General Materials and Methods
2.4 Virus quantification
2.4.1 ISAV quantification
2.4.1.1 End-point titration of ISAV from pre-formed cultures
SHK-1 and ASK-2 cells were seeded in 12-well plates to prepare pre-formed monolayers for virus inoculation. Cells were cultured as previously described in Section 2.2.1.2. One medium change was undertaken 24 h post seeding, then once the monolayers had reached a 50-60% confluence after 48 h, after which spent medium was decanted and 0.2 mL of 5-fold serially diluted ISAV in HBSS, 2% FBS was added to the wells. After adsorbing overnight at 15°C the cultures were supplemented with fresh medium, and cells were incubated for 2-3 weeks. Mock infected cells, which received non-infected culture medium, were included in every assay. Cells were scored for CPE after 7, 14 and then finally 21 dpi. Only cultures exhibiting a CPE of at least 50% were scored as positive before determining the TCID50
value by the method described in Section 2.4.3.1.
2.4.1.2 End-point titration of ISAV from simultaneous cultures
It proved difficult to undertake a simultaneous inoculation and back titration in 96-well tissue culture plates (Nunc, Denmark) with SHK-1 cells, therefore for simultaneous infection only ASK-2 cells were used. Eighty microlitres of HBSS (2% FBS) were mixed with 20µl of virus culture from Section 2.3.1 from left to right of the 96-well plate to produce a 5-fold dilution series of virus inoculum across the plate. The top and bottom rows of the plate only contained HBSS diluent. Confluent ASK-2 cells from a 25cm2 culture flask (Section 2.2.1.2) were trypsinised and 100µl of cell suspension added to every well of the 96-well plate, aspirating after the addition of the cells and incubating at 15°C without CO2. Cells were scored as
positive or negative depending on the level of CPE after 2 weeks and the end point titre was determined as described in Section 2.4.3.1.
2.4.2 KHV quantification
2.4.2.1 24-well plate end-point titration (pre-formed)
Similarly to the pre-formed titration carried out for ISAV (Section 2.4.1.1), KF-1 and CCB cells were cultured overnight at 22°C in 24 well tissue culture plates (Nunc, Denmark) to form a monolayer. After 50-60 % confluence had been obtained, medium was removed and the cells were inoculated with 100 µL 5-fold serially diluted KHV virus in HBSS, 2% FBS.
Mock infected cells received only culture medium with no virus. Absorption of the virus to the plate was undertaken for 1-2 h at 20°C before cells were resupplemented with fresh MEM medium containing 2% FBS. Cells were checked for the development of a CPE after 7 and 14 dpi. TCID50 was determined according to the method described in Section 2.4.3.1.
2.4.2.2 96-well plate end-point titration (Simultaneous/pre-formed)
Simultaneous inoculation of 96-well plates was undertaken as that described in Section 2.4.1.2 with KF-1 cells and CCB cells inoculated with 5-fold diluted KHV virus. However, the pre-formed method was preferred for KHV. One hundred microlitres of cell suspension was added to the wells of the 96-well plate (Nunc) and incubated overnight at 22°C in 4%
CO2. The following day, 5-fold dilutions of KHV were prepared and 100 µL of this was added to cells after removing the old culture medium. After 1-2 h adsorption at 20°C, the cells were re-supplemented with fresh MEM medium containing 2% FBS and cultured for 14 days, at which point the titre was determined according to the method described in Section 2.4.3.1.
2.4.2.3 Plaque assay
The plaque assay was predominantly used for confirmation of calculated TCID50 values. The plaque assay provides very sensitive, accurate and reliable quantitation of infectious virus particles, with each plaque being derived from a single infectious clone (Burleson et al., 1992). The assay was originally developed to determine titres of bacteriophages (Dulbecco and Vogt, 1953) and was applied here to detect KHV (Ronen et al., 2003; Hutoran et al., 2005). The used protocol was kindly provided by Dr. Maya Ilouze, The Hebrew University-Hadassah, Jerusalem, Israel).
The CCB cells were seeded at 1 x 105 cells well-1 into a 24-well plate and incubated overnight at 22°C. The following day the old medium was removed and the monolayers were washed twice with DPBS. Five or ten fold serial dilutions of virus were made in HBSS, 2%
FBS. Cells were then inoculated with 100µL KHV for 1-2 h at 20°C to allow adsorption of virus on to cells. Mock infected cells received only HBSS, 2% FBS. MEM media was prepared with 8% FBS and the usual supplements of 1% NEAA and 2mM L-glutamine. In some cases when CO2 was not available for the incubation, 15mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4) was added to the medium. The culture media was mixed 1:4 with 1.2% melted sterile agarose (Multi ABgarose, Thermo Scientific, UK) in PBS to form a media overlay containing 0.3% agarose. One millilitre of the MEM/agarose overlay gel was then added to the infected monolayers and the cells were incubated at 20°C with 4% CO2. After 5-7 dpi, the plaques were fixed with 1 mL 4%
formaldehyde for 10 min at RT and the cells were stained with 1.5% Gentian violet Gurr (Certistain®, VWR) in distilled H20 after careful removal of the overlay. The Gentian violet stains the intact cells of the monolayer allowing the plaques to be easily counted (Fig. 2.3).
Figure 2.3 Plaques obtained for quantification of koi herpesvirus (KHV) infectious particles on common carp brain (CCB) cells. The cells were fixed and stained 5 dpi and the plaques (arrows) counted. Row C = Control wells, row I = Infected wells.
2.4.3 Quantification of infectious virus particles
2.4.3.1 Spearman-Kärber method for virus titration
Mean log TCID50 (m) =
n
d r d
X 2
1
Where
X = log of the highest reciprocal dilution d = log of the dilution interval
r = number of test subjects not infected at any dilution.
n = number of test subjects inoculated at any dilution.
After Kärber, 1931
The Spearman-Kärber method (Kärber, 1931) provided a means for determining the dilution of virus (or virus sample) required to infect 50% inoculated cells. This is expressed as tissue culture infectious dose required for 50% infection of cells inoculated (i.e. TCID50). The TCID50 assay ultimately measures cytocidal virions (Burlesson et al., 1992).
After inoculation of cells with serial dilutions of virus the CPE was analysed and an all-or-nothing score was given for the culture: positive for a culture with ≥ 50% CPE of the monolayer and negative for a culture with < 50% CPE. The number of cultures infected at a specific viral dilution were then determined and TCID50 calculated using the formula defined above.
2.4.3.2 Plaque quantitation (plaque forming units; PFU)
PFU = Mean plaque number X reciprocal dilution X reciprocal of volume in mL
After Burleson et al. (1992).
Only wells with 20-100 plaques were counted at that reciprocal dilution. The number of plaques counted provides an estimate of the total number of infectious virions initiating infection. The infection titre of the plaque assay is expressed as plaque forming units (PFU) per mL and is calculated from the formula defined above.
2.4.3.3 Multiplicity of infection (MOI)
m = aN/C
Where
a = proportion of viral particles that initiate infection N = Total number of viral particles
C = Total number of cells
After Dulbecco and Ginsberg, (1988)
The multiplicity of infection is important in order to know the distribution and proportion of cells infected by virus particles which depends on the average number of viral particles per cell. MOI was calculated using the formula defined above.
Therefore, the number of PFU, which is a measure of the proportion of infectious particles, is divided by the total number of cells to give the MOI. Where plaque assays were not performed, TCID50 was converted to PFU by multiplying the antilog by 0.69. This takes into account the Poisson distribution which is a measure of the proportion of cells infected by a given number of virus particles (Dulbecco and Ginsberg, 1988), and has also been described elsewhere for determining the MOI for other virus infections (Wang et al., 2008;
Voronin et al., 2009).