Objective: To determine the copy number of hepatitis C virus (HCV) RNA, determined by nucleic acid amplification test (NAT) for screening blood units in Japan, that can transmit infection to chimpanzees. Methods: Fresh-frozen plasma with markers of HCV infection, as well as inocula pedigreed from 1 of them, were evaluated for the infectious activity in chimpanzees. Results: One unit each (273–282 ml) of fresh-frozen plasma from 2 blood donors or a pool from 13 donors to make a unit, which contained high-titered antibody to HCV but without HCV RNA detectable by NAT, did not infect any of 3 chimpanzees. Two chimpanzees were infected, however, when they were inoculated with 1 ml of serum from a blood donor in the ‘window period’ of HCV infection and containing 7.0 × 106 copies/ml of HCV RNA. The preacute phase serum from 1 of them harvested 7 weeks after the inoculation was titrated in 2 chimpanzees, and an inoculum containing approximately 2 × 101 copies of HCV RNA could transmit infection to both of them. Conclusion: Approximately 20 copies of HCV can transmit infection to recipients, which needs to be taken into consideration in planning the screening of blood units for HCV RNA by NAT. Although the sensitivity of present NAT could be improved further, there would be a limit of it in detecting a low-level HCV RNA in the window period of donors with the infectious capacity in recipients.
The sensitivity of the 50-sample pool MPX NAT system was higher than that of individual HBsAg screening by CLIA. By adopting this NAT-screening system, the JRC has improved the safety of the blood supply and maintained supply across Japan.
Two chimpanzees were inoculated with hepatitis C virus (HCV) and followed on a daily basis for 12 days. HCV RNA became detectable in their sera on day 5 by polymerase chain reaction with the detection limit of 102 copies/ml. Based on an exponential growth observed until 8 or 9 days after inoculation in their sera, the doubling time of HCV in the circulation was estimated at 6.3–8.6 h and log time (time required to grow 10-fold) at 31.3– 42.9 h. The exact doubling time of HCV determined in them would help plan an efficient strategy for screening out blood donors in the window period of infection between the exposure and the development of antibody to HCV in serum.
Hepatitis B e Ag (HBeAg) was isolated from pooled sera of carriers, without abnormalities in liver function, by affinity column chromatography with mAb against HBeAg. HBeAg polypeptide with an estimated molecular size of 20,000 Da (p20e) was detected, in addition to regular HBeAg polypeptides (p17e/p18e). p20e, as well as p17e/p18e, did not bind with mAb against the carboxyl-terminal domain of the C-gene product. p20e disclosed an N-terminal sequence of MQLFHLXLII- (X unknown), whereas p17e had that of SKLXLGXLXGMDIDPXKEFG- (X's unknown). By comparing them with the amino acid sequence encoded by the precore region and C gene of hepatitis B virus DNA, p20e was deduced to possess amino acids 1 to 19 of the precore-region product at the N-terminus, which contains signal sequence and usually removed before the secretion of HBeAg. p17e had amino acids 20 to 29 of the precore-region product that continued to the C-gene product. Inasmuch as p20e was invariably detected in HBeAg preparations from carriers without evidence for liver disease, it would not have been released into the circulation from destructed hepatocytes. HBeAg polypeptide bearing an uncleaved signal sequence would help in further understanding the mechanism of HBeAg secretion.
In the present study 5 patients with common variable hypogammaglobulinemia (CVH) and 4 patients with selective IgA deficiency (IgA-D) were analyzed for the cellular defects responsible for impaired Ig synthesis with use of peripheral blood lymphocytes stimulated with either PWM or EBV in vitro. By the use of co-culture with PWM, all the patients examined had intrinsic B cell defects restricted to the synthesis of Ig class corresponding to the low or absent Ig class(es) in the sera. Two types of excessive suppressor T activity were found, which were abrogated by irradiation. One was isotype-nonspecific and the other was IgA-specific. Moreover, failure of IgA-specific helper T activity was demonstrated. The use of EBV as an agent that polyclonally activates B cells independently of T cells and monocytes should allow a clearer delineation of the level of the B cell defects. When co-cultured with EBV, B cells from 3 patients with CVH produced normal to subnormal quantities of IgM although they could produce no IgM upon co-culturing with normal T cells and PWM. B cells from 2 patients with CVH could produce IgM normally by stimulation with either PWM or EBV; however, there was no restoration to produce IgG or IgA in these patients. In addition, B cells from 2 patients with IgA-D produced not only IgG and IgM but also IgA almost normally at 4 days after in vitro stimulation with EBV.
There are four polypeptides coded for by the region Pre-S and gene S on DNA of hepatitis B virus that carry the receptor for polymerized human serum albumin (poly-HSA), i.e., P31 and P39, as well as their glycosylated counterparts P35 and P43. With the use of monoclonal antibodies directed to Pre-S(1) sequence and Pre-S(2) sequence (bearing the receptor for poly-HSA), the content of these polypeptides, as well as their expression on the surface, was determined for hepatitis B particles of various categories. P39 and P43, carrying both Pre-S(1) and Pre-S(2) sequences, were contained abundantly in Dane and tubular particles, and to a much lesser extent in small spherical particles, all of which were purified from plasma containing hepatitis B e antigen (HBeAg). P31 and P35, carrying Pre-S(2) but not Pre-S(1) sequence, were contained comparably in these three categories of hepatitis B particles. In remarkable contrast, small spherical particles derived from plasma containing antibody to HBeAg were very low in the content of any Pre-S polypeptides. P31 and P39 showed higher activities for poly-HSA receptor than their glycosylated versions. When Dane particles were digested with trypsin, the poly-HSA receptor was deprived in parallel with the loss of antigenicity for Pre-S(2) sequence. The antigenicity for Pre-S(1) sequence was much less affected, and that for the product of gene S was virtually unchanged by the digestion.
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