Page 282 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 251

identifying a virus isolate. For identification of a
cytopathic virus, a known antibody (for example,
anti-bovine viral diarrhoea virus (BVDV) anti-
body) is added to tissue culture media to see if it
blocks the CPE of the subject virus (BVD). This
test system can also be used to detect the pres-
ence and titre of specific antibodies, for example,
a cytopathic virus (BVDV), is added at a known
concentration and antibody is titrated into tissue
culture wells to see if it can inhibit the CPE. If
sufficient BVD antibodies are present the CPE
will be inhibited. Details of how to perform the
test are given below.
Neutralization test (Inhibition of CPE):

1 Determine the virus infectivity (titre) prior
to the test (For a rapid identification one may
Figure 4.27 In SN assay, the unknown serum sam- select a dilution based on the rapidity with
ple is two-fold serially diluted and titrated against which it induces CPE).
a known quantity of virus. The serum blocks virus 2 Add equal volumes of a constant virus dilu-
infection at the 1 : 2, 1 : 4 and 1 : 8 dilutions, but not tion containing approximately 100 TCID
50
at all at 1 : 16. Each serum dilution has been tested per 0.1 ml to a known type-specific antiserum
in triplicate, which allows for more accuracy. In this at a concentration of 20 units and mix well.
sample, the SN titre would be 8, the reciprocal of (Note: one unit equals the highest serum
the last dilution at which infection was completely dilution that neutralizes 100 TCID of virus
blocked. See also Plate 16. Source: M. Sarjoon infectivity). 50
Abdul-Cader, University of Calgary, Canada.
3 Allow the virus-serum mixture to remain at
room temperature for 1 h; inoculate 0.2 ml of
be neutralized or rendered non-infective when the virus-serum mixture into each of two to
added to a ‘test system’. The amount of virus, four culture tubes or wells if using a multi-
the amount of serum and the test system (that well plate.
is, cytopathic effect in cell culture) must be mod- 4 For the virus control, inoculate 0.2 ml of each
ified to suit the conditions necessary for virus serial 10-fold dilution into a set of culture tubes
growth and to obtain satisfactory endpoints. or wells, two to four tubes or wells per dilution.
In VN tests the principle is similar but a given 5 Check all tubes or wells for CPE daily for 5–7
range of sera are used with an unknown (test) days.
virus. VN tests are highly specific and may also 6 Complete inhibition of CPE at a challenge
be used to serotype viral isolates. dose of 100 TCID by a known antiserum
50
type is considered a positive SN test and indi-
cates the identity of the virus. (It is always
A note on CPE inhibition
advisable to use serum pools, if available, for
The neutralization test performed in cell cul- preliminary identification of an isolate. Final
ture is one of the most sensitive and accurate identification can then be confirmed by using
methods for detecting specific antibody or for type-specific antiserum).

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