Physical, Chemical and Morphologic Dimensions of Human Hepatitis A Virus Strain CR326

Abstract

A previous report ( I ) from these laboratories described the recovery in S. mystax marmosets of CR326 and related strains of human hepatitis A virus from cases of hepatitis A that occurred in Costa Rica. The etiologic relationship of the CR326 virus to hepatitis in man was established based on the demonstration of physical-chemical properties considered characteristic of human hepatitis A virus (viz., small size and heat, ether and acid stability) (2), and on the development in patients with hepatitis A of neutralizing (2), and complement fixing (3) antibodies against the virus. The present report extends these findings to include electron microscopic demonstration of the virus intracellularly and in purified form, and a definition of the reaction of purified virus to heat (60" and loo"), ultraviolet irradiation, and formalin. These data, together with current evidence for RNA composition, fix hepatitis A as a likely enterovirus.Materials and Methods. Virus and host system. The origin, history, and marmoset propagation of CR326 strain human hepatitis A virus were described previously ( I , 2). These same reports described the tests for serum enzymes, and for infectious virus, and the conduct of the serum neutralization test.Isopycnic banding of hepatitis A virus and viral density determination. Three ml of CR326-infected serum from marmosets containing about 104-5 ID5o/ml was applied to 36 ml of cesium chloride gradient formed by conventional procedure and covering the density range as given in Fig. 1. Thirteen 3 ml fractions were collected after centrifugation at 23,000 rpm for 18 hr in a Beckman SW-27 rotor. The density of each fraction was measured by refractive index and the densities of certain of the fractions were also determined by direct weighing. Each gradient fraction was diluted 1:3000, a dilution at which the fraction tested would contain about 10 ID50/ml if the total amount of virus were present in that fraction. For testing gradient samples, groups of six marmosets were given 1.0 ml each, intravenously, and these were followed for serum enzyme elevations that indicated infection with the virus.Effect of heat, ultraviolet irradiation and formalin on purijed CR326 virus. Fractions 3 and 4 from a cesium chloride gradient (see Fig. 1) were pooled and successively dialyzed against phosphate buffered saline solution (PBS) and distilled water. The infectivity titer, in marmoset titration, was about 104.5 IDS0/ml and the protein content (4) was less than 6 pg/ml. This material was used to carry out the stability tests. Infectivity determinations were carried out in marmosets given 1 ml of test material intravenously. Appropriate positive and negative control animals were included. Heat, 60". The purified virus was diluted 1: 100 in distilled water and heated 1 hr in a sealed glass ampoule that was fully immersed in water at 60". A total of 24 S. mystax marmosets were given the heated preparation. A similar experiment had been carried out employing unpurified virus in marmoset serum diluted 1: 100 (2). Hea...

Small Virus Particles in Faeces of Patients with Infectious Hepatitis (Hepatitis A)

“…This observation contrasts with those of Feinstone et al (1973) and Provost et al (1975), who found 27-nm particles associated with hepatitis A. However, Thornton, Zuckerman and Almeida (1975) have recently described particles of variable diameter in faeces from a chimpanzee infected with MS-1 agent; their particles ranged in size from 24 to 29 nm.…”

Properties of a Hepatitis A Virus Candidate Vaccine Strain

“…In agreement with Provost et al (1975) we assume that the lower density value for HAV represents an association with lipids. , however, reported contradictory data: after extraction with chloroform, the buoyant density of light HAV particles (1.27 g/ml) propagated in BS-C-1 cells was unchanged.…”

Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation

“…These results suggest that HEV virions in serum possess the ORF3 protein on their surfaces, in association with lipids, similar to those in the supernatant of cultured cells (63), and would reconcile the apparently contradictory observation that IgM and IgA antibodies, which are capable of viral neutralization, and infectious virus coexist in acute-phase serum specimens, as reported for hepatitis A virus (HAV) (19,35). Provost et al (34) reported that the infectious HAV present in acute-phase marmoset sera banded at two densities in CsCl (1.34 g/ml and 1.15 g/ml), suggesting that the less-dense particles represent lipid-associated virions. A similar banding pattern was observed by Lemon and Binn (20), who reported that the HAV released from infected BS-C-1 cells was incompletely neutralized when it was incubated with a variety of convalescent-phase sera (nonneutralizable fraction of 17 to 32%) and that chloroform extraction of virus resulted in a substantial reduction of the nonneutralizable fraction (to Ͻ1%).…”

“…Although lipid-associated virions of other nonenveloped viruses, such as HAV, have been reported (20,34,37), the underlying mechanism by which HEV particles in the circulation can be propagated in cultured cells, similar to those in culture supernatant, despite the fact that they are seemingly "enveloped" and nonneutralizable by immune sera, needs to be clarified in future studies.…”