SINCE living cells are required for virus multiplication the earliest attempts to grow viruses were made, in the only source of living cells then available—living animals. These animals have now been replaced, for most viral studies, by embryonated hen's eggs and tissue cultures. For the moment we shall deal only with the foundation of virology using such animals as monkeys, ferrets and mice and mention the position of the laboratory animal today. The rabbit holds the dubious honour of being the first true laboratory animal. Before "The Viruses" were separated from the other disease causing "Microbes" Pasteur and his associates showed in 1881 that the intracerebral inoculation of brain homogenate from a rabid animal caused the death of these rabbits, and what was more important, they observed that the virus could be passed serially to other rabbits. This latter observation of continuing passage in a new host resulted in the attenuation of the virus for man and the later use of this material as a vaccine
In 1909 the rhesus monkey followed the rabbit and was used by Landsteiner and Popper in the growth of Poliovirus. Despite the expense of this monkey, both to obtain and maintain, limited experiments revealed the natural history of poliomyelitis. Again in 1928 the rhesus monkey figured in a new discovery, this time in the cultivation of yellow fever virus During the next two years the first important steps towards the understanding of the epidemiology of yellow fever were taken before the monkey was replaced by the mouse in 1930Unlike the rabbit the rhesus monkey is still in common use for determining the potential neuropathogenicity of viruses. Ferrets were the source of the last major advance associated with an animal, when in 1933 Smith et al discovered that influenza virus could be transmitted intranasally to ferrets using filtrates of gargle material from infected persons. Serial transmission was then maintained by passing extracts of ferret's turbinate tissue. The ferret proved to be a very useful animal because it has a very short lived immunity and can be readily re-infected with the same virus within a few months. Like poliomyelitis and yellow fever in monkeys, the symptoms of influenza in ferrets are similar to those in man
The Mouse
Swiss white mice6 are essential at present for the primary isolation of many viruses from infected humans. Wide use is also made of them in the study of the Arboviruses and the Coxsackie sub-group of the Picornaviruses, as well as the animal pathogens like Foot and Mouth Disease virus. Recently they have been the means of demonstrating the tumour forming properties of the PapovavirusesAlthough adult mice were used originally, today most work is performed on baby or suckling mice. This is largely dictated by ease of handling and cost. Before describing the principle uses of mice certain terms require definition. New-born mice are animals less than three days old. Baby mice are animals over three days old but still being suckled by their mothers. Young mice are those between weaning and adulthood. Most of the investigations on the Arboviruses are still made using mice for some stage in the work, usually the production of a stock of high titre virus or its initial isolation from clinical material. In general newborn mice are more sensitive and all the known Arboviruses can be propagated by intracerebral inoculation of new-born litters. In many instances satisfactory results follow inoculation by a peripheral route, that is by intramuscular, intraperitoneal, intranasal or sub-cutaneous injection. However, as the mice increase in age their susceptibility to peripheral inoculation decreases and with some types of virus even intracerebral inoculation produces poor and inconsistent results
Finally, in addition to isolation and propagation of the actual virus, new-born mice are used extensively for the detection of antibody in the mouse protection test.The Coxsackie A viruses are the only other viruses which require mice for reliable propagation and again new-born mice are essential, because most strains of these viruses do not produce recognisable illness in adult mice. On the other hand Coxsackie B viruses grow well in tissue cultures, but the type of illness produced in mice is still the only means of determining whether a Coxsackie virus belongs to group A or B
Other viruses studied in mice include Herpes Simplex, Foot and Mouth Disease, encephalomyocarditis and lymphocytic choriomeningitis viruses. The death of the mouse usually follows infection with any of these. It is possible to get an idea of the amount (titre) of virus in a given suspension by diluting the suspension in serial tenfold (log10) dilutions and inoculating each dilution into two or more litters of mice. The death of each mouse is recorded until it is obvious that no further deaths will occur. This usually takes about three weeks. The titre of virus can then be calculated as the 50% lethal dose (LD50) of virus, that is, the amount of virus suspension required to kill half the mice which have been inoculated. The simplest and most common method of determining this is the method of Reed and Muench and an example will now be given.
