Strains of myxoma virus associated with a case-mortality rate in wild rabbits of 90—95% can be distinguished from those associated with a case-mortality rate of more than 99% by the use of a standardized virulence test carried out in groups of five laboratory rabbits, and tests carried out on laboratory rabbits are closely correlated with the results obtained with Australian wild rabbits. The test, which attempts to mimic natural mosquito transmission, consists of the intradermal inoculation in one site of about 5 rabbit-infectious doses of the virus, and the subsequent observation of the rate of progress of clinical symptoms and ultimate fate of the rabbit. The mean survival time provides a figure which allows satisfactory classification of all known myxoma virus strains into one of four grades of virulence, very attenuated strains being recognized by their low case-mortality rates.

Using this test, 92 strains of myxoma virus have been compared. These comprised 6 laboratory strains, 62 strains recovered from Australian wild rabbits or from wild caught mosquitoes, between February, 1951 and March, 1955, 19 strains recovered from naturally infected rabbits in Europe between October, 1953 and August, 1955, 3 strains from South America, and 2 from California. Eight types have been described and illustrated, differentiation being based on their virulence and the clinical picture of the disease they cause in laboratory rabbits. The differences are summarized in Text-fig. 2. Four of the virus types produce recognizably different pocks on the chorioallantois of developing chick embryos.

In Australia the repeatedly introduced standard laboratory strain of virus has been replaced by a moderately virulent strain, which appears to be better adapted for survival than either the highly virulent standard laboratory strain, or less virulent variants of which only a few had been recovered up to 1955. Strains of the type dominant in 1953–55 have appeared independently in many widely separated parts of Australia. Examination of these attenuated variants by pure clone techniques showed that the variability in symptomatology associated with them is not due to a mixture of virus strains, but to the expression of differences in innate resistance of the host animals which are obscured by the overwhelming virulence of the highly virulent strains.

Only one introduction of a different strain of virus was made in Europe. All field strains recovered during the first two years after the introduction were of high virulence, but a few attenuated strains appeared in 1955, and they have since become widespread. One of them appears to be a relatively stable mixture of a highly virulent and a greatly attenuated strain.

The collection of the large numbers of strains of virus described in this paper has been made possible only by the generous collaboration of many people. In Australia we would like to acknowledge the great help afforded us by Mr F. N. Ratcliffe, officer-in-charge of the Wildlife Survey Section, Commonwealth Scientific and Industrial Research Organization and his officers, especially Messrs J. H. Calaby, G. Douglas, A. L. Dyce, B. V. Fennessy, E. W. Lines, K. Myers, R. Mykytowycz, W. E. Poole and E. Waterhouse. Messrs B. D. Robinson and T. Pearce of the Lands Department of Victoria have also provided us with specimens, as have Mr G. Edgar, Director of the Veterinary Research Station, Glenfield, New South Wales, Dr D. Surrey Dane of the Institute of Medical and Veterinary Science, Adelaide, Mr D. Gooding, Department of Agriculture, Western Australia, and Messrs T. B. Alexander and A. F. Ryan, Department of Agriculture, Tasmania.

We are indebted to Dr H. de B. Aragão of the Instituto Oswaldo Cruz, Rio de Janeiro, and Dr B. Szyfres, of the Laboratorio de Biologia Animal, Uruguay, for specimens from South America; Dr D. G. McKercher of the University of California School of Veterinary Medicine, Davis, California, for the Californian strains; Drs P. Lépine and H. Jacotot, of the Institut Pasteur, Paris, for strains from France; and Mr J. R. Hudson, of the Veterinary Laboratory, Weybridge, and Dr C. H. Andrewes of the National Institute for Medical Research, Mill Hill, for material from England.

We are grateful to Dr S. Fazekas de St Groth and Dr G. S. Watson of this University for advice on the statistical treatment of the data.

Mr V. Paral took the photographs.

The primary lesions produced in rabbits by the intradermal inoculation of the virulent French strain of myxoma virus were clearly distinguishable from those produced by the standard laboratory strain or by attenuated Australian field strains. Based on this fact a screening test was developed which allowed classification of large numbers of samples of virus into French or attenuated Australian types.

The virulent French type of virus was introduced into the wild rabbit population at Lake Urana by the inoculation of 242 rabbits over a period of 6 weeks, the attenuated Australian strain of virus having been recovered from a naturally infected rabbit at the beginning of the series of inoculations. A severe outbreak of myxomatosis occurred in which an estimated 70% of cases were caused by the French strain of virus. In spite of the early predominance of cases due to this introduced strain the majority of samples obtained in the latter half of the outbreak were of the attenuated Australian type, and this was probably the only strain which survived through the following winter and caused an intense epizootic in the spring.

A similar picture of early establishment of the artificially introduced virulent French strain, and its subsequent replacement by the naturally occurring attenuated Australian strain, was seen at two other study sites, North Urana Station and Merricumbene.

We are greatly indebted to colleagues on the staff of the Wildlife Survey Section at Albury, especially Mr M. P. Hines, for assistance in the field work, and to Mr P. Bentley, the manager of Cocketgedong Station, on which the experimental site is situated. We have profited from numerous discussions with Mr F. N. Ratcliffe, and are grateful to Dr G. S. Watson for assistance in the statistical treatment of the results.

1. Sera from an outbreak of about forty cases of dengue fever due to dengue-1 virus have been studied using the neutralization, haemagglutinin-inhibition, and complement-fixation tests.

2. The neutralization test was the most specific and the complement-fixation test the least so.

3. The neutralization test is essential for clear identification of the causal virus by serological means, and serial serum specimens from each patient must be examined.

4. The haemagglutinin-inhibition test can be used to screen patients in outbreaks where some cases have been fully identified by neutralization tests.

5. Homologous neutralizing antibodies persist in high titre for at least 30 weeks after infection, while heterologous antibodies have disappeared by that time.

6. Both haemagglutinin-inhibiting and complement-fixing antibodies to homologous and heterologous viruses usually persist for at least 30 weeks, although the homologous titres tend to be highest.

7. The implications of these findings in serological surveys are discussed.

8. Some evidence suggesting the occurrence of inapparent infections during the epidemic is presented.

I am greatly indebted to my staff for help with this work: especially to my senior technician, Che Ali bin Mohamed Amin, and to Che Mohamed bin Omar who drew the figures.

An analytical method based on survival-time response is described for use in survey neutralization tests with the neurotropic viruses in mice. The method is simple and has been shown to give consistent and comparable results, which enable tests which have been carried out with different doses of virus to be compared.

In October, November and early December 1952, an epidemic of poliomyelitis, with an attack rate exceeding 1 %, occurred in Ketchikan, Alaska, a community of approximately 6000 persons. Approximately half of the cases were paralytic.

Type 1 virus was regarded as the principal cause of the epidemic because Type 1 virus was isolated from one patient, and sera from eleven of twelve paralytic patients tested were positive for neutralizing antibodies to Type 1 virus. Three patients had antibodies to Type 2 virus and eight to Type 3.

Serological tests were made to determine the immune status of the general population at the time the epidemic terminated. Approximately one-third of twenty-five children from 6 months to 4 years of age had antibodies to Type 1 virus. Of the 105 children aged 5 to 9 years, approximately three-quarters were positive for antibodies to this virus. All but two of the twenty-six persons more than 9 years of age who were tested were similarly positive.

Spread of Type 2 and Type 3 viruses was more limited in Ketchikan than spread of Type 1 virus. Serological evidence is presented to show that Type 3 poliomyelitis virus was present in Ketchikan some time within the 3-year-period prior to the collection of serum in December 1952. Since the proportion of children showing antibodies to Type 3 virus did not increase with age from 3 to 9 years, it is surmised that virus of this type was not prevalent for a number of years prior to its last occurrence. It is further concluded that spread of Type 3 virus must have terminated when 40 % or less of the children of pre-school age and early school age had been infected and developed antibodies.

Type 2 virus apparently had not been prevalent for several years prior to December 1952. Only one of twenty-one specimens of serum from children under age 5 showed definite evidence of neutralizing antibodies and this child had resided elsewhere during the first 3 months of its life. The proportion of positive results in all ages to 9 years inclusive was well below that obtained in tests with Type 1 virus.

From a review of published data concerning the age distribution of cases in the 1916 epidemic in New York, it is concluded that termination of the spread of Type 1 virus in Ketchikan occurred when the proportion of susceptibles in the general population was comparable to that in New York in each of several non-epidemic years preceding 1916. It is further evident that spread of Type 2 virus in Baltimore during a period of low prevalence of poliomyelitic disease was comparable to that of Type 1 virus in Ketchikan in 1952, in that the proportion of susceptibles in the population at the end of the period of spread of virus was similar.

Serological studies of residents of Metlakatla, an Indian community near Ketchikan, showed evidence of essentially uniform infection with Type 1 virus, a very high incidence of antibodies to Type 3 virus, and a much lower incidence of antibodies to Type 2 virus.

The epidemiological aspects of poliomyelitis in Ketchikan cannot be reasonably attributed to susceptibility resulting from isolation of the community, since travel to and from Ketchikan was considerable and a relatively large proportion of persons with paralytic disease were adults who had travelled extensively, and resided for periods of years in various parts of the United States. It is noted that the municipal water supply showed frequent evidence of faecal pollution.

The willing co-operation and valuable assistance of the following persons is gratefully acknowledged: Mr Alfred Baker, Mrs Dixie M. Baade, Miss Geneva Hubbard and Miss Lorraine Singer of the Ketchikan office of the Alaska Territorial Health Department; and Dr C. Earl Albrecht, Director and Dr Charles R. Hayman, Chief of Section of Preventive Medical Services, of the Alaska Territorial Department of Health.

The National Foundation for Infantile Paralysis financed the initial visits to Ketchikan. Testing of specimens was accomplished through the support of the University of Washington Fund for Biological and Medical Research and the Office of Naval Research.

Data concerning numerous severe epidemics of poliomyelitis were surveyed for information useful in estimating the size of the immune component of the population by age groups of 1 year. The New York epidemic of 1916 and the Mauritius epidemic of 1945 were chosen as the most suitable for this purpose. It is shown that there was a regularly progressive decline in attack rates for successively older age groups from 2 to 8 years in New York. The attack rate in 8-year-olds was less than 10 % as large as that in 2-year-olds. It is noted that a difference of this magnitude could be accounted for by immunizing infections amounting to 30 % per year for 6 years.

A similar analysis of age specific attack rates during the Mauritius epidemic shows progressive declines of 28, 60, 50 and 53 % for the successive age groups 5–9. An average annual infection rate of 45 % over a 4 year period could account for the ten-fold difference in infection rates between 5-year-olds and 9-year-olds.

An immunizing infection rate of 30 % a year would lead to a pattern of immunity in which the seven youngest age groups had a total susceptible component of 30 % at the beginning of a ‘ poliomyelitis season’. Twenty per cent would remain susceptible at the end of the season when spread of virus terminated.

An annual immunizing infection rate of 45 % would bring about a situation in which 21 % of the five youngest age groups were susceptible at the start of a period of viral prevalence and 12 % at the end. Alternatively, one could consider that on Mauritius there may have been a continuous prevalence of virus in a population in which approximately 15 % of the five youngest age groups were susceptible at any time.

It is suggested that the essentially similar attack rates among children 3–5 years old on Mauritius may have reflected a 3-year period during which homotypic virus was not prevalent, in contrast to its great prevalence during the years prior to that time.

A more detailed discussion and analysis of additional data concerning age-specific attack rates in poliomyelitis will be found in the thesis submitted by Dr Sample to the University of Washington School of Medicine entitled, ‘Some observations on statistical and theoretical epidemiology of infectious diseases, principally poliomyelitis’, in 1955. This is obtainable by Inter-library Loan from the Health Sciences Library, University of Washington, Seattle.

We wish to express our sincere thanks to Dr Blair M. Bennett and Dr William E. Reynolds of the University of Washington for their advice on the statistical and epidemiologic aspects of this study.

Treatment of serum with the insoluble polysaccharide residue of yeast cells (zymosan) has been shown (a) to decrease the bactericidal power of serum for certain bacteria; (b) to remove from some sera a factor essential to the interaction of zymosan with the third component of complement. The adsorbed protein (properdin) was eluted from zymosan and found to contain α2, β and some γ globulins. It was shown to restore partially the normal characteristics to treated serum (RP).

Properdin levels have been estimated in the sera of eighteen burned patients and in twelve normal subjects. There was evidence of a reduced level in the patients' sera. The limitations of the assay method are discussed.

Bactericidal activity of normal serum containing properdin was tested against nineteen Gram-negative bacilli isolated from burns. Ps. pyocyanea, Proteus and Klebsiella, which are commonly found in burns, were with one exception resistant or slightly sensitive to the bactericidal action of this serum. Bact. coli, which is uncommon in burns, was usually highly sensitive.

From our preliminary studies reported here it would seem that the properdin system cannot be expected to kill the flora in established infections of burns, and properdin is therefore unlikely to prove useful as a therapeutic agent. However, it probably has an important role as one of the defence mechanisms of uninfected tissues.

We wish to thank Dr Derrick Rowley and Dr A. C. Wardlaw for helpful discussions, and Dr W. Weiner for his advice and co-operation. We are grateful to Miss Stella Baar, F.R.I.C., for N and P analyses.

1. The A.S.O. titre of cord blood is about 1½ times that in the mother's blood taken 4 weeks before delivery.

2. The distribution of A.S.O. levels in cord bloods from multiparae with children at school is not significantly different from that in multiparae whose children are under school age, or in primiparae.

3. Among these groups, only primiparae show a significantly increased A.S.O. level in winter.

4. The distribution of A.S.O. levels among the five social classes defined by the Registrar-General's occupation code showed a significant trend towards higher levels in the lower social grades.

We wish to thank Mr D. M. W. Maxwell, F.R.C.S., F.R.C.O.G. and Miss D. Slade, S.R.N., S.C.M. at Taplow, and Professor J. C. McC. Browne, F.R.C.S., F.R.C.O.G., Dr W. Hayes, D.Sc., F.R.C.P.I. and Miss M. Tweedle, S.R.N., S.C.M. at Hammersmith Hospital for their kind help.

The influenza virus serum flocculation previously reported (Belyavin, 1955) opened up a number of lines of investigation. One of obvious importance was extension of the reaction to other viruses belonging to both related and unrelated groups. Indeed, other workers in this laboratory have already achieved the flocculation of poliomyelitis viruses by specific antisera (Smith, Sheffield, Lee & Churcher, 1956) and flocculation of both mumps and Newcastle disease viruses is now reported in this communication. The ease with which the viruses of the mumps-influenza group can be flocculated by homologous rabbit antisera suggested that the technique may be applicable as a method of antigenic analysis. If so, it would have the advantage of being much simpler than the standard haemagglutination inhibition and complement-fixation tests. The exploration of this possibility forms the basis of this paper. A large-scale antigenic survey involving numerous virus strains has not been attempted, greater emphasis being placed on the examination of techniques and their applicability to the end in view. The investigation has also revealed new phenomena peculiar to the direct virus flocculation reaction.

1. Influenza virus can be labelled with 35S by cultivation in fertile eggs into which radioactive methionine has been introduced.

2. When virus labelled with 35S is fractionated by ether treatment the nucleoprotein soluble antigen fraction contains 15 % of the 35S, the haemagglutinin also contains 15%, while the denatured envelope protein contains 70% of the 35S.

3. When sulphur-labelled virus is introduced as a primary inoculum in fertile eggs and extracts of the chorioallantoic membrane are made 1–20 % of 35S is recovered as amino-acid, while the remainder is present as non-infective material of particle size similar to that of the original virus.

4. It is suggested that on entry into the cell the virus nucleoprotein is hydrolysed with the release of amino-acid and free nucleic acid, while the virus envelope protein and haemagglutinin remain on the cell surface.

The authors are indebted to the Medical Research Council for a grant in aid of expenses, and to Messrs. Eli Lilly and Co. for the generous provision of the Spinco centrifuge used in this work. The radioactive methionine was obtained from the Radiochemical centre at Amersham.