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Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108TCID50/mL and 7.5 × 107TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV.Key points•Cell contact-dependent oncolytic virus (OV) replicates in suspension cells.•Oncolytic potency is not encompassed during suspension cultivation.•Media composition, cell line, and MOI are critical process parameters for OV production.•The designed process is scalable and shows great promise for manufacturing clinical-grade material.

The adenovirus vector vaccines induce humoral and cellular immune responses and have been used to develop vaccines for effective prevention of life-threating viruses, such as Ebola and Coronaviruses. High demand of vaccines worldwide requires optimization of the production process. Perfusion process increases cell concentration and volumetric productivity, so that it becomes the commonly used strategy in vaccine production In this study, we optimized and developed a perfusion process for the adenovirus-based zoster vaccine production efficiently. We first tested different perfusion strategies in shake flasks, showing semi-continuous strategies for optimal HEK 293 cell growth. We then evaluated three empirical key process parameters (cell concentration at the time of infection (VCC), multiplicity of infection (MOI), virus production pH) by the design of experiment (DoE) method, from which the robust setpoint (VCC 1.04 × 10

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell–cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

The Ambr15 system is an automated, high-throughput bioreactor platform which comprises 24 individually controlled, single-use stirred-tank reactors. This system plays a critical role in process development by reducing reagent requirements and facilitating high-throughput screening of process parameters. However, until now, the system was used to simulate processes involving cells in suspension or growing on microcarriers and has never been tested for simulating cells growing on macrocarriers. Moreover, to our knowledge, a complete production process including cell growth and virus production has never been simulated. Here, we demonstrate, for the first time, the amenability of the automated Ambr15 cell culture reactor system to simulate the entire SARS-CoV-2 vaccine production process using macrocarriers. To simulate the production process, accessories were first developed to enable insertion of tens of Fibra-Cel macrocarries into the reactors. Vero cell adsorption to Fibra-Cels was then monitored and its adsorption curve was studied. After incorporating of all optimized factors, Vero cells were adsorbed to and grown on Fibra-Cels for several days. During the process, culture medium was exchanged, and the quantity and viability of the cells were followed, resulting in a typical growth curve. After successfully growing cells for 6 days, they were infected with the rVSV-ΔG-Spike vaccine virus. The present results indicate that the Ambr15 system is not only suitable for simulating a process using macrocarriers, but also to simulate an entire vaccine production process, from cell adsorption, cell growth, infection and vaccine virus production.

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 10

Infectious myonecrosis (IMN) is an important shrimp viral disease caused by infectious myonecrosis virus (IMNV). Based on previous reports, an attempt was made to propagate IMNV in apparently healthy C6/36 subclone of Aedes albopictus cell line. The confirmatory assays such as RT-PCR, real-time PCR and bioassay revealed that C6/36 cells were found to be susceptible to IMNV and these cells could be used easily for isolation and propagation of IMNV. The results of real-time PCR assay showed that a lower CT value of 22.25 in IMNV-infected cells was obtained on 10 day post-infection (d p.i.), whereas the higher CT value of 35.21 was obtained in IMNV-infected cells on 2 d p.i. There is no significant difference between CT values of IMNV production in vitro using C6/36 cell line and in vivo using shrimp. The IMNV propagated in C6/36 cells is capable of infecting shrimp and caused 100% mortality in shrimp. Clinical signs observed in shrimp injected with IMNV propagated in C6/36 cell line were found to be similar to naturally infected shrimp.

Objectives Respiratory syncytial virus (RSV) is a common cause of respiratory illness, health care visits, and hospitalizations. Arizona, which began conducting laboratory surveillance in 2004, has noted an increase in RSV cases (defined as a laboratory-positive result) among adults aged ≥65, concurrent with increasing reports from polymerase chain reaction (PCR) testing. We assessed whether the shift in the age distribution of reported RSV cases resulted from a change in RSV testing practices. Methods We used data on laboratory-confirmed RSV cases reported during 2013-2017 from the statewide surveillance system to assess the frequency of test types (rapid antigen, immunofluorescence assay, PCR, and viral culture) by age groups across RSV seasons, and we used logistic regression to estimate changes in odds of receiving a PCR test. We used statewide emergency department hospital discharge data for the same period to assess testing practices regardless of test result. Results The overall proportion of PCR tests among RSV cases increased significantly, from 22% in 2013 to 55% in 2017 ( P < .001). The percentage of RSV cases among adults aged ≥65 also increased significantly, from 4% in 2013 to 11% in 2017 ( P < .001) of RSV cases. Adults aged ≥65 had more than 8 times the odds of positive PCR results than children aged Conclusion Increasing RSV rates among adults aged ≥65 are likely a result of changes in testing practices. This age group may need more targeted intervention and future vaccination.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture–based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 μL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 μL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture–based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. Key points • First MDCK suspension cell–based perfusion process for IAV produciton was established. • “Cell density effect” was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 μL). Supplementary Information The online version contains supplementary material available at 10.1007/s00253-020-11050-8.

Abstract Kaposi’s sarcoma–associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi’s sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman’s disease). While in patients with late stage of Kaposi’s sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. Key messages In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system, in immunocompromised patients. Although PML used to be rare, recently the incidence of PML has risen due to an increase in immunosuppressive therapy. An in vitro JCPyV infection system could be used for anti-drug screening and investigation of tropism changes, but study of JCPyV in vitro has been limited due to the difficulty of efficiently propagating the virus in cultured cells. PML-type JCPyV efficiently propagates in primary human fetal and progenitor cell–derived astrocytes, but the preparation of cells from human fetuses is associated with severe ethical problems. In this study, human iPS cell–derived astrocytes were exposed to PML-type JCPyV. Infection, replication, and VP1 and T antigens of JCPyV were detected and confirmed in this culture. The non-coding control region (NCCR) of M1-IMRb was conserved in infected cells without point mutations. In addition, PML-type JCPyV genomic DNA in infected cells was detected as a single band of approximately 5.1 kbp, with no deletions. This is the first demonstration that human iPS cell–derived astrocytes efficiently support replication of PML-type JCPyV without production of defective interfering particles. These findings indicated that a culture system using human iPS cell–derived astrocyte would be useful for studies of PML, especially for screening anti-JCPyV drugs.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log(10)-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.

In our previous study, we found that a new type of Chikungunya virus particle with a complete capsid deletion (ΔC-CHIKV) is still infectious in BHK-21 cells and demonstrated its potential as a live attenuated vaccine candidate. However, the low yield as well as the disability to propagate in vaccine production cell line Vero of ΔC-CHIKV are not practical for commercial vaccine development. In this study, we not only achieved the successful propagation of the viral particle in Vero cells, but significantly improved its yield through construction of a chimeric VEEV-ΔC-CHIKV and extensive passage in Vero cells. Mechanistically, high production of VEEV-ΔC-CHIKV is due to the improvement of viral RNA packaging efficiency conferred by adaptive mutations, especially those in envelope proteins. Similar to ΔC-CHIKV, the passaged VEEV-ΔC-CHIKV is safe, immunogenic, and efficacious, which protects mice from CHIKV challenge after only one shot of immunization. Our study demonstrates that the utilization of infectious capsidless viral particle of CHIKV as a vaccine candidate is a practical strategy for the development of alphavirus vaccine. IMPORTANCE Chikungunya virus (CHIKV) is one of important emerging alphaviruses. Currently, there are no licensed vaccines against CHIKV infection. We have previously found a new type of Chikungunya virus particle with a complete capsid deletion (ΔC-CHIKV) as a live attenuated vaccine candidate that is not suitable for commercial vaccine development with the low viral titer production. In this study, we significantly improved its production through construction of a chimeric VEEV-ΔC-CHIKV. Our results proved the utilization of infectious capsidless viral particle of CHIKV as a safe and practical vaccine candidate.

Fixed-bed bioreactors packed with macrocarriers show great potential to be used for vaccine process development and large-scale production due to distinguishing features of low shear force, high cell adhering surface area, and easy replacement of culture media in situ. As an initial step of utilizing this type of bioreactors for Pseudorabies virus production (PRV) by African green monkey kidney (Vero) cells, we developed a tube-fixed-bed bioreactor in the previous study, which represents a scale-down model for further process optimization. By using this scale-down model, here we evaluated impacts of two strategies (use of serum-free medium and low cell inoculum density) on PRV production, which have benefits of simplifying downstream process and reducing risk of contamination. We first compared Vero cell cultures with different media, bioreactors and inoculum densities, and conclude that cell growth with serum-free medium is comparable to that with serum-containing medium in tube-fixed-bed bioreactor, and low inoculum density supports cell growth only in this bioreactor. Next, we applied serum-free medium and low inoculum cell density for PRV production. By optimization of time of infection (TOI), multiplicity of infection (MOI) and the harvesting strategy, we obtained total amount of virus particles ~ 9 log10 TCID50 at 5 days post-infection (dpi) in the tube-fixed-bed bioreactor. This process was then scaled up by 25-fold to a Xcell 1-L fixed-bed bioreactor, which yields totally virus particles of 10.5 log10 TCID50, corresponding to ~ 3 × 105 doses of vaccine. The process studied in this work holds promise to be developed as a generic platform for the production of vaccines for animal and human health.

Immunodominance is recognized as a key factor in the antigenic drift of seasonal influenza viruses. In the immunodominance model, each individual in a population predominantly responds to a single epitope among the five antigenic epitopes of the viral hemagglutinin (HA), driving escape mutations one at a time, and sequential mutations in multiple individuals who respond to different epitopes eventually generate a drifted strain with mutations in epitopes that are targeted by a majority of the population. A focused antibody response to the Sa epitope in people born between 1965 and 1979 was believed to contribute to a mutation at HA residue 163 and the first antigenic drift of the 2009 pandemic influenza A H1N1 virus. A serine-to-threonine mutation at HA residue 185 in the Sb epitope emerged in 2010 even before the 163 mutation. We show here that a large fraction of the population in 2010-2011 had responses to the Sb epitope, as shown by 47% of tested sera having altered titers to the S185T mutant. Responses to the Sb epitope showed an age-specific trend similar to that found for the response to Sa epitope in these subjects. Together, the focused responses to Sa and Sb epitopes may have driven the first antigenic drift of the 2009 pandemic H1N1 virus.

In recent years, there has been a strong interest in the development and production of gene therapy products, especially those utilizing adeno-associated virus (AAV) particles. This is evident with the growing number of clinical successes and agency approvals for AAV therapeutics. Due to this increased investment in this technology, a need exists for scalable commercial production methods to ensure adequate product supply as research in AAV shifts from bench-scale development to clinical production. The purpose of this review is to summarize current scalable purification techniques that can be employed during the commercial manufacturing of AAV as well as highlight certain development considerations, such as adventitious agent removal and process development using the principals of quality by design.

Introduction. Pestiviruses are the cause of reproductive problems, diseases of the gastrointestinal and respiratory tracts of animals. Three species are important for cattle: Pestivirus A, B, and H. Fast and reliable methods of differentiation of these pathogens are currently needed. Aims and objectives of the study: the development of multiplex real time PCR for the simultaneous detection and differentiation of three viruses. Material and methods. The nucleotide sequences of the conserved regions of the 5´-UTR genes of pestiviruses A, B, and H served as a target. Results. The reaction showed high specificity, sensitivity, reproducibility and was able to detect virus RNA at a concentration of not less than 0.6-1.2 lg TCID 50/cm 3 . Cross-reactions with other pestiviruses were not observed. Real time PCR confirmed the results obtained previously in RT-PCR with gel electrophoresis detection. In a parallel study of 1823 biological samples, the results of the two reactions were completely consistent. Pestivirus spp. was detected in 76 samples, Pestivirus A was present in 73 samples, Pestivirus B - in 3 samples, and Pestivirus H was not detected. Discussion. A two-step real time PCR was developed for the simultaneous detection and differentiation of three pestiviruses. Modified pan primers of S. Vilcek et al. were used for the first reaction, and primers and probes of our own design were used for virus typing, which resulted in high reaction efficiency. Conclusion. On the big dairy farms for livestock maintenance, there are favorable conditions for the circulation of pathogenic viruses. In this situation, rapid diagnostic methods are needed to quickly identify of several viruses. Real-time triplex analysis can be recommended as the rapid method for mass epidemiological studies, as well as for screening fetal calf serum used for virus cultivation in medicine and veterinary practice.

We have previously produced viral vectors (lentiviral vector, adenoviral vector, and adeno-associated viral vector) in small and in commercial scale in adherent cells using Pall fixed-bed iCELLis® bioreactor. Recently, a company called Univercells has launched a new fixed-bed bioreactor with the same cell growth surface matrix material, but with different fixed-bed structure than is used in iCELLis bioreactor. We sought to compare the new scale-X™ hydro bioreactor (2.4 m2) and iCELLis Nano system (2.67 m2) to see if the difference has any effect on cell growth or lentiviral vector and adenoviral vector productivity. Runs were performed using parameters optimized for viral vector production in iCELLis Nano bioreactor. Cell growth was monitored by counting nuclei, as well as by following glucose consumption and lactate production. In both bioreactor systems, cells grew well, and the cell distribution was found quite homogeneous in scale-X bioreactor. Univercells scale-X bioreactor was proven to be at least equally efficient or even improved in both lentiviral vector and adenoviral vector production. Based on the results, the same protocol and parameters used in viral vector production in iCELLis bioreactor can also be successfully used for the production in scale-X bioreactor system.

In December 2019, Wuhan, China suffered a serious outbreak of a novel coronavirus infectious disease (COVID) caused by novel severe acute respiratory syndrome-related coronavirus (SARS-CoV 2). To quickly identify the pathogen, we designed and screened primer sets, and established a sensitive and specific qRT-PCR assay for SARS-CoV 2; the lower limit of detection (LOD) was 15 (95% CI: 9.8–21) copies per reaction. We combined this qRT-PCR assay with an automatic integration system for nucleic acid extraction and amplification, thereby establishing an automatic integrated gene detection system (AIGS) for SARS-CoV 2. Cross reactive analysis performed in 20 other respiratory viruses and 37 nasopharyngeal swabs confirmed a 100% specificity of the assay. Using two fold diluted SARS-CoV 2 culture, the LOD of AIGS was confirmed to be 365 copies/ml (95% CI: 350–375), which was Comparable to that of conventional qRT-PCR (740 copies/ml, 95% CI: 690–750). Clinical performances of AIGS assay were assessed in 266 suspected COVID-19 clinical respiratory tract samples tested in parallel with a commercial kit. The clinical sensitivity of the AIGS test was 97.62% (95% CI: 0.9320–0.9951) based on the commercial kit test result, and concordance analysis showed a high agreement in SARS-CoV-2 detection between the two assays, Pearson R was 0.9623 (95% CI: 0.9523–0.9703). The results indicated that this AIGS could be used for rapid detection of SARS-CoV 2. With the advantage of simple operation and less time consuming, AIGS could be suitable for SARS-CoV2 detection in primary medical institutions, thus would do a great help to improve detection efficiency and control the spread of COVID-19.

The isolation of respiratory viruses, especially from clinical specimens, often shows poor efficiency with classical cell culture methods. The lack of suitable methods to generate virus particles inhibits the development of diagnostic assays, treatments, and vaccines. We compared three inoculation methods, classical cell culture, the addition of a JAK2 inhibitor AZD1480, and centrifugation-enhanced inoculation (CEI), to replicate human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). In addition, a combined method using AZD1480 treatment and CEI was used on throat swabs to verify that this method could increase virus isolation efficiency from human clinical specimens. Both CEI and AZD1480 treatment increased HRSV and HMPV genome replication. Also, the combined method using CEI and AZD1480 treatment enhanced virus proliferation synergistically. The combined method is particularly suited for the isolation of interferon-sensitive or slowly growing viruses from human clinical specimens.

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL.

Feline coronavirus infection can progress to a fatal infectious peritonitis, which is a widespread feline disease without an effective vaccine. Generating feline cells with reduced ability to respond to interferon (IFN) is an essential step facilitating isolation of new candidate vaccine strains. Here, we describe the use of Crispr/Cas technology to disrupt type I IFN signaling in two feline cell lines, AK-D and Fcwf-4 CU, and evaluate the replication kinetics of a serotype I feline infectious peritonitis virus (FIPV) within these cells. We report that polyclonal cell populations and a clonal isolate, termed Fcwf-4 IRN, exhibited significantly diminished IFN-responsiveness and allowed FIPV replication kinetics comparable to parental cells. Furthermore, we demonstrate that replication of FIPV is enhanced by ectopic expression of a host serine protease, TMPRSS2, in these cells. We discuss the potential of these cells for isolating new clinical strains and for propagating candidate vaccine strains of FIPV.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 106 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h−1 (doubling time = 32 h). Maximum virus titers in the range of 3.28–3.73 log10(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000–3500 virions/cell and productivities of 0.5–2.2 × 1012 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log10(HA units/100 µL), 1286 virions/cell and 1.4 × 1012 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

Background: Cell-based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation-associated antigenic changes observed in egg-based vaccines Seed viruses for cell-based vaccines can be prepared from clinical specimens by cell culture;however, clinical samples risk harboring respiratory viruses other than influenza virus Therefore, it is necessary to investigate the patterns of co-infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses Method(s): A total of 341 clinical specimens were collected from patients with influenza or influenza-like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses The patterns of co-infection were statistically analyzed with Fisher's exact test The samples with double or triple infections were passaged in suspension MDCK cells (MDCK-S), adherent MDCK cells (MDCK-A), and LLC-MK2D cells Cell-passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses Result(s): Double infections were detected in 8 5% and triple infections in 0 9% of the collected clinical specimens We identified four pairs of viruses with significant correlation For all samples with double and triple infection, MDCK-S and MDCK-A could selectively propagate influenza viruses, while eliminating all contaminating viruses In contrast, LLC-MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK-S and MDCK-A Conclusion(s): Both MDCK-S and MDCK-A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg-adaptation mutations Copyright © 2019 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd

Long-term culture of the human lung adenocarcinoma cell line A549 promotes the differentiation of these cells toward an alveolar type II cell phenotype. Here, we evaluated the susceptibility of long-term cultured A549 cells to human influenza viruses. A549 cells were cultured continuously for 25 days (D25-A549) or 1 day (D1-A549) in Ham’s F12K medium. Six human influenza A viruses grew much faster in D25-A549 cells than in D1-A549 cells; however, two influenza B viruses replicated poorly in both cell types. Two avian influenza viruses replicated efficiently in both cell types, with similar titres. Expression levels of human virus receptors were higher in D25-A549 cells than in D1-A549 cells. D25-A549 cells thus more efficiently support the replication of human influenza A viruses compared with D1-A549 cells. Our data suggest that long-term cultured A549 cells will be useful for influenza A virus research.

Suspension Madin-Darby canine kidney (MDCK) cells (MDCK-N), adherent MDCK cells (MDCK-C), and adherent rhesus monkey kidney LLC-MK2 cells (LLC-MK2D) were systematically evaluated for the preparation of influenza vaccine seed viruses for humans on the basis of primary virus isolation efficiency, growth ability, genetic stability of the hemagglutinin (HA) and neuraminidase (NA) genes, and antigenic properties in hemagglutination inhibition (HI) test of each virus isolate upon further passages. All the subtypes/lineages of influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), B-Victoria, and B-Yamagata) were successfully isolated from clinical specimens by using MDCK-N and MDCK-C, whereas LLC-MK2D did not support virus replication well. Serial passages of A(H1N1) viruses in MDCK-N and MDCK-C induced genetic mutations of HA that resulted in moderate antigenic changes in the HI test. All A(H1N1)pdm09 isolates from MDCK-C acquired amino acid substitutions at the site from K153 to N156 of the HA protein, which resulted in striking antigenic alteration. In contrast, only 30% of MDCK-N isolates showed amino acid changes at this site. The frequency of MDCK-N isolates with less than two-fold reduction in the HI titer was as high as 70%. A(H3N2) and B-Yamagata isolates showed high antigenic stability and no specific amino acid substitution during passages in MDCK-N and MDCK-C. B-Victoria isolates from MDCK-N and MDCK-C acquired genetic changes at HA glycosylation sites that greatly affected their antigenicity. When these cell isolates were applied to passages in hen eggs, A(H1N1), B-Victoria, and B-Yamagata viruses grew well in eggs, while none of the cell isolates of A(H3N2) viruses did. Thus, we demonstrate that MDCK-N might be useful for the preparation of influenza vaccine seed viruses.

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics. Successful cultivation is the gold standard for identifying a virus, according to the Koch postulates. However, this requires screening for a permissive cell line, which is traditionally time-, reagent- and labor-intensive. Here, a simple and easy-to-operate microfluidic chip, formed by seeding a variety of cell lines and culturing them in parallel, is reported for use in virus cultivation and virus-permissive host-cell screening. The chip was tested by infection with two known viruses, enterovirus 71 (EV71) and influenza virus H1N1. Infection with EV71 and H1N1 caused significant cytopathic effects (CPE) in RD and MDCK cells, respectively, demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection. Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a high-throughput, automatic, and unprecedentedly fast way.

A plethora of bat-associated lyssaviruses potentially capable of causing the fatal disease rabies are known today. Transmitted via infectious saliva, occasionally-reported spillover infections from bats to other mammals demonstrate the permeability of the species-barrier and highlight the zoonotic potential of bat-related lyssaviruses. However, it is still unknown whether and, if so, to what extent, viruses from different lyssavirus species vary in their pathogenic potential. In order to characterize and systematically compare a broader group of lyssavirus isolates for their viral replication kinetics, pathogenicity, and virus release through saliva-associated virus shedding, we used a mouse infection model comprising a low (102 TCID50) and a high (105 TCID50) inoculation dose as well as three different inoculation routes (intramuscular, intranasal, intracranial). Clinical signs, incubation periods, and survival were investigated. Based on the latter two parameters, a novel pathogenicity matrix was introduced to classify lyssavirus isolates. Using a total of 13 isolates from ten different virus species, this pathogenicity index varied within and between virus species. Interestingly, Irkut virus (IRKV) and Bokeloh bat lyssavirus (BBLV) obtained higher pathogenicity scores (1.14 for IRKV and 1.06 for BBLV) compared to rabies virus (RABV) isolates ranging between 0.19 and 0.85. Also, clinical signs differed significantly between RABV and other bat lyssaviruses. Altogether, our findings suggest a high diversity among lyssavirus isolates concerning survival, incubation period, and clinical signs. Virus shedding significantly differed between RABVs and other lyssaviruses. Our results demonstrated that active shedding of infectious virus was exclusively associated with two RABV isolates (92% for RABV-DogA and 67% for RABV-Insectbat), thus providing a potential explanation as to why sustained spillovers are solely attributed to RABVs. Interestingly, 3D imaging of a selected panel of brain samples from bat-associated lyssaviruses demonstrated a significantly increased percentage of infected astrocytes in mice inoculated with IRKV (10.03%; SD±7.39) compared to RABV-Vampbat (2.23%; SD±2.4), and BBLV (0.78%; SD±1.51), while only individual infected cells were identified in mice infected with Duvenhage virus (DUVV). These results corroborate previous studies on RABV that suggest a role of astrocyte infection in the pathogenicity of lyssaviruses., Author summary Globally, there are at present 17 different officially recognized lyssavirus species posing a potential threat for human and animal health. Bats have been identified as carriers for the vast majority of those zoonotic viruses, which cause the fatal disease rabies and are transmitted through infectious saliva. The occurrence of sporadic spillover events where lyssaviruses are spread from bats to other mammalian species highlights the importance of studying pathogenicity and virus shedding in regard to a potentially sustained onward cross-species transmission. Therefore, as part of this study, we compared 13 different isolates from ten lyssavirus species in a standardized mouse infection model, focusing on clinical signs, incubation periods, and survival. Based on the latter two, a novel pathogenicity index to classify different lyssavirus species was established. This pathogenicity index varied within and between different lyssavirus species and revealed a higher ranking of other bat-related lyssaviruses in comparison to the tested Rabies virus (RABV) isolates. Altogether, our results demonstrate a high diversity among the investigated isolates concerning pathogenicity and clinical picture. Furthermore, we comparatively analyzed virus shedding via saliva and while there was no indication towards a reduced pathogenicity of bat-associated lyssaviruses as opposed to RABV, shedding was increased in RABV isolates. Additionally, we investigated neuronal cell tropism and revealed that bat lyssaviruses are not only capable of infecting neurons but also astrocytes.

Stocks of vesicular stomatitis virus free of defective interfering particles were produced by serial clonal isolation. High-multiplicity infections with these stocks led to no interference or formation of defective interfering particles. Defective interfering particles were generated by three successive passages at high multiplicity.

African swine fever virus (ASFV) causes a virulent, deadly infection in wild and domestic swine and is currently causing a pandemic covering a contiguous geographical area from Central and Eastern Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in devastating economic losses to the swine industry. The most advanced vaccine candidates are live attenuated strains developed using a genetically modified virulent parental virus. Recently, we developed a vaccine candidate, ASFV-G-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV pandemic strain Georgia (ASFV-G). ASFV-G-ΔI177L is safe and highly efficacious in challenge studies using parental ASFV-G. Large-scale production of ASFV-G-ΔI177L has been limited because it can replicate efficiently only in primary swine macrophages. Here, we present the development of an ASFV-G-ΔI177L derivative strain, ASFV-G-ΔI177L/ΔLVR, that replicates efficiently in a stable porcine cell line. In challenge studies, ASFV-G-ΔI177L/ΔLVR maintained the same level of attenuation, immunogenic characteristics, and protective efficacy as ASFV-G-ΔI177L. ASFV-G-ΔI177L/ΔLVR is the first rationally designed ASF vaccine candidate that can be used for large-scale commercial vaccine manufacture. IMPORTANCE African swine fever is currently causing a pandemic resulting in devastating losses to the swine industry. Experimental ASF vaccines rely on the production of vaccine in primary swine macrophages, which are difficult to use for the production of a vaccine on a commercial level. Here, we report a vaccine for ASFV with a deletion in the left variable region (LVR). This deletion allows for growth in stable cell cultures while maintaining the potency and efficacy of the parental vaccine strain. This discovery will allow for the production of an ASF vaccine on a commercial scale.

Detecting severe acute respiratory syndrome coronavirus 2 in deceased patients is key when considering appropriate safety measures to prevent infection during postmortem examinations. A prospective cohort study comparing a rapid antigen test with quantitative reverse transcription PCR showed the rapid test's usability as a tool to guide autopsy practice.

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

The Epstein–Barr virus M81 strain, isolated from a nasopharyngeal carcinoma, induces potent spontaneous virus production in infected B cells. We found that the M81 non-coding Epstein–Barr-encoded RNA EBER2, which carries polymorphisms that are mainly restricted to viruses found in endemic nasopharyngeal carcinomas, markedly stimulated this process. M81 EBER2 increased CXCL8 expression, and this chemokine enhanced spontaneous lytic replication levels in M81-infected B cells. Both events resulted from the endocytosis of extracellular vesicles containing EBER2 that were generated by neighbouring M81-infected B cells, thereby generating a paracrine loop. These effects were strictly dependent on a functional Toll-like receptor 7 (TLR7), a sensor of single-stranded RNA located in the endosome of these cells. These unique properties of M81 EBER2 could be ascribed to its unusually high expression level and to the ability of its single-stranded region to activate TLR7; both of these properties were dependent on M81-specific polymorphisms. Thus, M81 induced chronic inflammation in its target cells and this resulted in increased virus production. These observations provide a mechanistic molecular link between M81 virus replication—a central viral function and a cancer risk factor—and the production of a chemokine involved in inflammation and carcinogenesis. The non-coding RNA EBER2 of the Epstein–Barr virus M81 strain potentiates virus lytic replication in B cells by generating a paracrine loop whereby the chemokine CXCL8 is released from infected cells via extracellular vesicles—which are taken up by neighbouring cells—thereby enhancing its own expression.

Summary Background The ongoing Ebola virus outbreak in the Ituri and North Kivu Provinces of the Democratic Republic of the Congo, which began in July, 2018, is the second largest ever recorded. Despite civil unrest, outbreak control measures and the administration of experimental therapies and a vaccine have been initiated. The aim of this study was to test the efficacy of candidate therapies and diagnostic tests with the outbreak strain Ituri Ebola virus. Lacking a virus isolate from this outbreak, a recombinant Ituri Ebola virus was compared with a similarly engineered Makona virus from the 2013–16 outbreak. Methods Using Ebola virus sequences provided by organisations in DR Congo and a reverse genetics system, we generated an authentic Ebola virus from the ongoing outbreak in Ituri and North Kivu provinces. To relate this virus to other Ebola viruses in DR Congo, we did a phylogenetic analysis of representative complete Ebola virus genome sequences from previous outbreaks. We evaluated experimental therapies being tested in clinical trials in DR Congo, including remdesivir and ZMapp monoclonal antibodies, for their ability to inhibit the growth of infectious Ituri Ebola virus in cell culture. We also tested diagnostic assays for detection of the Ituri Ebola virus sequence. Findings The phylogenetic analysis of whole-genome sequences from each Ebola virus outbreak suggests there are at least two Ebola virus strains in DR Congo, which have independently crossed into the human population. The Ituri Ebola strain initially grew slower than the Makona strain, yet reached similar mean yields of 3 × 107 50% tissue culture infectious dose by 72 h infection in Huh-7 cells. Ituri Ebola virus was similar to Makona in its susceptibility to inhibition by remdesivir and to neutralisation by monoclonal antibodies from ZMapp and other monoclonal antibodies. Remdesivir inhibited Ituri Ebola virus at a 50% effective concentration (EC50) of 12nM (with a selectivity index of 303) and Makona Ebola virus at 13nM (with a selectivity index of 279). The Zmapp monoclonal antibodies 2G4 and 4G7 neutralised Ituri Ebola virus with a mean EC50 of 0·24 μg/mL and 0·48 μg/mL, and Makona Ebola virus with a mean EC50 of 0·45 μg/mL and 0·2 μg/mL. The Xpert Ebola and US Centers for Disease Control and Prevention real-time RT-qPCR diagnostic assays detected Ituri and Makona Ebola virus sequences with similar sensitivities and efficiencies, despite primer site binding mismatches in the Ituri Ebola virus. Interpretation Our findings provide a rationale for the continued testing of investigational therapies, confirm the effectiveness of the diagnostic assays used in the region, and establish a paradigm for the use of reverse genetics to inform response activities in an outbreak. Funding US Centers for Disease Control and Prevention.

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.

Here, we report two novel enteroviruses, designated as SD-S67 and SD-S68, isolated from a goat farm. Their complete genome sequences were determined and found to be 7455 and 7465 nucleotides in length, respectively. Molecular characterization revealed that SD-S67 is closely related to bovine enterovirus strain 261 and that SD-S68 to caprine enterovirus strain CEV-JL14. Phylogenetic analysis showed that SD-S67 clustered with members of the species Enterovirus F, and that SD-S68 clustered with enteroviruses of goats and sheep. Recombination analysis showed that SD-S67 is likely to have undergone several recombination events in the process of its evolution. To the best of our knowledge, this is the first report of an enterovirus F isolate from a goat and of a coinfection with enteroviruses of different species in the same goat herd.

Highlights • We reported a hospital cluster combined with family cluster of H7N9 infection. A poultry farm was the initially infectious source of the H7N9 virus infection. Airborne transmission may result in the hospital cluster., Summary Objectives To identify human-to-human transmission of H7N9 avian influenza virus, we investigated a hospital cluster combined with family cluster in this study. Methods We obtained and analyzed clinical, epidemiological and virological data from the three patients. RT-PCR, viral culture and sequencing were conducted for determination of causative pathogen. Results The index case presented developed pneumonia with fever after exposure to chicken in a poultry farm. Case A presented pneumonia with high fever on day 3 after she shared a hospital room with the index case. Case B, the father of the index case, presented pneumonia with high fever on day 15 after he took care of the index case. H7N9 virus circulated in the local farm to which the index case was exposed. Full genomic sequence of virus showed 99.8–100% identity shared between the index case and case A or case B. Compared to the earliest virus of Anhui, a total of 29 amino acid variation sites were observed in the 8 segments. Conclusions A hospital cluster combined with family cluster of H7N9 avian influenza infection was identified. Air transmission resulted in the hospital cluster possibly. A poultry farm was the initially infectious source of the cluster.

Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved. To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 μg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.

Embryonating chicken eggs (ECEs) are among the most sensitive laboratory host systems for avian influenza virus (AIV) titration, but ECEs are expensive and require space for storage and incubation. Therefore, reducing ECE use would conserve resources. We utilized statistical modeling to evaluate the accuracy and precision of AIV titration with 3 instead of 5 ECEs for each dilution by the Reed–Muench method for 50% endpoint calculation. Beta-Poisson and exponential dose-response models were used in a simulation study to evaluate observations from actual titration data from 18 AIV isolates. The reproducibility among replicates of a titration was evaluated with one AIV isolate titrated in 3 replicates with the beta-Poisson, exponential, and Weibull dose-response models. The standard deviation (SD) of the error between input and estimated virus titers was estimated with Monte Carlo simulations using the fitted dose-response models. Good fit was observed with all models that were utilized. Reducing the number of ECEs per dilution from 5 to 3 resulted in the width of the 95% confidence interval increasing from ±0.64 to ±0.75 log10 50% ECE infectious doses (EID50) and the SD of the error increased by 0.03 log10 EID50. Our study suggests that using fewer ECEs per dilution is a viable approach that will allow laboratories to reduce costs and improve efficiency.

Newcastle disease is observed and detected on all the continents of the globe, except Australia, and causes great economic losses in poultry production. In the Kyrgyz Republic, Newcastle disease outbreaks were observed in 2015 and 2016. Regardless Newcastle’s pathogen is well explored, there are specific features of disease course and the problem of infection elimination is not solved. The authors explored the appropriate conditions for growing Newcastle’s virus in developing hen embryos. For this the researchers infected chicken embryos with different doses: 10-1 to 10-8 viral propagation was applied for 10-day developing chicken embryos into the allantoid cavity in the volume of 0.1 and 0.2 cm3 (doses of 6,104 to 0.6 50% embryonic infectious dose (EID50 ) per embryo and 1.2-105 to 1.2 50% embryonic infectious dose (EID50 ) per embryo, respectively). The degree of virus accumulation was estimated by means of hemagglutination test according to the titers of hemagglutinins in the allantoic fluid. The titers of hemagglutinins in solutions within 10-1 to 10-8 of virus-containing material shows that at volume of an inoculated virus-containing material of 0,1 cm3 accumulation of hemagglutinins of a Newcastle virus strain occurs at high level and does not essentially differ (Р>0,5); when applied for infecting developing chicken embryos of virus cultivation to 10-6 (a dose in this case ~ 60 50 % an embryonic infectious dose EID50 ).

Hepatitis E virus (HEV) is an enteric virus divided into eight genotypes. Genotype 1 (G1) and G2 are specific to humans; G3, G4 and G7 are zoonotic genotypes infecting humans and animals. Transmission to humans through water has been demonstrated for G1 and G2, mainly in developing countries, but is only suspected for the zoonotic genotypes. Thus, the water-related HEV hazard may be due to human and animal faeces. The high HEV genetic variability allows considering the presence in wastewater of not only different genotypes, but also quasispecies adding even greater diversity. Moreover, recent studies have demonstrated that HEV particles may be either quasi-enveloped or non-enveloped, potentially implying differential viral behaviours in the environment. The presence of HEV has been demonstrated at the different stages of the water cycle all over the world, especially for HEV G3 in Europe and the USA. Concerning HEV survival in water, the virus does not have higher resistance to inactivating factors (heat, UV, chlorine, physical removal), compared to viral indicators (MS2 phage) or other highly resistant enteric viruses (Hepatitis A virus). But the studies did not take into account genetic (genogroups, quasispecies) or structural (quasi- or non-enveloped forms) HEV variability. Viral variability could indeed modify HEV persistence in water by influencing its interaction with the environment, its infectivity and its pathogenicity, and subsequently its transmission by water. The cell culture methods used to study HEV survival still have drawbacks (challenging virus cultivation, time consuming, lack of sensitivity). As explained in the present review, the issue of HEV transmission to humans through water is similar to that of other enteric viruses because of their similar or lower survival. HEV transmission to animals through water and how the virus variability affects its survival and transmission remain to be investigated.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically-transmitted viral hepatitis worldwide. Increasing numbers of HEV infections, together with no available specific anti-HEV treatment, contributes to the pathogen's major health burden. A robust cell culture system is required for virologic studies and the development of new antiviral drugs. Unfortunately, like other hepatitis viruses, HEV is difficult to propagate in conventional cell lines. Many different cell culture systems have been tested using various HEV strains, but viral replication usually progresses very slowly, and infection with low virion counts results in non-productive HEV replication. However, recent progress involving generation of cDNA clones and passaging primary patient isolates in distinct cell lines has improved in vitro HEV propagation. This review describes various approaches to cultivate HEV in cellular and animal models and how these systems are used to study HEV infections and evaluate anti-HEV drug candidates.