Phenotypic characterization of cell culture-derived hepatitis E virus subjected to different chemical treatments: Application in virus removal via nanofiltration.

Safety evaluation for the hepatitis E virus (HEV) is required for plasma fractionation products. Plasma-derived HEV (pHEV) is quite unique in that it is associated with a lipid membrane, which, when stripped during manufacturing processes, induces morphological changes in the virus, making it difficult to select proper HEV phenotypes for clearance studies. We developed a convenient system for the preparation of a high titer cell culture-derived HEV (cHEV). In this system, PLC/PRF/5 cells transfected with the wild-type HEV genome generated lipid membrane-associated cHEV for a long period even after cryopreservation. We also examined how this lipid membrane-associated cHEV can be used to verify the robustness of pHEV removal via 19-nm nanofiltration. Sodium-deoxycholate and trypsin (NaDOC/T) treatment not only dissolved lipid but also digested membrane-associated proteins from pHEV and cHEV, making the resulting cHEV particle smaller in size than any pHEV phenotypes generated by ethanol or solvent-detergent treatment in this study. In both 19-nm and 35-nm nanofiltration, cHEV behaved identically to pHEV. These results indicate that cHEV is a useful resource for viral clearance studies in term of availability, and the use of NaDOC/T-treated cHEV ensured robust pHEV removal capacity via 19-nm nanofiltration.

Quantitative PCR evaluation of parvovirus B19 removal via nanofiltration.

When human parvovirus B19 (B19) is removed from plasma-derived products by nanofiltration, non-infectious fragmented B19 DNA in filtrate prevents quantitative real time PCR (qPCR) from accurately evaluating reduction of the virus particles. To determine optimal target sequence length for detection of full-length B19 genome in the viral particles by qPCR, we analyzed 4 different sequences ranging from 372 to 1,980 bp and found that a 989 bp sequence shows a well-balanced performance for the sensitivity and the run time. Nuclease treatment of filtrates prior to qPCR is also expected to decrease the influence of the residual B19 DNA, but extremely high protein concentration of plasma-derived products in filtrates may result in incomplete digestion of the B19 DNA. In this context, however, our analysis showed that even when B19 genome is incompletely digested, qPCR for the 989 bp sequence successfully eliminates the influence of the B19 DNA. Finally, we verified that when B19-spiked plasma products are subjected to nanofiltration with the resulting filtrates treated with nuclease, qPCR for the 989 bp sequence accurately evaluates B19 removal. These results demonstrate that qPCR for the 989 bp sequence combined with nuclease treatment enables convenient and accurate evaluation of B19 removal by nanofiltration.

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles.

Virus filtration with nanometer size exclusion membranes ("nanofiltration") is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18-26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled-down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.

Hepatitis E virus derived from different sources exhibits different behaviour in virus inactivation and/or removal studies with plasma derivatives.

Hepatitis E virus (HEV) causes viral hepatitis, and is considered a risk factor for blood products. Although some HEV inactivation/removal studies have been reported, detailed investigations of different manufacturing steps as heat treatment, partitioning during cold ethanol fractionation, low pH treatment, and virus filtration have yet to be reported for plasma-derived medicinal products. In this study, human serum- and swine faeces-derived HEVs, with and without detergent treatment, were used. The kinetic patterns of inactivation, log reduction value, or partitioning during the process were evaluated. In addition, the mouse encephalomyocarditis virus (EMCV) and canine and porcine parvoviruses (CPV/PPV) were also evaluated as model viruses for HEV. Small pore size (19 or 15 nm) virus filtration demonstrated effective removal of HEV. Middle pore size (35 nm) virus filtration and 60 °C liquid heating demonstrated moderate inactivation/removal. Ethanol fractionation steps demonstrated limited removal of HEV. Unpurified HEV exhibited different properties than the detergent-treated HEV, and both forms displayed differences when compared with EMCV, CPV, and PPV. Limited or no inactivation of HEV was observed during low pH treatment. Untreated plasma-derived HEV from humans showed different properties compared to that of HEV treated with detergent or derived from swine faeces. Therefore, HEV spike preparation requires more attention.

Caution in evaluation of removal of virus by filtration: Misinterpretation due to detection of viral genome fragments by PCR.

The testing of biological products at different stages of the manufacturing process currently involves quantitative polymerase chain reaction (Q-PCR)-based assays. Q-PCR techniques are able to detect not only the viral genome in viral particles but also fragments of degraded genome in samples. The ability of 15 and 19-nm filters to remove viruses was examined by conducting infectivity assays and Q-PCR assays using parvovirus B19 (B19), one of the smallest non-enveloped viruses. Although the filtered samples showed no infectivity, viral DNA was detected by Q-PCR. Interestingly, approximately 90% of the total viral genome in 15-nm filtrates had a detectable size of less than 0.5kb by the Q-PCR and as a consequence reduction factors were underestimated using Q-PCR. The reduction factors using Q-PCR might be underestimated due to the presence of a large amount of free B19 DNA which shows no infectivity in the tested filtrates. Therefore, the results of Q-PCR should be interpreted with caution. The careful design of primers is needed to eliminate amplification from fragments of viral DNA by Q-PCR.

Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells.

BACKGROUND: Rex1/Zfp42 has been extensively used as a marker for the undifferentiated state of pluripotent stem cells. However, its function in pluripotent stem cells including embryonic stem (ES) cells remained unclear although its involvement in visceral endoderm differentiation in F9 embryonal carcinoma (EC) cells was reported. RESULTS: We showed the function of Rex1 in mouse ES cells as well as in embryos using the conventional gene targeting strategy. Our results clearly indicated that Rex1 function is dispensable for both the maintenance of pluripotency in ES cells and the development of embryos. However, Rex1-/- ES cells showed the defect to induce a subset of the marker genes of visceral endoderm, when differentiated as embryoid body, as found in EC cells. CONCLUSION: Rex1 should be regarded just as a marker of pluripotency without functional significance like the activity of alkaline phosphatase.

Identification and characterization of subpopulations in undifferentiated ES cell culture.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells. The Rex1(-)/Oct3/4(+) and Rex1(+)/Oct3/4(+) populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells have characteristics similar to those of ICM and early-PrE cells, Rex1(+)/Oct3/4(+) cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1(-)/Oct3/4(+) cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.

Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells.

Although the POU transcription factor Oct3/4 is pivotal in maintaining self renewal of embryonic stem (ES) cells, little is known of its molecular mechanisms. We previously reported that the N-terminal transactivation domain of Oct3/4 is required for activation of Lefty1 expression (H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki, Mol. Cell. Biol. 22:1526-1536, 2002). Here we test whether Lefty1 is a direct target of Oct3/4. We identified an ES cell-specific enhancer upstream of the Lefty1 promoter that contains binding sites for Oct3/4 and Sox2. Unlike other known Oct3/4-Sox2-dependent enhancers, however, this enhancer element could not be activated by Oct3/4 and Sox2 in differentiated cells. By functional screening of ES-specific transcription factors, we found that Krüppel-like factor 4 (Klf4) cooperates with Oct3/4 and Sox2 to activate Lefty1 expression, and that Klf4 acts as a mediating factor that specifically binds to the proximal element of the Lefty1 promoter. DNA microarray analysis revealed that a subset of putative Oct3/4 target genes may be regulated in the same manner. Our findings shed light on a novel function of Oct3/4 in ES cells.

Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation.

Trophectoderm (TE), the first differentiated cell lineage of mammalian embryogenesis, forms the placenta, a structure unique to mammalian development. The differentiation of TE is a hallmark event in early mammalian development, but molecular mechanisms underlying this first differentiation event remain obscure. Embryonic stem (ES) cells can be induced to differentiate into the TE lineage by forced repression of the POU-family transcription factor, Oct3/4. We show here that this event can be mimicked by overexpression of Caudal-related homeobox 2 (Cdx2), which is sufficient to generate proper trophoblast stem (TS) cells. Cdx2 is dispensable for trophectoderm differentiation induced by Oct3/4 repression but essential for TS cell self-renewal. In preimplantation embryos, Cdx2 is initially coexpressed with Oct3/4 and they form a complex for the reciprocal repression of their target genes in ES cells. This suggests that reciprocal inhibition between lineage-specific transcription factors might be involved in the first differentiation event of mammalian development.