key: cord-0039762-t9tlijpp
authors: Williams, F.P.
title: Virus-like particles with T = 19 icosahedral symmetry in a human gastroenteritis stool
date: 2002-10-09
journal: nan
DOI: 10.1016/0739-6260(85)90061-9
sha: 3f8dae07ae3139ed366300d62d7e3bfa6e9ed28e
doc_id: 39762
cord_uid: t9tlijpp

Virus-like particles not previously described were observed in a human gastroenteritis stool using negative-stain TEM. The stool was among a number of acute-phase illness stools which had been collected in Egypt during 1980. The particles measured 65–70 nm in diameter, and it was possible to detect individual capsomeres on many of these particles. The capsomeric pattern identified on the particles corresponded to an icosahedrally symmetric T = 19 capsid. Distinctive five-fold vertices, usually appearing as darker spots on the capsid, were an additional feature of these particles. The capsid structure, which is skew, could readily be distinguished from the T = 25 capsid of adenovirus and the holey capsids of rotavirus and reovirus. Antibody to the particles was detected in both the shedding individual's acute- and convalescent-phase serum specimens using IEM, although an antibody increase was not demonstrated.

Direct TEM examination of the stool material been of fundamental importance in the detection was carried out using the following procedure. of gastroenteritis viruses, such as rotavirus, For each specimen, a moderately turbid suspenwhich eluded detection by routine cell-culture sion of stool was made in distilled water and a methods. In the examination of illness stool drop placed on a 200-mesh copper grid with specimens, TEM permits the detection and carbon support film. After 1 mm, excess material differentiation of a number of virus types based was removed with a piece of filter paper and the on the characteristic virus morphologies ob-grid rinsed with one or two drops of distilled served after negative-staining. Although other water. The grid was then stained with 2°/a detection methods have been developed for phosphotungstic acid (PTA), pH 7. Excess stain certain viruses (Yolken et a!., 1977;  Greenberg et was removed with the filter paper. After drying, al., 1978; Nakata et al., 1983) , negative-stain the grid was examined at 80 kV on a JEOL JEM-TEM retains the advantage of being able to IOOCX electron microscope for the presence of detect a range of antigenically unrelated viruses virus. One hundred and twenty-eight stool speciduring a single examination and includes the mens were examined in this manner. The specipossibility of observing new viruses. mens had been stored at -70°Csince collection. This method was employed in an examination Poliovirus was used as the reference standard for of the stools of individuals suffering respiratory virus-particle size determination. In addition to or gastrointestinal illness after swimming at a rotavirus and adenovirus particles observed in polluted beach near Alexandria, Egypt in 1980. some of the stools, cell-culture propagated Fe-Expected rotavirus and adenovirus particles ovirus (types 1-3), adenovirus (type 7), and were observed in some of the stools; however, in simian rotavirus (SA 11) particles were examined one stool a new type of virus-like particle was for morphological comparison to the newly observed. These particles, measuring 65-70 nm observed particles. in diameter, are described in this report.

Immune electron microscopy (IEM) was used to detect antibody to the new virus-like particles al b in the shedding individual's serum specimens. A distilled water suspension of the particle-___________ containing stool was carefully examined to assure the absence of antibody-coated or aggregated particles. Aliquots of 0.04 ml were _______ delivered by pipet into six wells of a microtiter plate. The individual's acute-and convalescentphase serum specimens were diluted 1: 10 in phosphate-buffered saline (PBS), pH 7.4, and ci dl 0.02 ml volumes were added to two of the wells. Acute-and convalescent-phase serum specimens (1:10 dilutions in PBS) from a rotavirus -_______________ ________________ shedding individual in the U.S. were also added -.

to two wells in 0.02 ml volumes. This individual's serum pair was known to demonstrate a seroresponse to rotavirus by both IEM and radioimmunoassay (RIA). To the remaining two wells were added 0.02 ml volumes of PBS without serum. The microtiter plate was briefly shaken, left at ambient temperature (24 C) for 15 mm, and then placed at 4°C overnight. The next ___________________________________ morning, the contents of the wells were examined with the electron microscope. A serum--stool mixture was considered positive if antibodycoated or aggregated particles were observed. Each of the mixtures was coded prior to examination and their identities not established until Fig Throughout the study, the grids were always revealing large area ofcapsomeric detail. (d) Same is (c)~tth inserted into the electron microscope with the the locations of the 6-coordinated capsomeres (while dots) specimen side nearest the electron source. For and the 5-coordinated capsorneres (white circles) indicated. printing, the EM plates were inserted into the The more darkly-stained areas atthe five-fold vertices suggest enlarger with the emulsion side away from the that the 5-coordinated (vertex) capsomeres are either more stain-penetrable, or more readily lost than the 6-coordinated photographic paper. Image enhancement of capsomeres. Bar=50 nm for (a d). )e) Image enhanced specific electron micrographs was performed micrograph (from Fig. 2b . n=3) with one capsomeric path using the rotation technique of Markham et al.

between five-fold vertices indicated. Bar =25 (1963) .

These particles were visually striking and more easily observable against stool background RESULTS material. The mean core diameter of these stain-The morphologically distinct particles which penetrated particles was 42 ±1 nm. The particles were observed in the acute-phase gastroenteritis resembled stain-penetrated particles of rotavirus. stool of a 5-year-old girl are shown in Fig. 1 . although the distinctive smooth rim of rotavirus Capsomeric detail was observable on many ofthe was not observed. particles. It was relatively easy to detect the On certain particles, virtually the entire capicosahedral nature of the capsid structure as the someric pattern of one side could be identified five-fold vertices appeared as darker spots on the ( Fig. Ic and d) . One capsomeric path between capsid (Fig. I a) . The capsomeres at these pos-neighbouring five-fold vertices is indicated on the itions appeared to be more stain-penetrable or image enhanced micrograph in Fig. Ic . All corthey were more readily lost than the other responding paths observed on other particles capsomeres. The mean diameter of 35 particles were the same. The observed structure cor-±SD was 67±2nm. Frequently, the particles responds to the icosahedral structure proposed were seen with stain-penetrated cores (Fig. ib) . by Caspar and KIug (1962) with a number=19. The structure is of skew class Fig. 2a, n=3 for Fig. 2b and n=5 for Fig. 2c . P=h 2+hk+k2=19 (h=3, k=2) with f=l. Rotations using other values of n resulted in Virus particles possessing this structure have not blurring of detail. previously been reported. Although the apparent Comparison of the T = 19 particles with rotahand indicated by the electron micrographs is virus and adenovirus particles observed in other levo, the absolute hand was not established, stools, and with reovirus from cell culture, is Figure 2 shows three particles viewed along shown in Fig. 3 . Although stain-penetrated axes of two-fold (a), three-fold (b) and five-fold T= 19 particles might be confused with stain-(c) rotational symmetry. Markham rotations for penetrated rotavirus particles, the more intact different values of n are shown below the original T= 19 particles are quite distinct from all three micrographs. Noted image enhancement was virus types. The holey capsids observed with observed at the expected values of n = 2 for rotavirus and reovirus particles ( Fig. 4a and b) l~c l~. Fig. 2 . The T= 19 particles viewed along axes of(a) two-fold, (b) three-fold and (C) five-fold rotational symmein. Markham rotations fordifferent values of n appear below the original micrographs. Slight image enhancement of (a) can be seen with n=2, but note the enhancement of (b) at n=3 and (c) at n=5. Bar=25 mm can be seen to be fundamentally different from DISCUSSION the capsid structure of the T= 19 particles. It~5

The 65-70 nm virus-like particles described in the holes which are the vertex points which have this report were distinct from all stool viruses been used to determine the T number for these previously observed in this laboratory. The two viruses (Palmer and Martin, 1982) . Although particles were readily distinguished from rota-T number determination for rotavitus and revirus, reovirus and adenovirus, and did not ovirus has not been clear-cut, recent studies appear to be an altered or degraded form of these suggest T= 13 icosahedral symmetry for both viruses. The T= 19 icosahedral symmetry of viruses (Roseto et a!., 1979; Metcalf. 1982 ; these particles has not been reported for other Khaustov et U!,, 1984) .

known viruses, and published micrographs of The capsid structure of adenovirus (Fig. 4c) pararotavirus (Espejo eta!., 1984) , Hantaan virus has been well-established for some time (Home (Lee and Cho, 1981; McCormick ef a!., 1982 McCormick ef a!., ). eta!., 1959 and the T=25 icosahedral symmetry and novel virus-like particles in canine enteritis of this virus is easily distinguished from the skew stools (Hill and Yang, 1984) also suggest parsymmetry of the T= 19 particles. tides different from those described here. The coded serum analysis by IEM revealed the The limited amount of specimen material presence of antibody activity against the T 19 available has prevented extensive IEM and other particles, only in the shedding individual's serum testing. Although the T= 19 particles would specimens. Antibody-coated and aggregated par-appear to represent a new virus to be recognized tides (Fig. 5) were observed in the reaction in stools, confirmation must await successful mixtures of both this individual's acute-and propagation of the agent. Unfortunately, atconvalescent-phase serum specimens, although tempts to propagate the particles in MA-104. 

2: 97 108. enhancement ofimage detail and accentuation of structure

Microtiter solid-phase radio-BioI., I : 84 86. immunoassay for detection of human calicis rut in stools

The structure of the internal capsid of

Structure of rotaviruses as studied h~the reeve-McCormick

Ln,',iiie-Bun~aviridae. Lancet, 1: 765 768. linked immunosorhent assay (ELISA) for detection of Macnaughton