- Volume 30, Issue 3, 1976

Additional antigenic sites, distinct from those present on spherical 20 nm diam. particles of hepatitis B surface antigen (HBsAg), are exposed on the surface of Dane particles and tubular forms of HBsAg. The immunological relationship of these sites to e-antigen, an antigen detected earlier in HBsAg-positive sera from patients with chronic hepatitis, cirrhosis or acute hepatitis but not in healthy HBsAg-carriers, was established by immune electron microscopy and affinity chromatography. These findings suggest that e-antigen may be potentially useful in active immunization against hepatitis B.

In the presence of poly-l-ornithine at 1 µg/ml, the uptake of [3H]-labelled particles of tobacco rattle virus (TRV) by tobacco mesophyll protoplasts during inoculation depended on the virus and protoplast concentrations in the inoculation mixture. Uptake decreased 15- to 30-fold in the absence of poly-l-ornithine. Substituting phosphate for citrate buffer in the inoculum greatly increased infection but had little effect on virus uptake. For infections detected by staining with fluorescent antibody to virus particles, the ID50 occurred at an uptake of 30 long TRV particles per protoplast using phosphate buffer, and 250 using citrate. Evidence was obtained that the phosphate-induced enhancement of infection is mediated through an effect on interaction of virus and poly-l-ornithine during pre-inoculation incubation. Batches of protoplasts differing in susceptibility to infection did not differ similarly in uptake of inoculum virus.

Electron microscopy of sections of freshly inoculated protoplasts revealed single TRV particles associated end-on with normal-looking plasmalemma, and aggregates that contained densely stained material plus TRV particles, associated with breaks in the plasmalemma, and adjacent to areas of intracytoplasmic vesiculation. Some aggregates entered the cytoplasm and some TRV particles entered the vesicles. The aggregates were on average smaller after inoculation with phosphate-containing than with citrate-containing inocula. It is suggested that mini-aggregates of the kind produced during incubation with phosphate play an important role in infection.

Four isolates of carnation ringspot virus showed a reversible aggregation which was dependent on the temperature and concentration of their preparations. At 7 mg/ml, preparations of two of these isolates aggregated at room temperature but at the same concentration the other two isolates required higher temperatures (40 °C) for aggregation. A fifth isolate formed aggregates of 12 virus particles and linked aggregates. Particles of the two isolates with less tendency to aggregate were almost completely dissociated by 0.4% sodium dodecyl sulphate (SDS) at pH 5 and by 0.1% SDS at pH 7. At pH 7, an RNA component of mol. wt. 0.5 × 106 was released by lower concentrations of SDS than was an RNA component of 1.5 × 106. At pH 5, the sedimentation rates of unaggregated particles of the remaining three aggregating strains were unaffected by up to 15% SDS. However, treatment with 0.0075 to 0.05% SDS at pH 7 produced protein, RNA and swollen virus particles. These swollen particles were not further affected by increasing the SDS concentration to 8%.

Isolated tobacco mesophyll protoplasts were inoculated in vitro with tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) either as a mixture or one after another. Fluorescent antibody staining demonstrated that about 80% of protoplasts produced both viruses following mixed inoculation, and 70% after sequential inoculation. Neither synergism nor antagonism was apparent between TMV and CMV in the establishment of infection. Double infection of protoplasts occurred also with potato virus X (PVX) and TMV, or with PVX and CMV. Inoculation with a mixture of TMV, CMV and PVX caused infection of some protoplasts by all three viruses.

Bovine enterovirus-1 (BEV-1) infection results in the production of a low amount of infective virus. A large number of non-infectious virus particles can be detected in BEV-1 lysates by haemagglutination. Attempts to isolate DI particles that might be responsible for this effect failed. However, infected cells were shown to contain large amounts of 80S particles as well as lesser amounts of 160S, 130S, 45S, 14S and 5S particles. The proportion of these subviral particles detectable by density gradient sedimentation depended on the ionic strength of the gradient buffer. At high ionic strength 130S particles were transformed into 160S particles, and 45S into 80S particles. The polypeptide composition of each virus particle was examined. Pulse-chase experiments confirmed that 80S particles were the predominant virus particles accumulating. No precursor-product relationship could be established for the 80S particle, although 5S and 14S particles were shown to be precursors of mature virus particles.

Persistent states of measles virus infection have been established in HeLa cells by using Edmonston strain virus and two types of measles virus vaccine (M-VAC and Schwarz). The absolute amount of surface viral antigens expressed on these cells infected separately with the three viruses has been assessed by a newly developed method which employs [125I]-labelled Fab fragments of immunoglobulin G (IgG) from immune human sera. This method was used to determine the level of viral antigenic expression on acutely infected HeLa cells harvested at a time when 95 to 100% of cells could be lysed by antiviral antibody and complement. From our data, more than 1 × 106 antibody molecules must bind to each cell infected with measles virus before complement dependent lysis can occur in a homologous test system. Persistently infected cells bind 2 to 3 times less antibody than acutely infected cells and correspondingly exhibit less susceptibility to humorally-mediated immune lysis.

The replication of rodent parvovirus X14 DNA has been studied in rat embryo tissue culture cells. Virus DNA was isolated from 1 m-NaCl-SDS-pronase supernatant fluids from 24 h after infection. The majority of this DNA was 1.7 µm in length and double-stranded, indicating that it was an intermediate in the replication cycle of this single-stranded DNA virus. Single-stranded DNA of equivalent length was isolated directly from X14 virions. The buoyant density of this DNA was 1.728 g/ml whereas the double-stranded form banded at 1.714 g/ml in caesium chloride gradients. Difficulties in detecting significant amounts of single-stranded viral DNA directly from infected cells would appear to indicate that progeny single-stranded DNA is rapidly encapsidated after synthesis.

An enterovirus (EVU-16) previously isolated from children with acute infectious lymphocytosis has been further characterized. The EVU-16 virus sediments as a 135S particle in sucrose gradients, has a density of 1.335 g/ml in CsCl, contains 4 polypeptides and has a single stranded RNA genome sedimenting at 35S. These structural features as well as the presence of a virus-related particle, the procapsid, are similar to those of other enteroviruses. However, the largest polypeptide of EVU-16 is 49 000 daltons, which is considerably larger than the corresponding polypeptide from poliovirus; the sizes of the other three viral polypeptides were similar in both viruses. Attempts to induce lymphocytosis by the inoculation of EVU-16 into various animals, including immunologically aberrant ‘nude’ mice, were unsuccessful.

A cytoplasmic polyhedrosis virus (CPV) isolated from larvae of Arctia caja contained at least 12 RNA segments when these were fractionated by poly-acrylamide gel electrophoresis. When the virus RNA was compared with RNA isolated from Nymphalis io CPV and from Spodoptera exempta CPV, it appeared that A. caja CPV could be interpreted as a mixture of these two viruses. The structural polypeptides of virus particles of A. caja CPV also resembled a mixture of proteins derived from the other two virus types, but two classes of particles were not separated by size or density measurements. Virus particles of S. exempta CPV and N. io CPV were unrelated serologically using antisera that did not react with double-stranded RNA. Particles of A. caja CPV were resolved into two fractions using the antiserum specific for S. exempta CPV; particles containing RNA segments characteristic of S. exempta CPV and particles containing RNA segments characteristic of N. io CPV. This confirmed that the A. caja CPV isolate was a naturally-occurring mixture of two virus types.

The human papovavirus, BK, appeared weakly oncogenic in newborn hamsters and was able to induce in vitro transformation of rat kidney cells. The infectivity of BK virus DNA was determined by employing the DEAE-dextran method. In human embryonic cells the infectivity was approx. 105 p.f.u./µg of DNA. The transforming ability of BK virus in primary rat kidney cells was measured by employing the calcium method and appeared to vary from 1 to 10 foci per µg of DNA.

The conclusion by Barker (1975) that the four proteins previously thought to occur only in virus-infected tobacco leaves are probably normal constituents is based on an erroneous interpretation of the banding pattern obtained by polyacrylamide gel electrophoresis. Patterns shown by Barker for non-infected and water-injected tobacco leaves are similar to those for non-inoculated or water-inoculated plants from our laboratory. ‘New’ protein components induced by virus infection relate to bands not observed under the above-mentioned conditions. This is illustrated by a comparison of protein patterns from water-inoculated and TMV-inoculated tobacco plants, obtained under various conditions and after staining with either amido black or Coomassie blue. Preferential extraction of the new protein components at low pH further supports the view that these components do not occur in non-infected plants.