The Effect of Incubation at 37C on the Neutralization Test with Various Encephalitis Viruses Including Lansing Strains of Poliomyelitis Virus
Authors: Olitsky Peter K. , Casals J. 
Published: January 2023

Discussion and Summary
It is of interest to trace back to the earliest experimental evidence presented for the use of preliminary incubation, before inoculation, of neurotropic viruses with serum for the purpose of enhancing the neutralization index. Kraus, Keller and Clairmont (28) in 1902 reported extensive but incomplete protocols on the comparison of the action of immune and of normal serum on rabies virus. Undiluted serum was added to virus from a state of “thick emulsion” to 1:100 dilution only, and the mixture held for 18 to 20 or 24 hours at room temperature before inoculation. They found that the immune serum was “rabicidal” whereas the normal serum was not, but if the normal serum-virus mixture was kept for 48 hours at room temperature, it also was “rabicidal.” No records were offered by them of a systematic study on the effect of immune and of normal serum on higher dilutions of virus, or of the influence of time and temperature on the intrinsic diminution in titer of the virus, or of the degree of enhancement of quantitatively measured specific neutralization that the prolonged contact brought about. The long period of incubation has, however, been practised since then in tests with this virus and is even now being employed (29) although it has been reduced in certain instances to as short a time as 1 hour at 37C (30). For the encephalitis viruses such as are now under investigation, namely those of Western and Eastern equine encephalitis; West Nile disease; St. Louis, Japanese B and Russian Far East encephalitis and poliomyelitis, varying periods of contact of serum with virus were permitted which with time were reduced to 1, or more often, 2 hours at room temperature or 37C. Here also systematic studies were either fragmentary or wholly lacking, and the procedure of preliminary incubation before inoculation in the neutralization test was generally based on custom rather than on controlled experiment.
The results of the present communication would appear to show that incubation for 2 hours at 37C as compared with nonincubation, brought about a greater reduction in the titer of virus. But a similar degree of reduction followed whether specific antiserum or normal serum was employed and a somewhat lesser reduction when hormone broth was tested. Moreover, and significant for the practice of the neutralization test in which a “negative” serum as control is usually added, the variation in the viral inhibition produced by different normal sera after incubation was marked, even by those sera derived from homologous species and kept under similar conditions. There was also variation in the LD50 titer of virus in different control sera in non-incubated serum-virus mixtures (table 1). Hence the results of a test varied appreciably depending whether reference was made to one or another “negative” control serum. In other words, in the absence of an ideal control system, the test can offer only a relative and not an exact value of the amount of specific neutralizing antibody.
For the practical neutralization tests with the viruses here studied, it would therefore seem advisable to omit, if possible, the preliminary incubation of serum-virus mixtures, or to mix the two and to inject the mixture immediately thereafter. In this way the hazard may be obviated of including as specific virus-neutralizing reactions certain of the nonspecific viral inactivation. If the procedure of incubation is followed, however, then nonspecific reactions should be anticipated.
One can only speculate on the mechanism of viral inhibition by means of control materials. That a certain amount of intrinsic deterioration of the viruses may take place at 37C is possible, as is a degree of inactivation through serum enzymatic action. The pH values, as the tests were carried out, apparently did not exert inhibiting action. The degree of diminution of the titer of the viruses was not dependent on their potency, whether high or low, nor on the species from which the serum was derived, nor on the form in which sera were used, such as stored in a refrigerator or in the frozen state, or freshly prepared. It was recently shown (31) that the lipid fraction of normal serum inactivates rapidly in vitro a large amount of certain encephalitis viruses; this factor may possibly reduce the titer of the viruses and should receive attention. Recently Koprowski, (32) and also Laemmert, Ferreira and Taylor, reported that sera from certain marsupials and rodents inactivated, after preliminary incubation of serum-virus mixtures for 1 hour at 37C, not only yellow fever but also one or more neurotropic viruses, Japanese B, St. Louis and West Nile. None of the 4 viruses apparently existed in the neotropic habitat of these animals; the reaction was therefore looked upon as nonspecific, and no explanation of the mechanism of the inhibition was given.