to replicate in embryonated chicken eggs (ECE), that represent the gold standard for virus isolation and replication, also used for vaccine manufacturing (Yeolekar and Dhere, 2012). ECE can support the growth of a broad range of Influenza viruses, although many field isolates do not readily grow. Also, availability of a sufficient number of high-quality ECE is a considerable limitation to their use. Furthermore, facts show that serial propagation of Influenza viruses in ECE can cause mutations in the hemagglutinin glycoprotein, leading to egg-adapted variants that may be antigenically different from the original field isolates and with variations in their glycosylation patterns (Schild et al., 1983; Audsley and Tannock, 2008). This may result in vaccine failure (poor inhibition of field virus replication and transmission by infected birds). In a scenario of a pandemic influenza outbreak, egg propagation system may not be adequate to face an emergency situation. In fact, it would be necessary to arrange a high number of vaccine doses in a limited period of time. For this reason, it seems to be useful to improve alternative diagnostic/productive tools, different from embryonated eggs (Liu et al., 2012). Another important aspect to consider working with ECE, is represented by the limited flexibility for increased vaccine

T. Lombardo et al. / Journal of Virological Methods 185 (2012) 82–88

manufacture, the possible abrupt interruption of the supply chain due to disease in layer flocks, the risks concerning sterility during processing of infected allantoic fluids and the poor growth of some reassortant vaccine strains in embryonated eggs (George et al., 2010; Moresco et al., 2010). At present, vaccine viruses are propagated in ECE, for this reason it would be important to consider the risk of egg-derived vaccines for individuals with allergies to ovalbumin and other egg proteins. Although the purification process is effective, the protein of the chicken embryo cannot be completely removed and may cause allergic reactions in vaccinated population. In order to overcome these problems, many attempts have been made to use cell culture technology as a suitable alternative for influenza virus isolation and vaccine production on a large scale, as cell cultures can be manipulated easily with reduced time expenditure (Ferrari et al., 2003; Lee et al., 2008; Chiapponi et al., 2010). In particular, use of continuous cell lines for influenza virus growth offers several advantages, including the opportunity to use fully characterized and standardized cells (Tree et al., 2001). Several types of primary, diploid and established cell lines are known to allow Influenza virus replication (Govorkova et al., 1996; Li et al., 2009); although the permissibility of most cell lines to Influenza virus infection is low and titres are lower than those detected by ECE (Hussain et al., 2010). Among them, Madin Darby Canine Kidney (MDCK) cell line was found to enable efficient viral growth and to support virus isolation from pathological specimens (Tobita et al., 1975; Meguro et al., 1979; Root et al., 1984; Voeten et al., 1999; Liu et al., 2009). Similar findings were obtained with African Green Monkey Kidney (VERO) cell line, which is now considered to be suitable for virus isolation from clinical specimens and growth of vaccine viruses (Kaverin et al., 1995; Govorkova et al., 1996; Kistner et al., 2007; Genzel et al., 2010). Other cell lines were investigated for permissiveness to Avian Influenza viruses (AIV), such as Embryonic Swine Kidney (ESK), Hamster Lung (HmLu-1) and Monkey Kidney (JTC-12) (Sugimura et al., 2000). MDCK cell line has been the most consistently used for culturing and propagating AIV (Moresco et al., 2010). Regarding Swine Influenza virus (SIV), chicken embryo is the most suitable biological method for virus isolation, on the other hand some studies have demonstrated the usefulness of different cell cultures as an alternative to ECE (Clavijo et al., 2002; Chiapponi et al., 2010). In these cases, authors underlined the possible link between viral hemagglutination and isolation rate on different substrates (MDCK, CACO-2 and ECE). In Chiapponi et al. (2010), it was studied the comparison among CACO-2, MDCK and ECE, in order to verify the real suitability of these biological tools during in vitro SIV isolation. The increased sensitivity of CACO-2 line than MDCK and ECE to isolate H1N1 and H1N2 subtypes. Otherwise, H3N2 virus detection was highest using ECE has been demonstrated. Clavijo et al. (2002) set forth the high variability of Swine Influenza virus; this aspect suggested, as the most efficacious diagnostic tool, the twin course use of both ECE and MDCK for the primary SIV isolation. In the present study, mammalian and avian cell lines were compared for their ability to support either virus isolation from infected target specimens, or growth of AIV and SIV subtypes; the aim was the selection of several cell lines that could be used as alternative to ECE. Furthermore, the Intravenous Pathogenicity Index (IVPI) was performed in ECE and cell cultures at the first and tenth passage of each Avian virus subtype, in order to evaluate the changes that could occur during serial cultivation. This first phase will be improved by further investigation regarding possible genomic virus changes during serial passages in cell cultures. This step is undertaken by genetic analysis.

83

2. Materials and methods 2.1. Cells The selected cell lines were: Newborn Swine Kidney (NSK, BS CL 177), Madin Darby Canine Kidney (MDCK, BS CL 64) and Chicken Embryo Fibroblasts (UMNSAH/DF1, BS CL 197). These cell lines were registered in the cell culture bank of IZSLER, Brescia, Italy. Both NSK and MDCK were propagated in Minimal Essential Medium (E-MEM, Sigma–Aldrich, Milano, Italy) free of antibiotics and supplemented with 10% (v/v) foetal bovine serum (FBS, Mascia Brunelli S.p.A., Milano, Italy) and l-glutamine (4 mM/l, Sigma–Aldrich, Milano, Italy). UMNSAH/DF1 were cultivated in Dulbecco’s modified Eagle medium (D-MEM, Sigma–Aldrich, Milano, Italy) supplemented with 10% (v/v) FBS. All cell lines were grown and maintained at 37 ◦ C in 5% CO2 . Sub-passages were performed when cell monolayers reached confluence. 2.2. Viruses and virus titration The following subtypes of AIV were selected: H5N1 LPAI A/Mallard/Italy/3401/05; H5N2 HPAI A/Chicken/Italy/8/A98; H7N1 HPAI A/Turkey/Italy/4580/99; H7N3 LPAI A/Turkey/Italy/2962/V03; they were field isolates. Finally, H9N2 LPAI A/Turkey Wisconsin/66 was a reference strain. All the samples were provided by Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy. SIV strains were: H1N1 A/Swine/Italy/25674/09, H1N1 A/Swine/Italy/186798/09, pandemic H1N1 A/Swine/Italy/290271/09, H1N2 A/Swine/BS2156, and H3N2 A/Swine/Oedenrode/1/96. They were field strains and provided by virus diagnosis laboratories (Parma and Brescia) of IZSLER. All samples were inoculated in ECE at least 2 times in order to isolate the subtypes. Regarding H9N2 LPAI A/Turkey/Wisconsin/66, the previous passages were unknown. Each virus was propagated in 10-day old, Specific Pathogen Free (SPF) embryos at 37 ◦ C over 3 days. Allantoic fluids were collected after centrifugation at 1540 × g for 10 min and their hemagglutinating titres were evaluated using chicken red blood cells (RBCs) (0.5%) in phosphate buffer solution (PBS). The infectious titres were measured following inoculation of 100 ␮l of serial 10-fold dilutions (10−1 to 10−7 ) into the allantoic cavity of three fertilized eggs. They were incubated for three days at 37 ◦ C, the allantoic fluid was collected and virus growth was confirmed by a hemagglutination (HA) test. Embryo vitality was checked daily, in order to quickly verify the death, especially for HPAI strains. In case of death, the procedure was performed (as aforementioned) immediately. Infectious titres were calculated as described by Reed and Münch (1938) and expressed as Embryo Infectious Dose 50% (EID50 ) per 0.1 ml. 2.3. Isolation of AIV from target tissues Twenty, 6-week-old susceptible SPF White Leghorn chickens, subdivided into 4 groups of 5 animals each, were infected by the ocular-nasal (o.n.) route with 100 ␮l of the H5N1 LPAI A/Mallard/Italy/3401/05, H5N2 HPAI A/Chicken/Italy/8/A98; H7N1 HPAI A/Turkey/Italy/4580/99, and H7N3 LPAI A/Turkey/Italy/2962/V03 viruses, with a EID50 of 103 /0.1 ml. These experimental infections were performed at Istituto Zooprofilattico, Padua, Italy. The infections were carried out in isolation facilities and all the procedures were performed according to the local guidelines on animal welfare and all groups were housed in cages set in isolators with water and food ad libitum. Birds were observed daily for clinical signs. At the time of death, after H5N2 and H7N1 HPAI virus infections, target tissues (intestine,

84

T. Lombardo et al. / Journal of Virological Methods 185 (2012) 82–88

lung and brain) were sterile collected, homogenized and the supernatants were used for virus isolation in either chicken embryos or MDCK, NSK and UMNSAH/DF1 cell lines. The same samples were collected 3 days post-infection from the animal groups without any clinical sign, infected with the LPAI viruses H5N1 and H7N3. In total 15 target tissues for each group were collected, homogenized 1:10 in PBS and supplemented with antibiotics (penicillin 5000 IU/ml, streptomycin 200 ␮g/ml, amphotericin B 10 ␮g/ml, Squibb, Roma, Italy). After an overnight incubation at 4 ◦ C, the supernatants were separated by centrifugation at 6160 × g for 30 min at 4 ◦ C, filtered through 0.45 ␮m membranes and used for virus isolation in either chicken embryos or selected cell lines. A 100 ␮l of each 10-fold virus dilution was inoculated in the allantoic cavity of 10-day-old ECE. After 72 h of incubation at 37 ◦ C, allantoic fluid was tested for hemagglutinating activity with 0.5% chicken RBCs, in order to evaluate virus titre. Three blind passages were made, when negative hemagglutinating response was observed.

2.4. Serial cultivation and titration of AIV grown in cell lines and chicken embryos Cell lines (MDCK, NSK, UMNSAH/DF1) and chicken embryos were inoculated with field and reference (as above mentioned) AIV subtypes: H5N1 LPAI A/Mallard/Italy/3401/05, H5N2 HPAI A/Chicken/Italy/8/A98, H7N1 HPAI A/Turkey/Italy/4580/99, H7N3 LPAI A/Turkey/Italy/2962/V03 and H9N2 LPAI A/Turkey/Wisconsin/66. All viruses were 10 times serially passaged. Cell samples were seeded into 25-cm2 flasks and incubated at 37 ◦ C in 5% CO2 for 48 h. Culture medium was removed, cell monolayers were rinsed once with buffered salt solution (SAL A) and 1 ml of each 1:10-diluted virus was used for infection. Cells were incubated at 37 ◦ C for 1 h, 9 ml of culture medium were added, supplemented with trypsin (MST, 5 ␮g/ml) and FBS (0.5%, v/v final). Cells were again incubated under the same conditions and virus replication was evaluated on the basis of daily observed cytopathic effect (CPE). When CPE reached 80% or failed to occur within 5 days of incubation, infected cell cultures were frozen at −40 ◦ C, thawed at 18 ◦ C, centrifuged at 1540 × g for 20 min at 4 ◦ C and the harvested supernatants were used for either a HA test or following passage. Virus replication was monitored by HA test, carried out at each passage and the median TCID50 /ml was calculated at the first and the last passage by the Reed and Münch (1938) method. All viruses were propagated either in the three cell lines or in chicken embryos for 10 serial passages; finally, virus infection titres were evaluated at each passage levels. Furthermore, 10-day-old ECE were inoculated into the allantoic cavity with 100 ␮l of each 1:10 diluted virus in accordance with the above procedure. For virus titration, cells were seeded into 24-well plastic plates and incubated at 37 ◦ C in 5% CO2 for 48 h. Culture medium was removed, cell monolayers were rinsed with 1 ml SAL A and wells were inoculated with virus dilutions (from 10−1 to 10−7 ) in FBSfree culture medium supplemented with trypsin. Three wells were inoculated with each virus dilution at 100 ␮l/well. Plates were incubated at 37 ◦ C for 1 h and then supplemented with 0.9 ml MST and 0.5% (v/v) FBS. Plates were kept at 37 ◦ C in 5% CO2 for 5 days and observed daily, in order to detect CPE. Plates were then frozen at −40 ◦ C and thawed at 18 ◦ C and samples of each dilution were pooled for a HA test. Infectious titres of viruses grown in ECE at the first and tenth serial passages were determined by the Reed and Münch (1938) method.

2.5. HA assay The HA assay was performed using freshly prepared 0.5% chicken RBCs in PBS. Culture supernatants, harvested 96 h post infection, or the allantoic fluids of the inoculated eggs, were tested for HA activity, in accordance with OIE guidelines procedures (OIE, 2008). HA results were recorded and titres were reported as log2 HA units/0.1 ml. 2.6. Evaluation of Intravenous Pathogenicity Index (IVPI) Changes in viral pathogenicity after serial cultivation in cell cultures and in chicken embryos, were evaluated following intravenous (i.v.) virus inoculation in groups of ten specificpathogen-free, 6-week-old White Leghorn chickens. They were housed in HEPA-filtered isolator cages and inoculated into the ulnar vein with 0.2 ml of a 1:10 dilution of each bacteria-free Influenza virus. Animal groups were treated with the first and the last passage of each virus subtype, propagated in all cell lines and chicken embryos. After the challenge, chickens were monitored at 24-h intervals for clinical signs over 10 days. Chickens were scored: (a) sick if they displayed one clinical sign, such as depression, cyanosis of the comb or wattles, respiratory distress, diarrhoea, face/head oedema and nervous signs; (b) severely sick if they displayed two or more clinical signs. Thus, at each observation, clinical status was scored as follows: “0” clinically healthy, “1” sick, “2” severely sick, and “3” dead. The IVPI was calculated as the mean score per bird per observation over the 10-day period according to OIE guidelines (OIE, 2008). 2.7. Serial passages and titration of SIV grown in cell cultures and chicken embryos H1N1 A/Swine/Italy/25674/09, H1N1 A/Swine/Italy/186798/09, pandemic H1N1 A/Swine/Italy/290271/09, H1N2 A/Swine/BS2156, and H3N2 A/Swine/Oedenrode/1/96 SIV were serially passaged (10 times) in the aforementioned cell lines and chicken embryos, titred as reported above. 3. Results 3.1. Isolation of Avian Influenza viruses (AIV) from target tissues Four viral subtypes were isolated from three different target tissues (intestine, lung and brain) of infected chickens. Fifteen samples of each virus were tested on four different biological systems (MDCK, NSK, UMNSAH/DF1 and ECE). H5N1 LPAI A/Mallard/Italy/3401/05 subtype virus was isolated from 7 target tissues in embryonated chicken eggs (ECE) at the first passage; 2 and 3 samples were positive at the second and third passage, respectively. At the first passage, 1 sample was positive in NSK, 1 in MDCK and none in UMNSAH/DF1 cells. At the second passage, positive samples were 2 both in NSK and MDCK cells, as opposed to none in UMNSAH/DF1. At the third passage 3 samples were positive in NSK and MDCK cells, whereas no virus was found in UMNSAH/DF1 cells. H5N2 HPAI A/Chicken/Italy/8/A98 virus was isolated at the first passage only in: ECE 14 samples and 10 MDCK cells. In contrast, all samples were negative in NSK and UMNSAH/DF1 cells. At the second passage 9 samples resulted positive in NSK; no virus was isolated in UMNSAH/DF1, MDCK cells and in ECE. At the third passage 4 samples were positive in NSK, 1 in MDCK and ECE, on the contrary none in UMNSAH/DF1 cells. From infected target tissues with H7N1 HPAI A/Turkey/Italy/4580/99, 6 samples were isolated at the first passage in ECE, 1 at the second and 8 at the third passage. Only

T. Lombardo et al. / Journal of Virological Methods 185 (2012) 82–88

85

Table 1 Viral isolation in different biological systems from target tissues of infected chicken.a

Chicken embryos

Passage no.

H5N1 LPAI A/Mallard/ Italy/3401/05

1 2 3

7 2 3

Total

NSK

1 2 3

Total

MDCK

1 2 3

Total

UMNSAH/DF1 Total

1 2 3

H5N2 HPAI A/Chicken/ Italy/8/A98

H7N1 HPAI A/Turkey/ Italy/4580/99

H7N3 LPAI A/Turkey/ Italy/2962/V03

14 0 1

6 1 8

8 3 3

12

15

15

14

1 2 3

0 9 4

3 4 6

1 3 2

6

13

13

6

1 2 3

10 0 1

1 4 8

1 1 0

6

11

13

2

0 0 0

0 0 0

0 0 0

0 0 0

0

0

0

0

Values indicated the number of tissues from which virus was obtained. Viral growth was confirmed by hemagglutinating test. a Target tissue (lung, brain and intestine) were collected and homogenized (1:10 in PBS with antibiotics). After an over-night incubation at 4 ◦ C, they were centrifuged and supernatant was filtered (0.45 ␮m membrane). 100 ␮l of each 10-fold virus dilution was inoculated in embryonated chicken eggs or in cell cultures.

3 samples were positive at the first passage in NSK, 1 in MDCK and none in UMNSAH/DF1 cells. At the second passage the positive samples increased to 4 in both NSK and MDCK cells and no virus growth was observed in UMNSAH/DF1. At the third passage, 6 samples were positive in NSK, 8 in MDCK and none in UMNSAH/DF1. Finally, H7N3 LPAI A/Turkey/Italy/2962/V03 was isolated from 8 samples at the first passage in ECE, 3 at the second and third passages. Virus was isolated from only 1 sample at the first passage in NSK and MDCK and no isolation occurred in UMNSAH/DF1 cells. At the second passage, 3 viruses were isolated in NSK and only 1 in MDCK. UMNSAH/DF1 cells remained constantly negative. At the third passage, 2 positive samples were detected in NSK cells only and no viral isolation was made in the other two cell lines. UMNSAH/DF1 cells, infected with the above-mentioned viruses, remained constantly negative despite three blind passages. In contrast, the four virus subtypes were isolated either in MDCK and NSK cell lines or in ECE as summarized in Table 1. 3.2. Serial cultivation and titration of AIV on cell cultures and chicken embryos Culture of reference and field strain AIV viruses obtained after 10 passages showed the following data. MDCK and NSK cell lines demonstrated their susceptibility to influenza viruses (reference and field strains), although at the first passage level infectious titres were lower in cell lines compared with ECE with the exception of H5N1 LPAI A/Mallard/Italy/3401/05; where NSK and MDCK infectious titres were higher or equal (106.24 , 104.50 , respectively) to ECE. In particular, cell infectious titres on the 1st passage in cell lines ranged between 101.74 and 106.24 TCID50 /0.1 ml, for all the AIV subtypes tested but H5N1 LPAI A/Mallard/Italy/3401/05. The titres in ECE demonstrated values between 104.50 and 107.50 EID50 /0.1 ml. In contrast, the UMNSAH/DF1 cell line did not allow replication of H9N2 LPAI A/Turkey/Wisconsin/66 strain despite five blind passages. The infectious titres determined at the 10th passage level in MDCK and NSK cells were higher than titres at the 1st passage; and titres in cell lines ranged between 104.74 TCID50 /0.1 ml and

107.50 TCID50 /0.1 ml. In ECE biological system, infectious titres were higher than in cells and values ranged between 106.50 EID50 /0.1 ml and 107.50 EID50 /0.1 ml in ECE (Table 2). 3.3. Calculation of Intravenous Pathogenicity Index (IVPI) These findings were obtained in animals infected with first passage viruses and in those injected with the viruses serially cultivated for 10 passages in both cell cultures and ECE. H5N1 LPAI A/Mallard/Italy/3401/05 gave an index score of 0.00 in all analysed biological systems. H7N3 LPAI A/Turkey/Italy/2962/V03 and H9N2 LPAI A/Turkey/Wisconsin/66 viruses demonstrated a positive result (IVPI 1.77) only in 1st passage ECE. In contrast, H5N2 HPAI A/Chicken/Italy/8/A98 IVPI was higher than 0.00 (from 0.20 MDCK to 2.81 ECE) in all biological substrates in first passage, but UMNSAH/DF1. Finally, H7N1 HPAI A/Turkey/Italy/4580/99 was characterized by an IVPI ranging between 0.45 and 2.75, otherwise MDCK were negative (0.00). Results were described in Table 3. Clinical evaluations showed healthy animals and no clinical signs were described even in 10-day observed humanely sacrificed chickens. 3.4. Serial cultivation and titration of SIV grown in cell cultures and chicken embryos All SIV subtypes used in the study were serially propagated either on cell culture or in ECE. In particular, each virus was cultivated for 10 passages and subsequently hemagglutination test was performed. All SIV subtypes grew on ECE, with different titres from first to tenth serial passage, with an increase for all the strains (from 102 EID/0.1 ml to 107 EID/0.1 ml), as described in Table 4. Regarding HA titre, it was noticed an increase from 1st to 10th passage for all the viral samples, with the exception of H1N1 A/Swine/Italy/186798/09, whose HA titres decreased (1:1024 to 1:512). An important result was observed during cell cultures cultivation. In fact, all swine strains grew on MDCK and NSK cell

86

T. Lombardo et al. / Journal of Virological Methods 185 (2012) 82–88

Table 2 Infectious (TCID50 , EID50 /0.1 ml) and hemagglutinating titres of Avian Influenza viruses grown in different biological systems.a Virus biotype

Biological system

First passage

Tenth passage

Infectious titres TCID50 or EID50 /0.1 ml (log10 )

HAd titres

Infectious titres TCID50 b or EID50 c /0.1 ml (log10 )

HA titres

H5N1 LPAI A/Mallard/Italy/3401/05

Chicken embryos NSK MDCK UMNSAH/DF1

4.50 6.24 4.50 2.74

1:256 1:128 1:64 1:16

7.50 6.50 6.50 7.50

1:4096 1:64 1:16 1:64

H5N2 HPAI A/Chicken/Italy/8/A98

Chicken embryos NSK MDCK UMNSAH/DF1

6.50 1.74 4.24 3.50

1:32 1:64 1:8 1:8

6.50 7.50 6.50 5.74

1:512 1:128 1:64 1:32

H7N1 HPAI A/Turkey/Italy/4580/99

Chicken embryos NSK MDCK UMNSAH/DF1

7.50 4.24 2.50 4.50

1:8 1:64 1:16 1:64

7.50 5.24 6.24 5.24

1:256 1:256 1:128 1:64

H7N3 LPAI A/Turkey/Italy/2962/V03

Chicken embryos NSK MDCK UMNSAH/DF1

6.50 5.24 3.50 5.74

1:4096 1:32 1:256 1:128

7.50 7.50 6.50 6.74

1:512 1:512 1:256 1:256

H9N2 LPAI A/Turkey/Wisconsin/66

Chicken embryos NSK MDCK UMNSAH/DF1

6.50 4.50 3.50 N.D.e

1:512 1:16 1:32 N.D.

7.50 5.50 4.74 N.D.

1:1024 1:128 1:16 N.D.

a Each 1:10 diluted virus was inoculated for cell lines infection. After incubation at 37 ◦ C each virus replication was evaluated by cytopathic effect observation. Infected cell cultures were frozen at −40 ◦ C, thawed at 18 ◦ C, centrifuged and supernatant was collected for HA test. Embyonated chicken eggs were inoculated with 0.1 ml of each 1:10 diluted virus into the allantoic cavity. HA titration was performed in order to verify virus replication and TCID50 /EID50 was calculated at the 1st and 10th passage. b Tissue Cell Infectious Dose 50, referred to titre in cell lines. c Egg Infectious Dose 50, referred to titre in embryonated chicken eggs. d Hemagglutination. e Not detected.

lines, with different infectious and HA titres. In contrast, UMNSAH/DF1 supported just H1N1 A/Swine/Italy/25674/09 growth with an infectious titre from 102.50 to 107.50 TCID50 /0.1 ml and a HA titre from 1:128 to 1:64. No other strains were Table 3 Intravenous Pathogenicity Index of Influenza virus subtypes before and after serial cultivation in different biological systems.a Virus

H5N1 LPAI A/Mallard/Italy/3401/05

Passage

Biological system

IVPIb

1st

Chicken embryo Chicken embryo NSK MDCK UMNSAH/DF1

0.00 0.00 0.00 0.00 0.00

Chicken embryo Chicken embryo NSK MDCK UMNSAH/DF1

1.77 0.00 0.00 0.00 0.00

Chicken embryo Chicken embryo NSK MDCK

1.77 0.00 0.00 0.00

Chicken embryo Chicken embryo NSK MDCK UMNSAH/DF1

2.81 2.17 2.31 0.20 0.00

Chicken embryo Chicken embryo NSK MDCK UMNSAH/DF1

2.49 0.45 1.70 0.00 2.75

10th

1st H7N3 LPAI A/Turkey/Italy/2962/V03

H9N2 LPAI A/Turkey/Wisconsin/66

10th

1st 10th 1st

H5N2 HPAI A/Chicken/Italy/8/A98

10th

1st H7N1 HPAI A/Turkey/Italy/4580/99

10th

a Each AIV subtype serial cultivated in cell culture and chicken embryos were inoculated (0.2 ml of 1:10 dilution) by intravenous route in 6-week-old White Leghorn chickens. Animals were monitored for clinical signs over 10 days. IVPI score showed: “0” clinically healthy, “1” sick, “2” severally sick, and “3” dead (OIE, 2008). b Intravenous Pathogenicity Index.

able to replicate in this substrate despite 5 blind passages (Table 4). MDCK infectious titre increased from a minimum of 103.50 TCID50 /0.1 ml to 107.50 TCID50 /0.1 ml, with the exception of H1N1 A/Swine/Italy/186798/09 (106.24 TCID50 /0.1 ml to 105.74 TCID50 /0.1 ml). HA titre was characterized by a high unevenness among the strains: it decreased in H1N1 A/Swine/Italy/186798/09 (from 1:1024 to 1:256) and H1N1 A/Swine/Italy/290271/09 (from 1:512 to 1:128), it increased in H1N1 A/Swine/Italy/25674/09 (from 1:4 to 1:512) and in H3N2 A/Swine/Oedenrode/1/96 (from 1:64 to 1:256). Finally, H1N2 A/Swine/BS2156 HA titre remained 1:128, as constant value. NSK cell line demonstrated different infectious values; they increased in H1N1 A/Swine/Italy/290271/09 (from 103.50 TCID50 /0.1 ml to 107.50 TCID50 /0.1 ml) and H3N2 A/Swine/Oedenrode/1/96 (from 106.50 TCID50 /0.1 ml to 107.50 TCID50 /0.1 ml), on the other hand, they decreased in H1N1 A/Swine/Italy/186798/09 (from 106.50 TCID50 /0.1 ml to 104.74 TCID50 /0.1 ml), H1N1 A/Swine/Italy/25674/09 (from 107.50 TCID50 /0.1 ml to 105.74 TCID50 /0.1 ml) and H1N2 A/Swine/BS2156 (from 106.74 TCID50 /0.1 ml to 106.50 TCID50 /0.1 ml). HA titres increased in all the strains (range from 1:16 at 1st passage to 1:2048 at 10th passage). The results were described in Table 4. 4. Discussion Successful replication of influenza viruses in eggs was first reported in 1940 (Burnet, 1940). Recovery and propagation of viable Influenza viruses still depend on the use of embryonated chicken eggs, which became an ideal substrate for influenza vaccines because of the high antigen yields. Several lines of evidence from previous studies support the use of cell cultures as host systems for primary isolation and cultivation of Influenza A viruses. In particular, cell lines with a different animal origin were employed in order to isolate influenza virus.

T. Lombardo et al. / Journal of Virological Methods 185 (2012) 82–88

87

Table 4 Infectious and hemagglutinating titres of Swine Influenza viruses grown in different biological systems.a Virus biotype

Biological system

First passage

Tenth passage

Infectious titres TCID50 b or EID50 c /0.1 ml (log10 )

HAd titres

Infectious titres TCID50 or EID50 /0.1 ml (log10 )

HAd titres

H1N1 A/Swine/Italy/186798/09

Chicken embryos NSK MDCK UMNSAH/DF1

2.00 6.50 6.24 N.D.e

1:1024 1:64 1:1024 N.D.

7.00 4.74 5.74 N.D.

1:512 1:1024 1:256 N.D.

H1N1 A/Swine/Italy/25674/09

Chicken embryos NSK MDCK UMNSAH/DF1

4.00 7.50 4.74 2.50

1:1024 1:512 1:4 1:128

7.00 5.74 6.24 7.50

1:4096 1:2048 1:512 1:64

H1N1 A/Swine/Italy/290271/09

Chicken embryos NSK MDCK UMNSAH/DF1

5.00 3.50 5.74 N.D.

1:32 1:16 1:512 N.D.

7.00 7.50 6.24 N.D.

1:256 1:64 1:128 N.D.

H1N2 A/Swine/BS2156

Chicken embryos NSK MDCK UMNSAH/DF1

5.00 6.74 3.50 N.D.

1:64 1:512 1:128 N.D.

7.00 6.50 7.50 N.D.

1:512 1:1024 1:128 N.D.

H3N2 A/Swine/Oedenrode/1/96

Chicken embryos NSK MDCK UMNSAH/DF1

4.00 6.50 3.50 N.D.

1:1024 1:32 1:64 N.D.

7.00 7.50 7.24 N.D.

1:2048 1:512 1:256 N.D.

a Each 1:10 diluted SIV subtypes was inoculated for cell lines infection. After incubation at 37 ◦ C each virus replication was evaluated by cytopathic effect observation. Infected cell cultures were frozen at −40 ◦ C, thawed at 18 ◦ C, centrifuged and supernatant was collected for HA test. Embyonated chicken eggs were inoculated with 0.1 ml of each 1:10 diluted virus into the allantoic cavity. HA titration was performed in order to verify virus replication and TCID50 /EID50 was calculated at the 1st and 10th passage. b Tissue Cell Infectious Dose 50, referred to titre in cell lines. c Egg Infectious Dose 50, referred to titre in embryonated chicken eggs. d Hemagglutination. e Not detected.

Several studies demonstrated the usefulness of MDCK (Clavijo et al., 2002; Zhang et al., 2011) and Intestinal CACO-2, as an important alternative to ECE (Chiapponi et al., 2010). Two factors seem to play a key role in efficient propagation of Influenza A viruses in cell culture: cell line selected (Moresco et al., 2010) and particular virus subtype used (Lee et al., 2008). This study represented the first step of a more complex research: possible use of alternative diagnostic methods to ECE will be delved by analysis of influenza virus genomic mutations, during serial passages either in ECE or in culture cell lines. Preliminary results showed the potential use of avian and mammalian cell cultures during serial replications of both Avian (AIV) and Swine Influenza viruses (SIV). Also, the analysis confirmed that ECE remained the gold standard assay for virus isolation from target tissues of birds experimentally infected with different AIV subtypes. Serial propagation of Influenza virus strains occurred in both NSK and MDCK mammalian cell lines, although significant differences were detected among the virus subtypes under study. As described by others (Lee et al., 2008), some difficulties for AIV isolation were shown, when mammalian cell lines were used. In fact, it is noticed a host specificity of the cell with a potential difference in target receptors. Lee et al. (2008) described DF1 as an important biological system in order to isolate AIV. In fact, they evaluated the use of DF1 and QT6 cells, another avian cell line used in AIV study instead of primary avian cells (Chicken Embryo Fibroblast, CEF) and MDCK cells. In conclusion, it is suggested that DF1 cells could represent another important method during Avian Influenza A virus studies. The main feature of avian-origin cell line was represented by its permissiveness to AIV even in trypsin absence. A particular sensitivity to trypsin was noted and, in order to obtain a correct value for this kind of cells during AIV isolation, different concentrations of this enzyme (0.75, 0.3 and 0.05 ␮g/ml) were investigated. This

aspect was considered a point of particular interest for the Italian Cell Culture Centre (IZSLER, Brescia, Italy). In fact, routinely the Centre investigates numerous cell lines under different points of view: cell features, quality standards, and growth conditions. In a previous study (data not published), UMNSAH/DF1, BS CL 197 were tested with the same trypsin concentrations and with two more: 5 and 2 ␮g/ml, respectively. These findings did not show the same results observed by Lee et al. (2008), the UMNSAH/DF1 monolayer did not demonstrate a particular sensitivity to trypsin, even at 5 ␮g/ml concentration. As a matter of fact, it was decided to use this last value in order to standardize all cell lines treatment. The results of this study indicated that mammalian cell lines were suitable candidates for studying and cultivating both AIV and SIV types. With respect to AIV: ECE, NSK and MDCK permitted viral isolation from target tissues of infected chickens either LPAI or HPAI strains. In addition, NSK cells appeared more susceptible than MDCK cells for primary isolation from clinical specimens. On the contrary, UMNSAH/DF1 did not guarantee viral replication. This aspect, would be considered a limit in UMNSAH/DF1 use during viral isolation procedure. In the second step of the study, it was demonstrated that the presence of AIV either in mammalian cell lines or ECE from 1st to 10th passage, confirmed by the HA test. In addition, all avian viral strains were isolated in UMNSAH/DF1, but H9N2 LPAI A/Turkey/Wisconsin/66, in accordance with Lee et al.’s (2008) research. The inability of H9N2 LPAI A/Turkey/Wisconsin/66 to replicate is not clear; it would be explained by the possible genomic mutation during serial passages, influencing virus adaptability to the different biological substrates. Virus replication was greatest in avian HPAI infected cell cultures and the same held true, although to a lesser extent, in the ECE system. In contrast, virus isolation was performed in both NSK and MDCK mammalian cell lines, although significant differences were

88

T. Lombardo et al. / Journal of Virological Methods 185 (2012) 82–88

detected among the different subtypes. The highest prevalence of virus isolation was observed with the HPAI viruses; this feature was also observed to a lesser extent in ECE. During serial virus growth in cell culture and in ECE, a Pathogenicity Index was carried out. These results showed lower values in cell line than in ECE. In the case of HPAI viruses the Pathogenicity Index appeared lower than detected in ECE at the first passage. An interesting exception was represented by the value of 2.75 in UMNSAH/DF1 for H7N1 HPAI A/Turkey/Italy/4580/99 despite other strains (0.00); this data will be delved, in order to better understand the possible genomic mutations during serial cultivation. Lee et al. (2008) described the use of avian (DF1) and mammalian (MDCK) cell lines, as a complementary substrate for avian-origin Influenza virus isolation. Furthermore, in the same paper, it was noticed a poor DF1 permissiveness regarding Swine lineage H1 and H3 subtype turkey isolates. Otherwise Swine H1N1 virus replicated in MDCK cells. These findings suggested a host restriction between avian cells and mammalian-origin virus. In contrast, in this study, SIV subtypes demonstrated a different behaviour on UMNSAH/DF1, in fact this cell line permitted the isolation only of H1N1 A/Swine/Italy/25674/09 from the 1st to the 10th passage. This outcome will be further investigated during the last step of the research. As mentioned above, ECE were confirmed to be the most susceptible biological system for virus isolation from target tissues of infected animals, with no significant differences among virus subtypes. In conclusion, the results of this study highlighted the capacity of two continuous mammalian cell lines, MDCK and NSK to support influenza virus isolation from infected target organs and to allow serial virus cultivation yielding high quantities of Influenza viruses. UMNSAH/DF1 demonstrated an irregular behaviour: no virus isolation from target tissues and only one subtype detection of AIV (H7N1 HPAI A/Turkey/Italy/4580/99) and SIV (H1N1 A/Swine/Italy/25674/09), respectively; it was not completely appropriate to compare outcome obtained by the use of this cell line with data observed in MDCK and NSK. Furthermore, variation in the Pathogenicity Index was only detected following serial cultivation of HPAI in either chicken embryos or NSK and MDCK cells. These results will be confirmed in a further study based on the genomic analysis of these viruses. Acknowledgements This work was supported by European Community grant, FLUTRAIN project of the sixth framework programme. The authors thank Dr. Massimo Amadori, Dr. Leonardo J. Vinco, and Dr. Chiara Tontini for practical support and collaboration. References Audsley, J.M., Tannock, G.A., 2008. Cell-based influenza vaccines: progress to date. Drugs 68, 1483–1491. Burnet, F.M., 1940. Influenza virus infections of the chick embryo lung. British Journal of Experimental Pathology 21, 147–153. Chiapponi, C., Zanni, I., Garbarino, C., Barigazzi, G., Foni, E., 2010. Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses. Journal of Virological Methods 163, 162–165. Clavijo, A., Tresnan, D.B., Jolie, R., Zhou, E., 2002. Comparison of embryonated chicken eggs with MDCK cell culture for the isolation of swine influenza virus. Canadian Journal of Veterinary Research 66, 117–121. Ferrari, M., Scalvini, A., Losio, M.N., Corradi, A., Soncini, M., Bignotti, E., Milanesi, E., Ajmone-Marsan, P., Barlati, S., Bellotti, D., Tonelli, M., 2003. Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories. Journal of Virological Methods 107, 205–212.

Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus haemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology 79, 2814–2822. Genzel, Y., Dietzsch, C., Rapp, E., Schwarzer, J., Reichel, U., 2010. MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation. Applied Microbiology and Biotechnology 88, 461–475. George, M., Farooq, M., Dang, T., Cortes, B., Liu, J., Maranga, L., 2010. Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process. Biotechnology and Bioengineering 106, 906–917. Govorkova, E.A., Murti, G., Meignier, B., De Taisne, C., Webster, R.G., 1996. African Green Monkey Kidney (Vero) cells provide an alternative host cell system for Influenza A and B viruses. Journal of Virology 70, 5519–5524. Hussain, A.I., Cordeiro, M., Sevilla, E., Liu, J., 2010. Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells. Vaccine 28, 3848–3855. Kaverin, N.V., Webster, R.G., 1995. Impairment of multicycle influenza virus growth in Vero (WHO) cells by loss of trypsin activity. Journal of Virology 69, 2700–2703. Kistner, O., Howard, M.K., Spruth, M., Wodal, W., Brühl, P., Gerencer, M., Crowe, B.A., Savidis-Dacho, H., Livey, I., Reiter, M., Mayerhofer, I., Tauer, C., Grillberger, L., Mundt, W., Falkner, F.G., Barrett, P.N., 2007. Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross protective immune responses. Vaccine 25, 6028–6036. Lee, C.W., Jung, K., Jadhao, S.J., Suarez, D.L., 2008. Evaluation of chicken-origin (DF1) and quail origin (QT-6) fibroblast cell lines for replication of avian influenza viruses. Journal of Virological Methods 153, 22–28. Li, I.W., Chan, K.H., To, K.W., Wong, S.S., Ho, P.L., Lau, S.K., Woo, P.C., Tsoi, H.W., Chan, J.F., Cheng, V.C., Zheng, B.J., Chen, H., Yuen, K.Y., 2009. Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses. Journal of Clinical Virology 46, 325–330. Liu, J., Shi, X., Schwartz, R., Kemble, G., 2009. Use of MDCK cells for production of live attenuated influenza vaccine. Vaccine 27, 6460–6463. Liu, K., Yao, Z., Zhang, L., Li, J., Xing, L., Wang, X., 2012. MDCK cell-cultured influenza virus vaccine protects mice from lethal challenge with different influenza viruses. Applied Microbiology and Biotechnology 94, 1173–1179. Massin, P., Kuntz-Simon, G., Barbezange, C., Deblanc, C., Oger, A., Marquet-Blouin, E., Bougeard, S., Van Der Werf, S., Jestin, V., 2010. Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells. Veterinary Microbiology 142, 232–241. Meguro, H., Bryandt, J.D., Torrence, A.E., Wright, P.F., 1979. Canine Kidney cell line for isolation of respiratory viruses. Journal of Clinical Microbiology 9, 175–179. Moresco, K.A., Stallknecht, D.E., Swayne, D.E., 2010. Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses. Avian Diseases 54, 622–626. OIE (Ed.), 2008. OIE Manual of Standards of Diagnostic Tests and Vaccines for Terrestrial Animals. , sixth edition. Office International of des Epizooties, Paris. Olsen, C.W., Brown, I.H., Easterday, B.C., Van Reeth, K., 2006. Swine Influenza. In: Straw, B.E., Zimmerman, J.J., d’Allaire, S., Taylor, D.J. (Eds.), Diseases of Swine. Blackwell Publishing, Oxford, U.K., pp. 469–482. Reed, L.J., Münch, H., 1938. A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27, 493–497. Root, R., Orlich, M., Klenk, H.D., Wang, M.L., Skehel, J.J., Wiley, D.C., 1984. Studies on the adaptation of influenza viruses to MDCK cells. EMBO Journal 3, 3329–3332. Schild, G.C., Oxford, J.S., De Jong, J.C., Webster, R.G., 1983. Evidence for host cell selection of influenza virus antigenic variants. Nature 303, 706–709. Sugimura, T., Murakami, Y., Ogawa, T., 2000. The susceptibility of culture cells to avian influenza viruses. Journal of Veterinary Medical Science 62, 659–660. Tobita, K., Sugiura, A., Enomoto, C., Furuyama, M., 1975. Plaque assay and primary isolation of influenza A viruses in an established line of Canine Kidney Cells (MDCK) in the presence of trypsin. Medical Microbiology and Immunology 162, 9–14. Tree, J.A., Richardson, C., Fooks, A.R., Clegg, J.C., Looby, D., 2001. Comparison of largescale mammalian cell culture systems with egg culture for the production of influenza virus a vaccine strains. Vaccine 19, 3444–3450. Van Reeth, K., 2007. Avian and swine influenza viruses: our current understanding of the zoonotic risk. Veterinary Research 38, 243–260. Voeten, J.T.M., Brands, R., Palache, A.M., Van Scharrenburg, G.J.M., Rimmelzwaan, G.F., Osterhaus, A.D.M.E., Claas, E.C.J., 1999. Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium. Vaccine 17, 1942–1950. Yeolekar, L.R., Dhere, R.M., 2012. Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine. Biologicals 40, 146–150. Zhang, W., Xue, T., Wu, X., Zhang, P., Zhao, G., Peng, D., Hu, S., Wang, X., Liu, X., Liu, W., Liu, X., 2011. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine 29, 8032–8041.