Takeru Urayama

Extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters

Background and Objective  To investigate the physico‐chemical properties of hepatitis E virus (HEV) with regard to inactivation/removal, we have studied four isolates with respect to sensitivity to heat during liquid/dry‐heating as well as removal by nanofiltration.

Variability of parvovirus B19 to inactivation by liquid heating in plasma products

Background and Objectives  Previously, we reported that although human parvovirus B19 in albumin and intravenous immunoglobulin preparations was rapidly inactivated during liquid heating, in contrast to other parvoviruses such as canine parvovirus, sensitivity to heat was highly dependent on the composition of the solution. In this study, we aimed to further elucidate the sensitivity to heat of B19 in haptoglobin and antithrombin (previously named antithrombin III) preparations during liquid heating.

Inactivation of parvovirus B19 by liquid heating incorporated in the manufacturing process of human intravenous immunoglobulin preparations

Several reports have suggested the possible transmission of human parvovirus B19 (B19) through the administration of plasma derivatives that had undergone virus inactivation by various types of heat treatment. However, none of the reports evaluated and discussed the inactivation of B19 by the heat treatment that is implemented in the individual manufacturing processes of such products. The present study evaluated the ability to inactivate B19 of liquid‐heat treatment at 60°C for 10 h that was incorporated in the manufacturing process of intravenous human immunoglobulin preparations. The results showed that B19 was rapidly inactivated under the conditions used for the liquid‐heat treatment.

Significant neutralizing activity of human immunoglobulin preparations against pandemic 2009 H1N1

The influenza-like illness that began in the Unites States and Mexico was first reported by the World Health Organization (WHO) on 24 April, 2009, and declared a phase 6 pandemic on 11 June. As of 6 July 2009, over 90 000 cases and more than 400 deaths in some 120 countries had been confirmed (WHO, 2009). Importantly, on July 8th, the WHO announced that oseltamivir (Tamiflu)-resistant viruses had been identified in Denmark, Japan and Hong Kong (WHO, 2009). The pandemic virus 2009 H1N1 was a triple reassortant of human-, swine- and avian-derived influenza A virus segments and the HA gene was classified as being of swine-origin (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, (2009). Evidence is accumulating that specific IgG antibodies against this virus are present in certain populations, especially the elderly (Itoh et al, 2009). However, Katz et al (2009) reported that cross reactive IgG against a pandemic influenza virus (A/California/04/2009) was found in no serum specimens of children aged 6 months–9 years old, 8% of samples from 5- to 9-year olds, 9% of samples from 18- to 64-year olds, 6% of samples of 18- to 40-year olds and 33% of samples of those over 60 years old, suggesting that immunoglobulin preparations derived from pooled plasma from over 10 000 healthy donors could contain such cross reactive IgG. The present study evaluated haemagglutinin-inhibition (HI) and virus neutralization (VN) activities against 2009 H1N1 and seasonal H1N1 as a positive control in intravenous human immunoglobulin (IVIG) preparations manufactured in 1999 and 2008. An influenza A/H1N1 vaccine strain (A/New Caledonia/20/99), a clinical isolate of A/H1N1 (A/Osaka/16/2008), a classical swine isolate of A/H1N1 (A/Swine/Hokkaido/2/1981) and a pandemic influenza isolate of A/H1N1 (A/Osaka/168/2009 H1N1 pdm) were used in this study. Three lots (Lot. A, B and C) of IVIG derived from pooled plasma collected in Japan and manufactured in 2008 (IVIG2008JP, ‘Kenketsu Venoglobulin®-IH Yoshitomi; Benesis Corp., Osaka, Japan’) were also used. In addition, two lots of IVIG that were manufactured in 1999, derived from plasma pooled collected in Japan and the USA (IVIG1999JP ‘Kenketsu Venoglobulin®-IH’, IVIG1999US ‘Venoglobulin®-IH; Yoshitomi Pharmaceutical Industries, Ltd. at the time, currently Benesis Corp.’), were used. The viruses were propagated in Madin-Darby canine kidney (MDCK) cells or in the allantoic cavity of chicken embryonated eggs. The culture media and the allantoic fluids were stored at −80°C prior to use. Infectivity, as infectious focus-forming units (FFU) per ml, was titrated in MDCK cells using peroxidase and an anti-peroxidase (PAP) staining technique (Okuno et al, 1990). The haemagglutinin-inhibition (HI) test using 0·75% guinea pig red blood cells was carried out as described previously (Okuno et al, 1993). The results were expressed as the reciprocal of the highest dilution of the culture medium to show inhibition. The virus neutralization (VN) test was carried out as described (Okuno et al, 1990). Briefly, IVIG was diluted twofold with serum-free medium. The diluted IVIG (50 μl) was mixed with 100 FFU (50 μl) of virus, then applied to MDCK cells in a 96-well microplate. After culturing, the cells were fixed with ethanol and stained by PAP as above. The results were expressed as the reciprocal of the dilution giving 50% neutralization. Intravenous human immunoglobulins were manufactured using plasma pooled from over 10 000 healthy donors. The HI and VN activities of IVIGs were titrated against pandemic, seasonal human and swine influenza A viruses (Table I). Of note, both the 1999 and 2008 IVIGs were shown to have anti pandemic and classical swine influenza A/H1N1 virus titres with HI (×4–×8) and VN (×32–×64). The 2008 IVIGs showed titres against the vaccine strain A/New Caledonia/20/99, which was isolated in 1999, with HI (×160–×320) and VN (×640–×1280), while the 1999 IVIGs showed titres with HI (×10–×40) and VN (×32–×128). These results suggested that the IVIG derived from the pooled plasma contained a certain amount of functional IgG, including IgG against pandemic or classical swine influenza A/H1N1. Of note, such IgG titres were slightly higher in the IVIG2008JP products compared with IVIG1999JP. However, the titres were slightly higher in IVIG1999US than in IVIG1999JP. Higher titres against the vaccine and clinical strains were observed in IVIG1999US than IVIG1999JP. Interestingly, the difference in the increase in titres against the vaccine strain was much greater between the products manufactured in 2008 and 1999 than between the others. This difference seems to be an outcome of vaccination. Our preliminary results showed a HI titre >×40 in 1·2% (7/580), ×20 in 3·1% (18/580) and ×10 in 4·3% (25/580), indicating the possible production of hyperimmune globulin with these sources of plasma collected in 2008, Japan. Table I Cross reactivity of several lots of IVIG against pandemic 2009, classical swine and seasonal H1N1 viruses.

Heat sensitivity of a SARS-associated coronavirus introduced into plasma products

Background and Objectives  Various measures to inactivate/remove viruses have been implemented for manufacturing plasma‐derived products. Here, we examined the heat inactivation ability of an agent of the severe acute respiratory syndrome (SARS), SARS coronavirus (CoV).

Possible removal of prion agents from blood products during the manufacturing process

Blood products prepared from human blood theoretically risk contamination with infectious pathogens. Since recent reports now confirm the likely transmission of pathogenic prions through blood transfusion, effective measures to prevent transmission are required globally, although the prevalence of variant Creutzfeldt–Jakob disease outside of the UK is extremely low. Many studies evaluating the manufacturing process have been conducted for the potential removal of the prion protein from plasma derivatives. In this review, we discuss the possibility of removing prions via several processing steps, especially depth and virus-removal filtration. Through a discussion of the limitations and issues associated with such studies, we hope our review will be of help for better study design in the future.

Variability of parvovirus B19 genotype 2 in plasma products with different compositions in the inactivation sensitivity by liquid-heating

Background and Objectives  Our previous report showed that parvovirus B19 genotype 1 in different solutions derived from plasma preparations showed different heat‐sensitivity patterns during liquid‐heating. In this study, we similarly examined B19 genotype 2.

Protective Role of Human Intravenous Immunoglobulin from Influenza A Virus Infection in Mice

Intravenous immunoglobulin (IVIG) has been manufactured from pooled plasma of 10,000 or more units from healthy donors. Recently, we reported that the IVIG manufactured even before the 2009 influenza pandemic contained antibodies reactive to seasonal H1N1 and pandemic H1N1 2009 (H1N1 pdm) viruses. In this study, we used an animal model to evaluate the efficacy of IVIG against influenza infections. A seasonal influenza H1N1 strain (New Caledonia, A/NC/20/99) and an H1N1 pdm strain (A/Osaka/168/2009) were used. The BALB/c and severe combined immuno- deficiency mice (SCID; C.B-17/lcr-scid/scid) were also used. Mice inoculated with A/NC/20/99 or A/Osaka/168/2009 were administrated IVIG and monitored for 3 weeks. The administration of IVIG 48 h before and after inoculation with a mouse-adapted seasonal H1N1 virus, resulted in survival rates of 80 and 88%, respectively. The rate among control mice was 30%. In addition, infectivity in lungs from IVIG-treated mice also decreased significantly. Similar effects of IVIG on the survival rate were obtained with H1N1 pdm. Thus, IVIG was shown to be effective against both viruses in mice.

Full-length sequences of one genotype 4 and three genotype 3 Hepatitis E viruses in fecal samples from domestic swine in Japan.

The Hepatitis E virus (HEV) induces zoonotic infections and causes hepatitis. In Japan, HEV occurs in deer, wild boar and swine, and genotype (G)3 and G4 have been isolated from domestic swine. We previously reported that HEV isolates from a total of 320 swine fecal samples from 32 farms in Japan could be predominantly classified into four clusters: three G3 (G3JP, G3SP and G3US) and one G4 (G4JP). In this study, we performed full-length sequencing of four representative HEVs, one from each of the clusters. We found significant nucleotide variation throughout the sequences within a genotype, but not within each cluster. However, we found few variations at the amino acid level. Most of the highly conserved regions within genotypes were concentrated in the overlapping region of open reading frame (ORF)2 and ORF3, while most of the variable regions were within the ORF1 V region. This region was variable even at the amino acid level. Essentially, this region was highly conserved among G3 clusters, with some more dissimilarities between G3SP and the other two clusters, G3JP and G3US. The regions conserved and variable across genotypes had virtually the same positions as those within genotypes, but were much narrower and wider, respectively. For the latter, ORF1 V and P regions were especially variable. Finally, we focused on the sequence conservation in the region widely used for primer and probe sets to detect HEV infections.

Sequence Variation in Hepatitis E Virus Genotypes 3 and 4 from Swine Fecal Samples in Japan

Hepatitis E virus (HEV) is a causative agent for hepatitis. HEV is transmitted via the fecal-oral route through contaminated drinking water and induces zoonotic infections through eating uncooked and undercooked meat of deer, wild boar, and swine. In Japan, genotypes 3 (G3) and 4 (G4) are prevalent in domestic swine. Here, we examined the genetic variation among HEVs derived from swine fecal samples in Japan. A total of 320 samples were collected at 32 commercial farm facilities (1 fecal sample from each of 10 pig houses in individual farms). Viral RNA amplification at open reading frame (ORF) 3 was possible in 159 (49.7%) of the fecal samples. For genotyping, the same samples were subjected to amplification at ORF2 and the resulting amplicons were sequenced. The results revealed that all the HEVs in each farm belonged to the same cluster of G3 and G4: G3JP in 8 farms, G3SP in 4 farms, G3US in 6 farms, and G4JP in 2 farms, unclassified G3 in 2 farms, unable to decide due to a low rate of amplification in 5 farms, and no detection in 5 farms. Interestingly, the HEVs from one farm were more homogeneous than those of the same cluster that was derived from other farms. Thus, the efficiency of farm-to-farm transmission of HEVs is likely to be low and HEV seems to have evolved independently at each farm in Japan.