Host-derived lactic acid bacteria alleviate short beak and dwarf syndrome by preventing bone loss, intestinal barrier disruption, and inflammation.

Short-beak and dwarf syndrome (SBDS) is caused by novel goose parvovirus (NGPV) infection, which leads to farm economic losses. Our research aimed to investigate the potential of administering isolated lactic acid bacteria (LAB) in alleviating SBDS in ducks. Eight wild LAB strains were isolated from duck feces and their biosecurity was investigated in both duck embryo fibroblast (DEF) and live ducks. Moreover, the LAB strains exhibited no detrimental effects on bone metabolism levels and facilitated the tight junction proteins (TJPs) mRNA expression, and contributing to the mitigation of inflammation in healthy ducks. Subsequently, we conducted in vitrol and in vivo experiments to assess the impact of LAB on NGPV infection. The LAB strains significantly reduced the viral load of NGPV and downregulated the mRNA levels of pro-inflammatory factors in DEF. Additionally, LAB treatment alleviated SBDS in NGPV-infected ducks. Furthermore, LAB treatment alleviated intestinal damage, and reduced the inflammatory response, while also mitigating bone resorption in NGPV-infected ducks. In conclusion, the LAB strains isolated from duck feces have favorable biosecurity and alleviate SBDS in ducks, and the mechanism related to LAB improves intestinal barrier integrity, alleviates inflammation, and reduces bone resorption. Our study presents a novel concept for the prevention and treatment of NGPV, thereby establishing a theoretical foundation for the future development of probiotics in the prevention and treatment of NGPV.

Continuous surveillance of pathogens detects excretion of avian orthoreovirus and parvovirus by several wild waterfowl: possible wild bird reservoirs.

Migratory wild birds can carry various pathogens, such as influenza A virus, which can spread to globally and cause disease outbreaks and epidemics. Continuous epidemiological surveillance of migratory wild birds is of great significance for the early warning, prevention, and control of epidemics. To investigate the pathogen infection status of migratory wild birds in eastern China, fecal samples were collected from wetlands to conduct pathogen surveillance. The results showed that duck orthoreovirus (DRV) and goose parvovirus (GPV) nucleic acid were detected positive in the fecal samples collected from wild ducks, egrets, and swan. Phylogenetic analysis of the amplified viral genes reveals that the isolates were closely related to the prevalent strains in the regions involved in East Asian-Australasian (EAA) migratory flyway. Phylogenetic analysis of the amplified viral genes confirmed that they were closely related to circulating strains in the regions involved in the EAA migration pathway. The findings of this study have expanded the host range of the orthoreovirus and parvovirus, and revealed possible virus transmission between wild migratory birds and poultry.

Subclinical infection and potential shedding routes of equine parvovirus-hepatitis among hospitalized horses in Austria.

BACKGROUND: Equine parvovirus hepatitis (EqPV-H) can cause Theiler's disease and subclinical hepatitis in horses. OBJECTIVES: Assess the frequency of subclinical EqPV-H infection in hospitalized horses and to study viral transmission by investigating potential shedding routes. ANIMALS: One hundred sixteen equids, that presented to the University Equine Hospital of the University of Veterinary Medicine Vienna between February 2021 and March 2022, for causes other than hepatopathy. METHODS: In this cross-sectional study, samples (serum, feces, nasal, and buccal swabs) of hospitalized horses were collected. Sera were screened for the presence of anti-EqPV-H antibodies by a luciferase immunoprecipitation system assay. Quantitative PCR was used for the detection of EqPV-H DNA in the samples and a nested PCR was used for further validation. RESULTS: Seroprevalence was 10.3% (12/116) and viremia occurred in 12.9% (15/116) of the serologically positive horses. The detected viral load in serum varied from non-quantifiable amount to 1.3 × 106 genome equivalents per milliliter of serum. A low viral load of EqPV-H DNA was detected in 2 nasal swabs and 1 fecal sample. CONCLUSION AND CLINICAL IMPORTANCE: EqPV-H DNA was detected in nasal secretions and feces of viremic horses, which could pose a risk to naive hospitalized horses. It is advisable to screen hospitalized horses that are potential donors of blood or plasma to reduce the risk of iatrogenic EqPV-H transmission.

Detection of Equine Parvovirus-Hepatitis Virus and Equine Hepacivirus in Archived Sera from Horses in France and Australia.

Reports of newly discovered equine hepatotropic flavi- and parvoviruses have emerged throughout the last decade in many countries, the discovery of which has stimulated a great deal of interest and clinical research. Although commonly detected in horses without signs of disease, equine parvovirus hepatitis (EqPV-H) and equine hepacivirus (EqHV) have been associated with liver disease, including following the administration of contaminated anti-toxin. Our aim was to determine whether EqPV-H and EqHV are present in Australian horses and whether EqPV-H was present in French horses and to examine sequence diversity between strains of both viruses amongst infected horses on either side of the globe. Sera from 188 Australian horses and 256 French horses from horses with and without clinical signs of disease were collected. Twelve out of 256 (4.7%) and 6 out of 188 (3.2%) French and Australian horses, respectively, were positive for the molecular detection of EqPV-H. Five out of 256 (1.9%) and 21 out of 188 (11.2%) French and Australian horses, respectively, were positive for the molecular detection of EqHV. Australian strains for both viruses were genomically clustered, in contrast to strains from French horses, which were more broadly distributed. The findings of this preliminary survey, with the molecular detection of EqHV and EqPV-H in Australia and the latter in France, adds to the growing body of awareness regarding these recently discovered hepatotropic viruses. It has provided valuable information not just in terms of geographic endemicity but will guide equine clinicians, carers, and authorities regarding infectious agents and potential impacts of allogenic tissue contamination. Although we have filled many gaps in the world map regarding equine hepatotropic viruses, further prospective studies in this emerging field may be useful in terms of elucidating risk factors and pathogenesis of these pathogens and management of cases in terms of prevention and diagnosis.

Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Serologic Cross-reactivity of Murine Parvovirus Capsid Antigens.

Genomic sequence analysis of autonomous parvoviruses within the genus Protoparvovirus generates 2 groups that are principally of mouse origin: the minute virus of mice (MVM) strains (MVMp, MVMi, MVMc, MVMm) and the mouse parvovirus (MPV)-like strains (MPV-1, MPV-2, MPV-3, MPV-4, MPV-5, HaPV, LuIII). Baculovirus-expressed recombinant capsid protein (rVP2) from each of these 11 parvovirus strains were produced, purified, and demonstrated to form virus-like particles. Each rVP2 preparation was then used as antigen in a multiplex fluorescent immunoassay and to immunize 5 different strains of mice. Sera from immunized mice, mice experimentally monoinfected with various MVM or MPV isolates, and mice naturally infected with murine parvoviruses were evaluated with the multiplex fluorescent immunoassay rVP2 panel. Results for sera from immunized mice indicate that homologous antigen-antisera interactions produced the strongest seroreactivity. All MVM antigens were highly cross-reactive with heterologous MVM antisera, while more variability was observed in heterologous antigen-antisera reactions among the MPV-like strains. MPV-1, MPV-3, HaPV, and LuIII were highly cross-reactive with each other, MPV-2 and MPV-5 were highly cross-reactive with each other, and MPV-4 displayed modest cross-reactivity with certain MPV-like strains. Serologic cross-reactivity patterns similar to those in immunized mice were observed in mice experimentally infected with MVMp, MVMm, MPV-1, MPV-5, or HaPV, and in sera from mice naturally infected with MVM and MPV. Serologic cross-reactivity spectrums suggest a small panel of rVP2 antigens (MVM, MPV-1, MPV-2, MPV-4) combined with the generic murine parvovirus recombinant nonstructural protein 1 (rNS1) antigen are sufficient for qualitative detection of currently known MVM and MPV-like strains.

Stability and integrity of self-assembled bovine parvovirus virus­like particles (BPV­VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.

BACKGROUND: Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences. METHODS: In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism. RESULTS: Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs. CONCLUSIONS: In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

Opportunistic sampling of yellow canary (Crithagra flaviventris) has revealed a high genetic diversity of detected parvoviral sequences.

Parvoviruses are known to be significant viral pathogens that infect a wide range of species globally. However, little is known about the parvoviruses circulating in Australian birds, including yellow canaries. Here, we present four parvoviral sequences including three novel parvoviruses detected from 10 yellow canaries (Crithagra flaviventris), named canary chaphamaparvovirus 1 and -2 (CaChPV1 and CaChPV2), canary dependoparvovirus 1 and -2 (CaDePV1 and CaDePV2). The whole genome sequences of CaChPV1, CaChPV2, CaDePV1, and CaDePV2 showed the highest identity with other parvoviruses at 76.4%, 75.9%, 84.0%, and 59.1%, respectively. Phylogenetic analysis demonstrated that CaChPV1 and CaChPV2 were clustered within the genus Chaphamaparvovirus. Meanwhile, CaDePV1 and CaDePV2 fall within the genus Dependoparvovirus and have the closest evolutionary relationship to the bird-associated dependoparvoviruses. Overall, this study enriched our understanding of the genetic diversity among avian parvoviruses within the Parvoviridae family.

For better or worse: crosstalk of parvovirus and host DNA damage response.

Parvoviruses are a group of non-enveloped DNA viruses that have a broad spectrum of natural infections, making them important in public health. NS1 is the largest and most complex non-structural protein in the parvovirus genome, which is indispensable in the life cycle of parvovirus and is closely related to viral replication, induction of host cell apoptosis, cycle arrest, DNA damage response (DDR), and other processes. Parvovirus activates and utilizes the DDR pathway to promote viral replication through NS1, thereby increasing pathogenicity to the host cells. Here, we review the latest progress of parvovirus in regulating host cell DDR during the parvovirus lifecycle and discuss the potential of cellular consequences of regulating the DDR pathway, targeting to provide the theoretical basis for further elucidation of the pathogenesis of parvovirus and development of new antiviral drugs.

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Antiviral alternatives against important members of the subfamily Parvovirinae: a review.

Parvoviruses are responsible for multiple diseases, and there is a critical need for effective antiviral therapies. Specific antiviral treatments for parvovirus infections are currently lacking, and the available options are mostly supportive and symptomatic. In recent years, significant research efforts have been directed toward understanding the molecular mechanisms of parvovirus replication and identifying potential targets for antiviral interventions. This review highlights the structure, pathogenesis, and treatment options for major viruses of the subfamily Parvovirinae, such as parvovirus B19 (B19V), canine parvovirus type 2 (CPV-2), and porcine parvovirus (PPV) and also describes different approaches in the development of antiviral alternatives against parvovirus, including drug repurposing, serendipity, and computational tools (molecular docking and artificial intelligence) in drug discovery. These advances greatly increase the likelihood of discoveries that will lead to potent antiviral strategies against different parvovirus infections.

Parvovirus B19 and Human Parvovirus 4 Encode Similar Proteins in a Reading Frame Overlapping the VP1 Capsid Gene.

Viruses frequently contain overlapping genes, which encode functionally unrelated proteins from the same DNA or RNA region but in different reading frames. Yet, overlapping genes are often overlooked during genome annotation, in particular in DNA viruses. Here we looked for the presence of overlapping genes likely to encode a functional protein in human parvovirus B19 (genus Erythroparvovirus), using an experimentally validated software, Synplot2. Synplot2 detected an open reading frame, X, conserved in all erythroparvoviruses, which overlaps the VP1 capsid gene and is under highly significant selection pressure. In a related virus, human parvovirus 4 (genus Tetraparvovirus), Synplot2 also detected an open reading frame under highly significant selection pressure, ARF1, which overlaps the VP1 gene and is conserved in all tetraparvoviruses. These findings provide compelling evidence that the X and ARF1 proteins must be expressed and functional. X and ARF1 have the exact same location (they overlap the region of the VP1 gene encoding the phospholipase A2 domain), are both in the same frame (+1) with respect to the VP1 frame, and encode proteins with similar predicted properties, including a central transmembrane region. Further studies will be needed to determine whether they have a common origin and similar function. X and ARF1 are probably translated either from a polycistronic mRNA by a non-canonical mechanism, or from an unmapped monocistronic mRNA. Finally, we also discovered proteins predicted to be expressed from a frame overlapping VP1 in other species related to parvovirus B19: porcine parvovirus 2 (Z protein) and bovine parvovirus 3 (X-like protein).

Viral clearance capability of monoclonal antibody purification.

Viral clearance steps are routinely included in monoclonal antibody purification processes to safeguard product from potential virus contamination. These steps are often experimentally studied using product-specific feeds and parameters for each project to demonstrate viral clearance capability. However, published evidence suggests that viral clearance capability of many of these steps are not significantly impacted by variations in feed material or process parameter within commonly used ranges. The current investigation confirms robust retrovirus inactivation by low pH treatment and parvovirus removal by second-generation virus filters, independent to individual antibody molecules. Our results also reveal robust retrovirus removal by flowthrough anion exchange chromatography, inside the limits of protein load and host cell protein content. The cumulative viral clearance capability from these steps leads to an excess clearance safety factor of 10,000-fold for endogenous retrovirus-like particles. These results further justify the use of prior knowledge-based modular viral clearance estimation as opposed to repetitive experimentation.

A chromosome-level genome assembly of the Rhus gall aphid Schlechtendalia chinensis provides insight into the endogenization of Parvovirus-like DNA sequences.

The Rhus gall aphid, Schlechtendalia chinensis, feeds on its primary host plant Rhus chinensis to induce galls, which have economic importance in medicines and the food industry. Rhus gall aphids have a unique life cycle and are economically beneficial but there is huge gap in genomic information about this group of aphids. Schlechtendalia chinensis induces rich-tannin galls on its host plant and is emerging as a model organism for both commercial applications and applied research in the context of gall production by insects. Here, we generated a high-quality chromosome-level assembly for the S. chinensis genome, enabling the comparison between S. chinensis and non-galling aphids. The final genome assembly is 344.59 Mb with 91.71% of the assembled sequences anchored into 13 chromosomes. We predicted 15,013 genes, of which 14,582 (97.13%) coding genes were annotated, and 99% of the predicted genes were anchored to the 13 chromosomes. This assembly reveals the endogenization of parvovirus-related DNA sequences (PRDs) in the S. chinensis genome, which could play a role in environmental adaptations. We demonstrated the characterization and classification of cytochrome P450s in the genome assembly, which are functionally crucial for sap-feeding insects and have roles in detoxification and insecticide resistance. This genome assembly also revealed the whole genome duplication events in S. chinensis, which can be considered in comparative evolutionary analysis. Our work represents a reference genome for gall-forming aphids that could be used for comparative genomic studies between galling and non-galling aphids and provides the first insight into the endogenization of PRDs in the genome of galling aphids. It also provides novel genetic information for future research on gall-formation and insect-plant interactions.

Canine bufavirus (Carnivore protoparvovirus-3) infection in dogs with respiratory disease.

Canine bufavirus (CBuV) or Carnivore protoparvovirus-3, a nonenveloped DNA virus belonging to the genus Protoparvovirus, family Parvoviridae, has been identified in dogs with respiratory and enteric diseases. Although CBuV detection has been reported in multiple countries, descriptions of pathologic findings associated with infection have not yet been provided. In this study, the authors necropsied 14 dogs (12 puppies and 2 adult dogs) from a breeding colony that died during multiple outbreaks of respiratory diseases. Postmortem investigations revealed extensive bronchointerstitial pneumonia with segmental type II pneumocyte hyperplasia in all necropsied puppies but less severe lesions in adults. With negative results of common pathogen detection by ancillary testing, CBuV DNA was identified in all investigated dogs using a polymerase chain reaction (PCR). Quantitative PCR demonstrated CBuV DNA in several tissues, and in situ hybridization (ISH) indicated CBuV tissue localization in the lung, tracheobronchial lymph node, and spinal cord, suggesting hematogenous spread. Dual CBuV ISH and cellular-specific immunohistochemistry were used to determine the cellular tropism of the virus in the lung and tracheobronchial lymph node, demonstrating viral localization in various cell types, including B-cells, macrophages, and type II pneumocytes, but not T-cells. Three complete CBuV sequences were successfully characterized and revealed that they clustered with the CBuV sequences obtained from dogs with respiratory disease in Hungary. No additional cases were identified in small numbers of healthy dogs. Although association of the bufavirus with enteric disease remains to be determined, a contributory role of CBuV in canine respiratory disease is possible.

Protoparvovirus carnivoran 1 infection of golden jackals Canis aureus in Serbia.

Parvoviruses are among the major animal pathogens that can cause considerable health disorders ranging from subclinical to lethal in domestic and wild animals. Golden jackal (Canis aureus), an expanding European species, is a reservoir of many pathogens, including vector-borne diseases and zoonoses. Given the importance of parvovirus infections in dogs and cats, this study aimed to unfold the virus prevalence and molecular characterisation in the golden jackal population in Serbia. The spleen samples from 68 hunted jackals during 2022/2023 were tested for the VP2-specific genome region of Protoparvovirus carnivoran 1 by PCR. BLAST analysis of partial VP2 sequences obtained from three animals (4.4%) revealed the highest similarity to Protoparvovirus carnivoran 1, genogroup Feline panleukopenia virus, which is the second report on FPV infection in jackals. Based on specific amino acid residues within partial VP2, the jackals' Protoparvovirus carnivoran 1 was also classified as FPV. One jackal's strain showed two synonymous mutations at positions 699 and 1167. Although species cross-transmission could not be established, jackals' health should be maintained by preventing the transmission of viruses to native species and vice versa. Although jackals are considered pests, their role as natural cleaners is of greater importance. Therefore, further monitoring of their health is needed to understand the influence of infectious diseases on population dynamics and to determine the relationship between domestic predators and jackals and the direction of cross-species transmission.

Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work.

Impact of proteins and protein fouling on virus retention during virus removal filtration.

Virus filtration is a crucial step in ensuring the high levels of viral clearance required in the production of biotherapeutics produced in mammalian cells or derived from human plasma. Previous studies have reported that virus retention is often reduced in the presence of therapeutic proteins due to membrane fouling; however, the underlying mechanisms controlling this behavior are still not well understood. Experimental studies were performed with a single layer of the commercially available dual-layer PegasusTM SV4 virus removal filter to more easily interpret the experimental results. Bacteriophage ФX174 was used as a model parvovirus, and human immunoglobulin (hIgG) and Bovine Serum Albumin (BSA) were used as model proteins. Data obtained with 5 g/L solutions of hIgG showed more than a 100-fold reduction in virus retention compared to that in the protein-free solution. Similar effects were seen with membranes that were pre-fouled with hIgG and then challenged with ФX174. The experimental data were well-described using an internal polarization model that accounts for virus capture and accumulation within the virus filter, with the hIgG nearly eliminating the irreversible virus capture while also facilitating the release of previously captured virus. These results provide important insights into the performance and validation of virus removal filters in bioprocessing.

Integration of Planova filters in manufacturing processes of biologicals improve the virus safety effectively: A review of publicly available data.

The capacity to remove viruses by Planova filters produced by Asahi Kasei, primarily by small virus-retentive filters, were compiled from data in peer-reviewed publications and, partly, publicly available data from presentations at conferences (Planova workshops). Data from more than 100 publications and presentations at conferences covering Planova filters were assessed. The data were grouped according to the different virus filters regarding mean pore sizes and viruses of different sizes for plasma and cell culture derived products. Planova 15N and 20N filters removed parvoviruses below the limit of detection of viruses in the filtrate in approx. 50% of all studies and mean LRFs (log reduction factors) for viruses detected in the filtrate were above 4, demonstrating effective parvovirus reduction. Parvovirus removal capacity increased for Planova BioEX filters as well as for 2 Planova 20N in series. Large viruses as retroviruses (e.g., HIV and MuLV), herpesviruses, flaviviruses and togaviruses were removed effectively by Planova 15N, 20N and BioEX filters and also by Planova 35N filters. Flow interruption, transmembrane pressure, volume and protein concentration per filter area had had no substantial impact on virus removal capacity at manufacturing specification. In conclusion, the incorporation of Planova filters in manufacturing processes of biologicals remove, depending on the filter pore size, small and large viruses from the feed stream reliably. This virus reduction step with an orthogonal mechanism integrated in the manufacturing processes of biologicals, based primarily on size exclusion of viruses, improves the virus safety of these biopharmaceutical products considerably.

Development and application of a multiplex PCR method for the simultaneous detection of goose parvovirus, waterfowl reovirus, and goose astrovirus in Muscovy ducks.

A multiplex polymerase chain reaction (PCR) method was developed to detect and distinguish goose parvovirus (GPV), waterfowl reovirus (WRV), and goose astrovirus (GAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of these enteric viruses and were used to specifically amplify targeted fragments of 493 bp from the viral protein 3 (VP3) gene of GPV, 300 bp from the sigma A-encoding gene of WRV, and 156 bp from the capsid protein-encoding gene of GAstV. The results showed that the primers can specifically amplify target fragments, without any cross-amplification with other viruses, indicating that the method had good specificity. A sensitivity test showed that the detection limit of the multiplex PCR method was 1 × 103 viral copies. A total of 102 field samples from Muscovy ducks with clinically suspected diseases were evaluated using the newly developed multiplex PCR method. The ratio of positive samples to total samples for GPV, WRV, and GAstV was 73.53% (75/102) for multiplex PCR and was 73.53% (75/102) for routine PCR. Seventy-five positive samples were detected by both methods, for a coincidence ratio of 100%. This multiplex PCR method can simultaneously detect GPV, WRV, and GAstV, which are associated with viral enteritis, thereby providing a specific, sensitive, efficient, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.