Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system.

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10 kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface.

The first report of porcine parvovirus 7 (PPV7) in Colombia demonstrates the presence of variants associated with modifications at the level of the VP2-capsid protein.

There are a wide variety of porcine parvoviruses (PPVs) referred to as PPV1 to PPV7. The latter was discovered in 2016 and later reported in some countries in America, Asia, and Europe. PPV7 as a pathogenic agent or coinfection with other pathogens causing disease has not yet been determined. In the present study, we report the identification of PPV7 for the first time in Colombia, where it was found retrospectively since 2015 in 40% of the provinces that make up the country (13/32), and the virus was ratified for 2018 in 4/5 provinces evaluated. Additionally, partial sequencing (nucleotides 380 to 4000) was performed of four Colombian strains completely covering the VP2 and NS1 viral genes. A sequence identity greater than 99% was found when comparing them with reference strains from the USA and China. In three of the four Colombian strains, an insertion of 15 nucleotides (five amino acids) was found in the PPV7-VP2 capsid protein (540-5554 nt; 180-184 aa). Based on this insertion, the VP2 phylogenetic analysis exhibited two well-differentiated evolutionarily related groups. To evaluate the impact of this insertion on the structure of the PPV7-VP2 capsid protein, the secondary structure of two different Colombian strains was predicted, and it was determined that the insertion is located in the coil region and not involved in significant changes in the structure of the protein. The 3D structure of the PPV7-VP2 capsid protein was determined by threading and homology modeling, and it was shown that the insertion did not imply a change in the shape of the protein. Additionally, it was determined that the insertion is not involved in suppressing a potential B cell epitope, although the increase in length of the epitope could affect the interaction with molecules that allow a specific immune response.

Osmolyte enhanced aqueous two-phase system for virus purification.

Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations. The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system.

Clearance of porcine circovirus and porcine parvovirus from porcine-derived pepsin by low pH inactivation and cation exchange chromatography.

The contamination of oral rotavirus vaccines by porcine circovirus (PCV) raised questions about potential PCV contamination of other biological products when porcine trypsin or pepsin is used in production process. Several methods can be potentially implemented as a safety barrier when animal derived trypsin or pepsin is used. Removal of PCV is difficult by the commonly used viral filters with the pore size cutoff of approximately 20 nm because of the smaller size of PCV particles that are around 17 nm. It was speculated that operating the chromatography step at a pH higher than pepsin's low pI, but lower than pIs, of most viruses would allow the pepsin to flow through the resin and be recovered from the flow through pool whilst the viruses would be retained on the resin. In this study, we investigated low pH inactivation of viruses including PCV Type 1 (PCV1) and PCV1 removal by cation exchange chromatography (CEX) in the presence of pepsin. Both parvovirus and PCV1 could be effectively inactivated by low pH and PCV1 could be removed by POROS 50HS CEX. The POROS 50HS method presented in this article is helpful for designing other CEX methods for the same purpose and not much difference would be expected for similar product intermediates and same process parameters. While the effectiveness needs to be confirmed for specific applications, the results demonstrate that both low pH (pH 1.7) and CEX methods were successful in eliminating PCV1 and thus either can be considered as an effective virus barrier.

Virus Isoelectric Point Determination Using Single-Particle Chemical Force Microscopy.

Virus colloidal behavior is governed by the interaction of the viral surface and the surrounding environment. One method to characterize the virus surface charge is the isoelectric point (pI). Traditional determination of virus pI has focused on the bulk characterization of a viral solution. However, virus capsids are extremely heterogeneous, and a single-particle method may give more information on the range of surface charge observed across a population. One method to measure the virus pI is chemical force microscopy (CFM). CFM is a single-particle technique that measures the adhesion force of a functionalized atomic force microscope (AFM) probe and, in this case, a virus covalently bound to a surface. Non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV) were used to demonstrate the use of CFM for viral particles with different surface properties. We have validated the CFM to determine the pI of PPV to be 4.8-5.1, which has a known pI value of 5.0 in the literature, and to predict the unknown pI of BVDV to be 4.3-4.5. Bulk measurements, ζ-potential, and aqueous two-phase system (ATPS) cross-partitioning methods were also used to validate the new CFM method for the virus pI. Most methods were in good agreement. CFM can detect the surface charge of viral capsids at a single-particle level and enable the comparison of surface charge between different types of viruses.

Tie line framework to optimize non-enveloped virus recovery in aqueous two-phase systems.

Viral particle purification is a challenge due to the complexity of the broth, the particle size, and the need to maintain virus activity. Aqueous two-phase systems (ATPSs) are a viable alternative for the currently used and expensive downstream processes. This work investigated the purification of two non-enveloped viruses, porcine parvovirus (PPV), and human rhinovirus (HRV) at various ATPS tie lines. A polyethylene glycol (PEG) 12 kDa-citrate system at pH 7 was used to study the behavior of the partitioning on three different thermodynamic tie line lengths (TLLs). It was experimentally determined that increasing the TLL, and therefore increasing the hydrophobic and electrostatic driving forces within the ATPS, facilitated higher virus recoveries in the PEG-rich phase. A maximum of 79% recovery of infectious PPV was found at TLL 36 w/w% and tie line (TL) ratio 0.1. Increased loading of PPV was studied to observe the change in the partitioning behavior and similar trends were observed for all the TLs. Most contaminants remained in the citrate-rich phase at all the chosen TLLs, demonstrating purification of the virus from protein contaminants. Moderate DNA removal was also measured. Net neutral charged HRV was studied to demonstrate the effects of driving forces on neutrally charged viruses. HRV recovery trends remained similar to PPV on each TLL studied, but the values were lower than PPV. Recovery of viral particles in the PEG-rich phase of the PEG-citrate system utilized the difference in the surface hydrophobicity between virus and proteins and showed a direct dependence on the surface charge of each studied virus. The preferential partitioning of the relatively hydrophobic viral particles in the PEG-rich phase supports the hypothesis that both hydrophobic and electrostatic forces govern the purification of viruses in ATPS.

Porcine parvovirus capsid protein expressed in Escherichia coli self-assembles into virus-like particles with high immunogenicity in mice and guinea pigs.

Porcine parvovirus (PPV) is a causative agent of reproductive failure in pregnant sows. Classical inactivated vaccine is extensively used to control PPV infection, but problems concerning safety, such as incomplete inactivation may occur. In this study, a novel subunit vaccine against PPV based on virus-like particles (VLPs) formed from the complete PPV VP2 protein expressed in a prokaryotic system with co-expressed chaperones is reported. The VLPs have a similar size, shape, and hemagglutination property to the PPV. Immunization with these VLPs stimulated the neutralization antibody and hemagglutination inhibition (HI) antibody responses in mice and guinea pigs. The lymphocyte proliferation response and cytokine secretion was also induced in immunized guinea pigs comparable to those immunized with PPV inactivated vaccine. In addition, immunization with VLPs also significantly reduced the PPV content in the spleen of guinea pigs 14 days after the challenge with intact virus. These studies suggest that PPV VLPs created as described here could be a potential candidate for vaccine development.

Separation of porcine parvovirus from bovine serum albumin using PEG-salt aqueous two-phase system.

Vaccine production faces a challenge in adopting conventional downstream processing steps that can efficiently purify large viral particles. Some major issues that plague vaccine purification are purity, potency, and quality. The industry currently considers 30% as an acceptable virus recovery for a vaccine purification process, including all downstream processes, whereas antibody recovery from CHO cell culture is generally around 80-85%. A platform technology with an improved virus recovery would revolutionize vaccine production. In a quest to fulfill this goal, we have been exploring aqueous two-phase systems (ATPSs) as an optional mechanism to purify virus. ATPS has been unable to gain wide implementation mainly due to loss of virus infectivity, co-purification of proteins, and difficulty of polymer recycling. Non-enveloped viruses are chemically resistant enough to withstand the high polymer and salt concentrations that are required for effective ATPS separations. We used infectious porcine parvovirus (PPV), a non-enveloped, DNA virus as a model virus to test and develop an ATPS separation method. We successfully tackled two of the three main disadvantages of ATPS previously stated; we achieved a high infectious yield of 64% in a PEG-citrate ATPS process while separating out the main contaminate protein, bovine serum albumin (BSA). The most dominant forces in the separation were biomolecule charge, virus surface hydrophobicity, and the ATPS surface tension. Highly hydrophobic viruses are likely to benefit from the discovered ATPS for high-purity vaccine production and ease of implementation.

Non-enveloped virus reduction with quaternized chitosan nanofibers containing graphene.

Membranes are an accepted technology for water purification. Membrane filtration can remove pathogens, including bacteria and viruses, by size. For small viruses that can have a diameter <25nm, removal by size leads to large membrane areas, high transmembrane pressures, low water flux, and frequent changing of membranes. In this work, we discovered that electrospun nanofibers made of chitosan and functionalized with a quaternary amine (HTCC) have the ability to adsorb a model non-enveloped virus, porcine parvovirus (PPV). To improve the virus removal of HTCC, we added graphene. Graphene both enhanced the ability to form nanofibers with HTCC and improved the virus removal. The hydrophobicity of graphene and the high charge of the HTCC create a system that can bind 95% of PPV. The HTCC/graphene nanofibers could be incorporated into microfiltration membranes and remove virus by adsorption. This would create a low pressure system that is more likely to benefit areas in need of fresh water.

Impact of virus preparation quality on parvovirus filter performance.

Virus-removal filtration technology is commonly used in the manufacturing process for biologics to remove potential viral contaminants. Virus-removal filters designed for retaining parvovirus, one of the smallest mammalian viruses, are considered an industry standard as they can effectively remove broad ranges of viruses. It has long been observed that the performance of virus filters can be influenced by virus preparations used in the laboratory scale studies (PDA, 2010). However, it remains unclear exactly what quality attributes of virus preparations are critical or indicative of virus filter performance as measured by effectiveness of virus removal and filter capacity consistency. In an attempt to better understand the relationship between virus preparation and virus filter performance, we have systematically prepared and analyzed different grades of parvovirus with different purity levels and compared their performance profiles on Viresolve® Pro parvovirus filters using four different molecules. Virus preparations used in the studies were characterized using various methods to measure DNA and protein content as well as the hydrodynamic diameter of virus particles. Our results indicate that the performance of Viresolve® Pro filters can be significantly impacted depending on the purity of the virus preparations used in the spike and recovery studies. More importantly, we have demonstrated that the purity of virus preparations is directly correlated to the measurable biochemical and biophysical properties of the virus preparations such as DNA and protein content and monodispersal status, thus making it possible to significantly improve the consistency and predictability of the virus filter performance during process step validations.

Porcine parvovirus removal using trimer and biased hexamer peptides.

Assuring the microbiological safety of biological therapeutics remains an important concern. Our group has recently reported small trimeric peptides that have the ability to bind and remove a model nonenveloped virus, porcine parvovirus (PPV), from complex solutions containing human blood plasma. In an effort to improve the removal efficiency of these small peptides, we created a biased library of hexamer peptides that contains two previously reported trimeric peptides designated WRW and KYY. This library was screened and several hexamer peptides were discovered that also removed PPV from solution, but there was no marked improvement in removal efficiency when compared to the trimeric peptides. Based on simulated docking experiments, it appeared that hexamer peptide binding is dictated more by secondary structure, whereas the binding of trimeric peptides is dominated by charge and hydrophobicity. This study demonstrates that trimeric and hexameric peptides may have different, matrix-specific roles to play in virus removal applications. In general, the hexamer ligand may perform better for binding of specific viruses, whereas the trimer ligand may have more broadly reactive virus-binding properties.

The epitope of the VP1 protein of porcine parvovirus.

Porcine parvovirus (PPV) is the major causative agent in a syndrome of reproductive failure in swine. Much has been learned about the structure and function of PPV in recent years, but nothing is known about the epitopes of the structural protein VP1, which is an important antigen of PPV. In this study, the monoclonal antibody C4 against VP1 of PPV was prepared and was used to biopan a 12-mer phage peptide library three times. The selected phage clones were identified by ELISA and then sequencing. The amino acid sequences detected by phage display were analyzed, and a mimic immuno-dominant epitope was identified. The epitope of VP1 is located in the N-terminal and contains the role amino acid sequence R-K-R. Immunization of mice indicated that the phage-displayed peptide induces antibodies against PPV. This study shows that peptide mimotopes have potential as alternatives to the complex antigens currently used for diagnosis of PPV infection or for development of vaccines.

SAT: a late NS protein of porcine parvovirus.

The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT(-)), with an unmodified VP sequence, showed a "slow-spreading" phenotype. These knockout mutants could be complemented with VP2(-) SAT(+) mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.

The structure of porcine parvovirus: comparison with related viruses.

The structure of baculovirus-expressed porcine parvovirus (PPV) capsids was solved using X-ray crystallography and was found to be similar to the related canine parvovirus (CPV) and minute virus of mice (MVM). The PPV capsid protein has 57 % and 49 % amino acid sequence identity with CPV and MVM, respectively, but the degree of conservation of surface-exposed residues is lower than average. Consequently, most of the structural differences are on the surface and are the probable cause of the known variability in antigenicity and host range. The NADL-2 and Kresse strains of PPV have distinct tissue tropisms and pathogenicity, which are mediated by one or more of the amino acid residues 381, 386, and 436. These residues are on or near the surface of the virus capsid, where they are likely to be associated with virus-cell interactions.