Engineered CHO cells as a novel AAV production platform for gene therapy delivery.

The Herpes simplex virus (HSV)-based platform for production of recombinant adeno-associated viral vectors (rAAVs) yields higher titers and increased percentage of full capsids when compared to the triple transient transfection (TTT) method. However, this platform currently faces two major challenges. The first challenge is the reliance on commercial media, sometimes supplemented with serum, leading to costly manufacturing and a high risk for introduction of adventitious agents. The second challenge is that the production of HSV-1 relies on adherent complementing Vero cells (V27), making it difficult to scale up. We engineered serum-free-adapted CHO cells expressing key HSV-1 entry receptors, HVEM and/or Nectin-1 to address the first challenge. Using high-throughput cloning methods, we successfully selected a HVEM receptor-expressing clone (CHO-HV-C1) that yields 1.62 × 109, 2.51 × 109, and 4.07 × 109 viral genome copies/mL with rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors respectively, within 24 h post rHSV-1 co-infection. Moreover, CHO-HV-C1-derived rAAVs had comparable in vitro transduction, infectivity, and biodistribution titers to those produced by TTT. The second challenge was addressed via engineering CHO-HV-C1 cells to express HSV-1 CP27. These cells successfully produced rHSV-1 vectors, but with significantly lower titers than V27 cells. Taken together, the CHO/HSV system provides a novel, scalable, reduced cost, serum-free AAV manufacturing platform.

Genetic characterization of upper respiratory tract virome from nonvaccinated Egyptian cow-calf operations.

Bovine respiratory disease (BRD) is the costliest complex disease affecting the cattle industry worldwide, with significant economic losses. BRD pathogenesis involves several interactions between microorganisms, such as bacteria and viruses, and management factors. The present study aimed to characterize the nasal virome from 43 pooled nasal swab samples collected from Egyptian nonvaccinated cow-calf operations with acute BRD from January to February 2020 using metagenomic sequencing. Bovine herpesvirus-1 (BHV-1), first detection of bovine herpesvirus-5 (BHV-5), and first detection of bovine parvovirus-3 (BPV-3) were the most commonly identified in Egyptian cattle. Moreover, phylogenetic analysis of glycoprotein B revealed that the BHV-1 isolate is closely related to the Cooper reference strain (genotype 1.1), whereas the BHV-5 isolate is closely related to the reference virus GenBank NP_954920.1. In addition, the whole-genome sequence of BPV-3 showed 93.02% nucleotide identity with the reference virus GenBank AF406967.1. In this study, several DNA viruses, such as BHV-1 and first detection BHV-5, and BPV-3, were detected and may have an association with the BRD in Egyptian cattle. Therefore, further research, including investigating more samples from different locations to determine the prevalence of detected viruses and their contributions to BRD in cattle in Egypt, is needed.

RNA-Seq Analysis of Influenza A Virus-Induced Transcriptional Changes in Mice Lung and Its Possible Implications for the Virus Pathogenicity in Mice.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA), where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

Evolutionary insights into the furin cleavage sites of SARS-CoV-2 variants from humans and animals.

The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.

Nanoparticles as a novel and promising antiviral platform in veterinary medicine.

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

An overview of current COVID-19 vaccine platforms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic that emerged in December 2019 in Wuhan city, China. An effective vaccine is urgently needed to protect humans and to mitigate the economic and societal impacts of the pandemic. Despite standard vaccine development usually requiring an extensive process and taking several years to complete all clinical phases, there are currently 184 vaccine candidates in pre-clinical testing and another 88 vaccine candidates in clinical phases based on different vaccine platforms as of April 13, 2021. Moreover, three vaccine candidates have recently been granted an Emergency Use Authorization by the United States Food and Drug Administration (for Pfizer/BioNtech, Moderna mRNA vaccines, and Johnson and Johnson viral vector vaccine) and by the UK government (for University of Oxford/AstraZeneca viral vector vaccine). Here we aim to briefly address the current advances in reverse genetics system of SARS-CoV-2 and the use of this in development of SARS-CoV-2 vaccines. Additionally, we cover the essential points concerning the different platforms of current SARS-CoV-2 vaccine candidates and the advantages and drawbacks of these platforms. We also assess recommendations for controlling the COVID-19 pandemic and future pandemics using the benefits of genetic engineering technology to design effective vaccines against emerging and re-emerging viral diseases with zoonotic and/or pandemic potential.

Characterization and genetic analysis of recent and emergent virulent newcastle disease viruses in Egypt.

Extensive vaccination against Newcastle disease virus (NDV) induced more selective immune pressure from hosts that enhanced the evolutionary process of NDV. Herein, we characterized 13 recently isolated NDV isolates from vaccinated chicken flocks during 2014-2017. Sequence analysis of F gene showed the presence of 112 RRQKRF117 velogenic cleavage motif in 11 isolates, whereas the other two isolates (Ck/ME3/Eg/16 and Ck/ME5/Eg/16) showed the monobasic motif 112 GGRQGRL117 . Interestingly, isolate Ck/ME5/Eg/16 showed 100% and 99.82% nucleotide identity with the LaSota F gene hypervariable region and full-length gene, respectively. On the other hand, isolate Ck/ME3/Eg/16 revealed natural recombination with strains NDV/Ck/Egypt/3/2006 and NDV/Teal/VRLCU/Egypt/2015 that indicates re-emergence of that old strain. Interestingly, all 13 isolates showed high intracerebral pathogenicity (ICPI) and mean death time (MDT) despite the presence of lentogenic motif in both Ck/ME5/Eg/16 and Ck/ME3/Eg/16 isolates. Comparative analysis of F antigenic epitopes in our isolates with other published sequences from Egypt revealed high sequence conservation; recent isolates had one fixed amino acid substitution (K78R) and a novel V168I substitution, whereas a D170N substitution was detected in older strains (NDV-EG-35-2014 and NDV-KFR-B7-2012). Taken together, our results support the first isolation of virulent NDV isolates with a lentogenic motif; isolate Ck/ME5/Eg/16 might be generated in nature from LaSota live vaccine, whereas isolate Ck/ME3/Eg/16 is emergent from NDV/Ck/Egypt/3/2006. We conclude that the current diagnostic evaluation of the virulence of NDV isolates by characteristic amino acid residues at the F protein cleavage site is insufficient. There is a need to link virologic and epidemiologic data together, and routine and emergency LaSota vaccination protocols should be carefully and optimally applied, with regards to the timing and presence of co-infecting agents in the field.

Combined H5ND inactivated vaccine protects chickens against challenge by different clades of highly pathogenic avian influenza viruses subtype H5 and virulent Newcastle disease virus.

AIM: The aim of the current study was to evaluate the efficacy of a trivalent-inactivated oil-emulsion vaccine against challenge by different clades highly pathogenic avian influenza (HPAI) viruses including HPAI-H5N8 and the virulent genotype VII Newcastle disease virus (NDV) (vNDV). MATERIALS AND METHODS: The vaccine studied herein is composed of reassortant AI viruses rgA/Chicken/Egypt/ME1010/2016 (clade 2.2.1.1), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 2.2.1.2), and "NDV" (LaSota NDV/CK/Egypt/11478AF/11); all used at a concentration of 108 EID50/bird and mixed with Montanide-ISA70 oil adjuvant. Two-week-old specific pathogen free (SPF) chickens were immunized subcutaneously with 0.5 ml of the vaccine, and hemagglutination inhibition (HI) antibody titers were monitored weekly. The intranasal challenge was conducted 4 weeks post-vaccination (PV) using 106 EID50/0.1 ml of the different virulent HPAI-H5N1 viruses representing clades 2.2.1, 2.2.1.1, 2.2.1.2, 2.3.4.4b-H5N8, and the vNDV. RESULTS: The vaccine induced HI antibody titers of >6log2 against both H5N1 and NDV viruses at 2 weeks PV. Clinical protection against all HPAI H5N1 viruses and vNDV was 100%, except for HPAI H5N1 clade-2.2.1 and HPAI H5N8 clade-2.3.4.4b viruses that showed 93.3% protection. Challenged SPF chickens showed significant decreases in the virus shedding titers up to <3log10 compared to challenge control chickens. No virus shedding was detected 6 "days post-challenge" in all vaccinated challenged groups. CONCLUSION: Our results indicate that the trivalent H5ND vaccine provides significant clinical protection against different clades of the HPAI viruses including the newly emerging H5N8 HPAI virus. Availability of such potent multivalent oil-emulsion vaccine offers an effective tool against HPAI control in endemic countries and promises simpler vaccination programs.

Co-expression of the Hemagglutinin and Neuraminidase by Heterologous Newcastle Disease Virus Vectors Protected Chickens against H5 Clade 2.3.4.4 HPAI Viruses.

Avian influenza remains an important zoonotic disease with a significant global impact. The spread of H5 highly pathogenic avian influenza (HPAI) viruses (clade 2.3.4.4) by migratory birds has caused outbreaks in wide geographic regions (Asia, Europe, and North America) with great economic losses during 2014-2015. Efficient vaccines and vaccination approaches are needed to enhance protective immunity against HPAI viruses. Although several vaccination strategies have been developed, none has been satisfactory. Our strategy has been to use avirulent vaccine strain of Newcastle disease virus (NDV) as a vaccine vector for HPAI viruses. For poultry vaccination, we previously generated a new platform of chimeric NDV vector to overcome preexisting maternal antibodies to NDV in poultry. In this study, we have generated vaccine candidates targeting H5 clade 2.3.4.4 HPAI viruses by using our chimeric NDV and conventional NDV strain LaSota vectors for a heterologous prime-boost immunization approach. Co-expression of the HA and NA proteins by our vaccine vectors induced enhanced HPAI virus specific immune responses in specific-pathogen free and broiler chickens prior to challenge. Further, these vaccine candidates efficiently protected broiler chickens from mortality, clinical signs, and shedding of homologous and heterologous H5 HPAI viruses and highly virulent NDV, thus providing a dual vaccination approach in the field.

Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses.

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses.

Effects of PB1-F2 on the pathogenicity of H1N1 swine influenza virus in mice and pigs.

Although several studies have exploited the effects of PB1-F2 in swine influenza viruses, its contribution to the pathogenicity of swine influenza viruses remains unclear. Herein, we investigated the effects of PB1-F2 on the pathogenicity of influenza virus using a virulent H1N1 A/swine/Kansas/77778/2007 (KS07) virus, which expresses a full-length PB1-F2, in mice and pigs. Using reverse genetics, we generated the wild-type KS07 (KS07_WT), a PB1-F2 knockout mutant (KS07_K/O) and its N66S variant (KS07_N66S). KS07_K/O showed similar pathogenicity in mice to the KS07_WT, whereas KS07_N66S displayed enhanced virulence when compared to the other two viruses. KS07_WT exhibited more efficient replication in lungs and nasal shedding in infected pigs than the other two viruses. Pigs infected with the KS07_WT had higher pulmonary levels of granulocyte-macrophage colony-stimulating factor, IFN-γ, IL-6 and IL-8 at 3 and 5 days post-infection, as well as lower levels of IL-2, IL-4 and IL-12 at 1 day post-infection compared to those infected with the KS07_K/O. These results indicate that PB1-F2 modulates KS07 H1N1 virus replication, pathogenicity and innate immune responses in pigs and the single substitution at position 66 (N/S) in the PB1-F2 plays a critical role in virulence in mice. Taken together, our results provide new insights into the effects of PB1-F2 on the virulence of influenza virus in swine and support PB1-F2 as a virulence factor of influenza A virus in a strain- and host-dependent manner.

Recombinant Newcastle disease virus expressing H9 HA protects chickens against heterologous avian influenza H9N2 virus challenge.

In order to produce an efficient poultry H9 avian influenza vaccine that provides cross-protection against multiple H9 lineages, two Newcastle disease virus (NDV) LaSota vaccine strain recombinant viruses were generated using reverse genetics. The recombinant NDV-H9Con virus expresses a consensus-H9 hemagglutinin (HA) that is designed based on available H9N2 sequences from Chinese and Middle Eastern isolates. The recombinant NDV-H9Chi virus expresses a chimeric-H9 HA in which the H9 ectodomain of A/Guinea Fowl/Hong Kong/WF10/99 was fused with the cytoplasmic and transmembrane domain of the fusion protein (F) of NDV. Both recombinant viruses expressed the inserted HA stably and grew to high titers. An efficacy study in chickens showed that both recombinant viruses were able to provide protection against challenge with a heterologous H9N2 virus. In contrast to the NDV-H9Chi virus, the NDV-H9Con virus induced a higher hemagglutination inhibition titer against both NDV and H9 viruses in immunized birds, and efficiently inhibited virus shedding through the respiratory route. Moreover, sera collected from birds immunized with either NDV-H9Con or NDV-H9Chi were able to cross-neutralize two different lineages of H9N2 viruses, indicating that NDV-H9Con and NDV-H9Chi are promising vaccine candidates that could provide cross-protection among different H9N2 lineage viruses.

Characterization of uncultivable bat influenza virus using a replicative synthetic virus.

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

Studies on genetic diversity of bovine viral diarrhea viruses in Danish cattle herds.

Scandinavian countries have successfully pursued bovine viral diarrhea virus (BVDV) eradication without the use of vaccines. In Denmark, control and eradication of BVDV were achieved during the last two decades, but occasionally new BVDV infections are detected in some Danish cattle herds. The aim of this study was to determine recent BVDV subtypes isolated from 4 Danish herds (A, B, C, and D) isolated in 2009-2012 and to analyze the genetic variation of these isolates within the same herd and its relation with those of other herds. The results showed that three herds (B, C, D) were BVDV 1-b and only one herd (herd A) was BVDV 1-d, no other subtypes were detected. The deduced E2 amino acids result showed a high identity percent (99-100 %) between isolates originating from the same herd, but with higher variation compared to isolates of the other herds. Some of these new Danish strains have closer relationship to BVDVs from outside Denmark than to older Danish strains indicating that these are new introductions to Denmark. In conclusion, BVDV-1 subtypes recently detected in Denmark were only subtypes 1b and 1d, and BVDV infections established in a herd is genetically stable over a long time period.