RABIES VIRUS IN McCOY CELL LINE: PART

: The McCoy cellline has showed high sensivity to rabies vírus. This cell line presents cythopatic effect (CPE), when infected with rabies vírus. Three strains of rabies virus were tested: CVS, ERA, and PVl and three street vírus were isolated: one from a patient bitten by bat, other from a rabid dog and another from bovine. This cellline was used for virus titration purposes due to the presence of cytophatic effect, two techniques for observation of the CPE were used and were compared to the classical method of intracerebral ínoculation in mice.


INTRODUCTION
Several authors studied a series of methods for antíbody or vírus titration using microtechniques for rabies virus detection.Blancou  Perrin et aW 6 used rapid enzyme immunodiagnosis (RREID) as a simple and rapid diagnosis method for detection of rabies virus in the laboratory routine, despite the necessary of a specific conjugate with peroxidase.
Webster!! used the infection of tissue culture technique (a suspension of murine neuroblastom cells) using a multi-plate of 96 wells and the infection was nut in evidence with immunofluorescent technique.This technique proved to be equivalent to the mouse inoculation method.
Rudd et aZ!8 used the murine neuroblastom cell line (C-1300; clone NA) for isolation of small quantities of street virus.Thus it was assumed by Wiktor et al 12 the slow adaptation of rabies vírus into tissue culture.ln 1978, Smith et alii lO showed the easy adaptation of rabies virus to cellline CER (murine neuroblastom) and compared it to the infection with BHK-21, clone 13, sensitized with DEAE -dextran and both celllines showed eficiency for isolation of rabies virus in vitro, Other group of authors, Mannen et aW 4 , used the mieroteehnique based on an enzime immunoassay similar to the fluorescent foeus inhibition assay, but they used enzyme assay method (RAMIN), that is an authomatized method which permits a rapid test of many samples and the results were similar to the rapid fluoreseent inhibition test (REFIT).
, Observing CPE in loco TITRATION Ten-fold dilution of the virus was prepared for titration in Eagles medium plus 2% ofFCS, 0,5 ml of each dilution were inoculated in each well of multi-well plates Limbro Flow, with 24 wells.
The plates were incubated with 5% of C02 at 33°C, during 30 minutes for virus adsorption.Then each well was completed with 1.5 m! ofmaintenance medi um (Eagle's plus 2% FCS), and again incubated at 33°C.CPE was observed daily, and highest dilution ofvirus causing CPE (++) in 50% ofwells was considered the titration end-point.

Observing CPE Using Karaya Gum as Overlay CELL CULTURES
The same cellular suspension of 4.0xl0 5 .0 cells/ml was used in each well, on the multi-well plates (24 wells) with Eagle 's medium plus 5% of FCS for cellular growth.

VIRUS
It was used 0.2 ml of inocula ofthe same dilution of the 6th passage ofERA strain in McCoy cellline already referred to.

PROCEDURE
After the incubation period of 48 hours, the cells were fixed during 30 minutes with solution of formalin 20% in alcoholic so!ution 20%, the plates were washed in distilled water and stained with 18 a1coholic solution 20% and violet crystal 0.5%, again the p!ates were washed and dried.
A uni que large plaque was evidenced with different kinds of stain, the cells showed CPE and there was a great difference in the morphology ofthe cells in different stains.
Forty-eight hours were necessary for the occurence of CPE and the end-point considered was the highest diIution were 50% of the area was affected in 50% of the wells, figures not shown.

Titration in Baby Mice
Five-to-nine-day-old Swiss-Webster baby-mice were used, The virus titration followed the same dilutions ofthe 6th passage ofERA strain already referred to.The animals were inoculated intracerebrally with 0.03 m! of each dilution ten-fold performed.
Control animaIs with the same age were inoculated intracerebra!ly with 0.03 m! of sterile saline solution, and observed during twenty-one days.

Preliminary studies showed the CPE caused in
McCoy cellline by rabies virus (Nogueira"), this celular lineage is now use for titration purposes ofrabies virus.For each dilution onIy one point was plotted (50% of infection).
When Karaya gum was used as overlay the rapid action of virus in the cell monolayer was observed and 48 hours later the presence of CPE was also obsered.Figure 2 shows the difference of cell mor-phology caused by virus action.In this case lhe percentage of infectivity was evident, and the cri teria used was the same.
Figure 3 shows the observation os CPE at a dif-where morphological alterations caused by rabies ferent magnitude in the microscope observed with virus, as welI as stains differences, can be seen.plannar objective 6.3/0.16 in IM-Zeiss microscope  shown in figure 4. Their results were equivalent when the values were adjusted to the same volume, as it can be seen in table L   * The values of the titres were adjusted to the same inoeulum volume, evidencing the similarity of data.
Three different methods of observation were used.The titers were obtained by the end point teehnique aeeording to Reed-Mueneh's method.

DISCUSSION
Data obtained in these preliminary studies show the possibilities of the use of McCoy cell line for titration of rabies virus.The use of Karaya gum as overlay speeded the aetion of the virus, and the results using 0.2 ml of inoeulum per 2 ml of total volume was eomparable to the results obtained with intracerebral inoculation ofbaby mice (0.03 ml).
On the other hand, the direct observation of CPE is efficient to obtain an increase of the titre during viral replication.When the logarithm of each di lution is plotted against th days of CPE observation (with more than 50% ofinfectivity) a sigmoid curve 20 is obtained and its inflection is equivalent to the result obtained with mice inoculation on the eight dai;' of CPE observation and with a dilution of about 10 .0(see Nogueira.").It was observed that the obtainment of CPE in McCoy cellline after innoculation with rabies virus can detect small amounts of antige and is as sensitive and efficient as another techniques such as RIFT (Rapid Inhibition Focus Test) on murine neuroblastom cells.1oBesides, it has the advantage that the intermediary reaction of IFA or enzyme linked reaction or UV microscope are unnecessary.The costs decreased due to the absenee os laboratory mice as well as another kinds of reagents.
In spite of the relatively small nunber of samples the isolation of street virus (patient bitten by bat;

FIGURE 1
FIGURE 1 Percentage of Infection Observed With The Presence of Cytopathic Effect During the Observation Days.

FIGURE 3
FIGURE 3 Differences ofMorphology with Inoculation ofRabies Virus (ERA strain) Where the Action ofthe Virus on McCoy Cell Line is Observed.The dilution observed is the same as figure 2, but observed with other magnitude.A -Dilution 10_ 4 ; B -Dilution 10); C -Dilution 10_ 6 and D -Control celIs.This observation was performed with plannar objective 6.3/0.16 with IM -Zeiss microscope.

FIGURE 4
FIGURE 4 Titration ofRabies Virus (ERA strain) Observed with Two Teehniques (A AND B) Compared to the Classie Intracerebral Inoeulation in Baby Miee (C).
, Heberling et alii: developed a new assay based on Dot Immunobiding Assay (DIA) for the routine detection of antibody to various viruses including rabies virus.
th passage which titre was 10 6 . 2 LDso/O.03ml was inoculated intracerebrally in baby mice and the end-point was calculated by the Reed-Muenchmethod 9 .

TABLE 1 Titres
Observed with the 6 th Passage ofRabies Virus (ERA strain), on the MeCoy Cell Line.