494 DISCUSSION AND PRELIMIKARY REPORTS

Baermann funnel extracts from which all the L. elongatus had been removed. RRV was transmitted by both larval and adult L. elongatus, but the numbers of transmissions were too small to establish whether they differ in efficiency as virus vectors: trans- mission of RRV occurred in 2 out of 14 pots that received larvae and 1 out of 13 pots that received adult nematodes.

The results of two furt.her experiments suggest that L. elongatus can retain infec- tivity for some weeks. The first of these was made with Glendevon soil which had been stored in the laboratory for about 3 months and which contained large numbers of L. elongatus. RRV was detected in only 2 out of 10 sample pots, but 8 other pots filled with soil from the same batch were all infective after cucumber, systemically infected with a stock isolate of RRV, had been grown in them for 11 weeks. The cu- cumbers were carefully uprooted from the pots and pregerminated spinach seed was sown in them 13 days later. Tests of the spinach seedlings after 4 weeks’ growth showed that each of the 8 pots still con- tained RRV. When a second batch of spin- ach was sown 14 days after the first had been harvested, RRV was detected in 6 out of the 8 pots. As there were no weeds in the pots at any time and no virus was detected in samples of root debris taken from the pots 3 days after the removal of the cucum- ber infectors, it is concluded that L. elon- gatus can retain the virus for at least 14 days in the absence of any host plants and perhaps for 63 days, although the first crop of spinach may have acted as a source of virus. In another experiment, lots of 50 L. elongatus were added to pots of sterilized compost containing RRV-infected cucum- bers and spinach bait plants. Tests of the hypocotyls of the spinach bait plants showed that transmission of RRV had oc- curred in 17 out of 30 pots; no virus trans- mission was detected in any of 10 control pots in which spinach bait plants were grown with infected cucumbers but which received no L. elongatus. The infective pots divided into four lots, the cucumbers were removed, and spinach bait seedlings were planted after intervals of 1, 10, 20, and 42

days. Each batch of seedlings was allowed to grow for 4 weeks before assaying for virus. The results showed that transmission of virus had occurred in 4/6, l/4, l/4, and 2/3 pots, respectively, for the different treatments, indicating that L. elongatus can retain the virus for at least 42 days in the absence of host plants and a source of virus; this behavior is comparable to that of grapevine fanleaf virus in Xiphinema index Thorne Rr Allen (5) and of arabis mosaic virus in X. diversicaudatum (Mico- letzky) (6, 7).

REFERENCES

1. HARRISON, B. D., Ann. appl. Biol. 46, 571-584 (1958).

2. CADMAN, C. H., J. Hod. s’ci. 31, 111-118 (1956). 3. HARRISON, B. D., MOWAT, IV. P., and TXYLOH,

C. E., Virology 14, 480-485 (1961). /t. SEINHORST, J. W., Nematologica 1, 249-265

(1956). 5. RASKI, D. J., and HEWITT, IV. B., .Ajmnrrtologiccc

5, 166-170 (1960). 6. JHA, A., and POSNETTE, A. F., Virology 13, 119-

123 (1961). 7. HARRISON, B. D., and WINSLOW, R. D , Am

appl. Biol. 49,621-633 (1961). C. E. TAI-LOR

Scottish Horticultural Research Institute Invergowrie, Dundee Scotland

Received March 1, 1862

Human Blood Group Activity Associated with

a Myxovirus’

The DA virus is a recently described (1) member of the SV-5 group of myxoviruses. During the course of studies on infection of primates with this agent, it was noted that a chimpanzee inoculated intravenously with supernatant fluid from virus-infected rhesus monkey kidney tissue responded by developing antibodies directed not only against the virus particle, but also against human group B erythrocytes. The chimpan- zee serum anti-B agglutinin titer rose from less than 1: 10 before inoculation to 1: 8000

’ Supported in Iwt, by :I I’HS Frllow~hil) .1I-lt3- 13,881 from the National Institutes of Health, United States Public Health Service.

DISCUSSION AND PRELIMIN.1RY REPORTS 495

one month after administration. In order to test the reproducibility of this phenomenon and its possible relation to the DA virus, further immunizations were carried out under a variety of conditions and the re- sults form the basis of the preliminary re- port.

Methods of DA virus growth and assay in monkey kidney cell cultures and determina- tion of neutralizing and hemagglutination- inhibiting antibodies have been previously described (1 ,I. Egg-adapted virus was ob- tained by serial passage in the amniotic cavity of lo- to 11-day-old embryonated eggs. Human erythrocytes were collected and stored in Alsever’s solution and washed three times with phosphate-buffered saline (PBS) prior to use. Serum anti-B agglutinin titers were determined by mixing serial serum dilutions in PBS with an equal vol- ume of 1% human group B erythrocytes. The mixtures were shaken and allowed to settle at room temperature for 45 minutes; end points were determined by gross in- spection of the pattern of the settled eryth-

rocytes. Primates used were adult chimpan- zees (Pan troglodytes) , rhesus monkeys (Macacas mulatta), patas monkeys (Eryth- rocebus patas), and African green monkeys (Cercopithecus aethiops) . Each animal was bled at least twice prior to experimental manipulation and at weekly intervals there- after.

The anti-B agglutinin titers obtained were independent of the Rh status of the tested erythrocytes, and no significant dif- ference in serum titers were noted in com- parative tests using erythrocytes from eighteen randomly selected type B donors. No significant change in agglutinin titers were found after absorption of sera with human Al, A,, or 0 (H) erythrocytes, whereas incubation with purified group B substance from horse stomach (Knicker- bocker) removed the anti-B agglutinins in all sera tested. Results of immunizations with DA virus obtained from a variety of sources are summarized in Table 1. Two chimpanzees possessing erythrocytes of a group corresponding to human A were in-

TABLE 1

ANTI-B AGGLTJTININ RESPONSE IN PRIMATES FOLLOWING DA VIRUS INFECTION

Species tested

Chimpanzee

Patas monkey

African green monkey

Rhesus monkey

Agent

DA Echo-11

I>A

NIjV

supernat,ant fluid

l>A

1)A

B substance

-

Source

Rh. kidney Rh. kidney

Rh. kidney Patas kidney natural infect,ion

Rh. kidney

Normal Rh. kid-

ney

Rh. kidney Egg amnion

Rh. kidney

Horse stomach

1 Number , tested

\Tumber with IA antibody

risea

-

lr\ a

-

-

Jumber with Inti-B agglu- tinin risea

0

0

a Eightfold or greater rise bet,ween serum taken immediately prior to infection and serum collected 1 month later.

396 DISCUSSION A1SD PRELIMINARY REPORTS

fected with rhesus kidney-grown DA virus, one by nasopharyngeal swabbing and one by intravenous inoculation of 2.0 ml of supernatant fluid. Both animals showed a significant rise in titer of antibodies to the DA virus and to human group B erythro- cytes 3 weeks after administration. Three chimpanzees were inoculated intravenously with supernatant fluid from rhesus kidney tissue infected with ECHO-11 virus, a he- magglutinating enterovirus. No response to human B erythrocytes was found in serial sera collected up to 4 months after inocula- tion. Patas and African green monkeys were also found to respond to rhesus monkey kidney grown DA virus with the develop- ment of anti-B agglutinins and viral anti- bodies. In addition, virus grown in patas monkey kidney tissue and in the amniotic cavity of embryonated eggs was found to be capable of stimulating an anti-B response. The egg-adapted virus, however, was not effect,ive when given as a single intravenous inoculation, but required repeated intrader- ma1 and intramuscular inoculations before a response was obtained. Two patas mon- keys developed spontaneous infection with DA virus during the course of their cap- tivity as evidenced by virus isolation and the appearance of neutralizing antibodies in serial serum samples. In both instances anti- B agglutinins appeared in parallel with viral antibodies. In contrast, two patas monkeys whose serial serum samples showed no significant change in viral antibody dur- ing a 6-month span of captivity also failed to show a change in titer of anti-B aggluti- nins. No change in anti-B agglutinin level was found in patas monkeys inoculated with either supernatant fluid from normal rhesus kidney cultures or from cultures infected with Newcastle disease virus (NDV). Rhc- sus monkeys failed to develop anti-B ag- glutinins after DA virus infection despite the appearance of high-titered viral anti- body. This failure to respond is consistent with the finding that organs of these mon- keys contain a B-like substance (2) and the monkeys would thus be unable to produce anti-B antibody. This was substantiated by the demonstration that, parenteral inocula-

tion of purified B substnncc induced no anti-B agglutinins in a rhesus monkey.

Since substances with blood group B ac- tivity are widespread in nature, the finding that supernatant fluid from virus infected tissue cultures induced an anti-B response after parenteral inoculation did not in itself demonstrate a relationship bet,ween the DA virus and B antigen. It was possible that the B antigen existed as an independent entity in the inoculum with no relation to the virus particle or that the B antigen was present in the tissue culture cells and was incorporated intact into the virus particle during maturation and/or liberation of the virus. These possibilities appear inadequate to explain the present findings since an ag- glutinin response was found after admin- istration of virus grown in different cell systems and appeared after either naso- pharyngeal swabbing or during the course of natural infection. It was also shown that supernatant fluid from rhesus kidney cul- tures either uninfected or infected with un- related viruses failed to induct an anti-B response after inoculation into susceptible animals. Preliminary results have indicated that the in vitro blood group activity of DA virus preparations may be sedimented along with the intact particle. The data thus sug- gest that the blood group B activity is an inherent property of the DA virus and pro- vide further evidence for the immune origin of certain “natural” hemagglutinins. Al- though the rata do not bear directly on the origin of human blood group isoagglutinins, it should be noted that the DA virus was initially isolated from a human whole blood sample.

REFERENCES

1. HSIUNO, G. D., ISACYON, P., and MCCOLLUM, R.

W., J. Immunol. 88, 284-290 (1962).

2. WIENER, A. s., CANDELA, P. B., and GOSS, 1,. J., J.

Immunol. 45,229-235 (1942).

PETER ISACSON

DAVID M. HOLDEN

Department of Microbiology Yale University School of Medicine New Haven, Connecticut

Received April 12, 196.2