BHK-21 Research Articles

Infectious clone of a contemporary Tembusu virus and replicons expressing reporter genes or heterologous antigens from poultry viruses.

The novel mosquito-borne Tembusu virus (TMUV, family Flaviviridae) was discovered as the cause of a severe outbreak of egg-drop syndrome affecting ducks in Southeast Asia in 2010. TMUV infection can also lead to high mortality in various additional avian species such as geese, pigeons, and chickens. This study describes the construction of an infectious cDNA clone of a contemporary duck-isolate (TMUV WU2016). The virus recovered after transfection of BHK-21 cells shows enhanced virus replication compared to the mosquito-derived MM1775 strain. Next, the WU2016 cDNA clone was modified to create a SP6 promoter-driven, self-amplifying mRNA (replicon) capable of expressing a range of different reporter genes (Renilla luciferase, mScarlet, mCherry, and GFP) and viral (glyco)proteins of avian influenza virus (AIV; family Orthomyxoviridae), infectious bursal disease virus (IDBV; family Bunyaviridae) and infectious bronchitis virus (IBV; family Coronaviridae). The current study demonstrates the flexibility of the TMUV replicon system, to produce different heterologous proteins over an extended period of time and its potential use as a platform technology for novel poultry vaccines.

Fabrication of water-dispersible and cell-stimulating calcium phosphate nanoparticles immobilizing basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a therapeutic protein that can enhance angiogenesis, wound healing, and tissue regeneration; however, it is extremely unstable even under a normal physiological environment. Biocompatible calcium phosphate (CaP) nanoparticles (NPs) co-immobilizing bFGF, heparin, and ferucarbotran would be useful as a multifunctional delivery carrier of bFGF. In this study, such NPs were successfully fabricated by a coprecipitation process, using a labile supersaturated CaP solution containing bFGF, heparin, and ferucarbotran. The NPs showed relatively high negative zeta potential (-12mV) because of the negatively charged heparin, which enabled their stable dispersion in water. The hydrodynamic diameter of the NPs was around 200nm. Immunoreactive bFGF was released from the NPs in an acellular medium dose-dependently. The NPs promoted proliferation of baby hamster kidney fibroblasts (BHK-21 cells) and mouse osteoblastic MC3T3-E1 cells at a certain dose range, although they inhibited proliferation of rat pheochromocytoma (PC-12) cells. These results demonstrated that the effect of the NPs on cell proliferation was dependent on the cell type and dose, the details of which should be investigated in a future study.

Preparation of mcl-polyhydroxyalkanoate nanoparticles stabilized by the Tween 80 nonionic surfactant

Nanoparticles based on biodegradable polymers find numerous applications in medicine as substances for intracellular drug delivery. Biosynthetically produced poly-3-hydroxyalkanoates (P3HAs) are among the most promising polymers of a lipid nature. In particular, polyhydroxybutyrate and polyhydroxyvalerate (scl-poly-3-hydroxyalkanoates) are widely used compounds, which are soluble only in organochlorine solvents. The use of organochlorine solvents faces several obstacles, since such chemicals may exhibit carcinogenic effects on the human body. However, the P3Has compounds consisting of hydroxybutyric acid residues with 6–14 carbon atoms in the main chain (mcl-poly-3-hydroxyalkanoates) are soluble not only in CHCl3 or CH2Cl2 but also in paraffins. These hydrocarbons, such as n-hexane, can be easily separated from aqueous solutions and are not known to exhibit high cytotoxicity. Consequently, the application of mcl-poly-3-hydroxyalkanoates can prevent the contamination of prepared dosage forms with organochlorine compounds. To this end, a methodology for the synthesis of mcl-P3HA nanoparticles stabilized with the Tween 80 nonionic surfactant was proposed. The ratio between the concentration of P3HA and the detergent was optimized. The present study revealed that the obtained particles have an average size of 200±90 nm and a zeta potential of -17±5 mV. Upon investigating the stability of the particle solution at 4 °C, it was found that the nanoparticles did not alter their size and zeta potential for 90 days. The fluorescence microscopy method showed that they could be delivered into BHK-21 cells within 2 h. In addition, the nanoparticles synthesized had no toxic effect on BHK-21 cells at a concentration of up to 200 μg/mL.

Antiviral mechanisms of sorafenib against foot-and-mouth disease virus via c-RAF and AKT/PI3K pathways.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that poses a significant threat to the global livestock industry. However, specific antiviral treatments against FMDV are currently unavailable. This study aimed to evaluate the antiviral activity of anticancer drugs, including kinase and non-kinase inhibitors against FMDV replication in BHK-21 cells. Sorafenib, a multi-kinase inhibitor, demonstrated a significant dose-dependent reduction in FMDV replication. It exhibited a half maximal effective concentration (EC50) value of 2.46µM at the pre-viral entry stage and 2.03µM at the post-viral entry stage. Further intracellular assays revealed that sorafenib effectively decreased 3Dpol activity with a half maximal inhibitory concentration (IC50) of 155nM, while not affecting 3Cpro function. The study indicates that sorafenib influences host protein pathways during FMDV infection, primarily by potentiating the c-RAF canonical pathway and AKT/PI3K pathway. Molecular docking analysis demonstrated specific binding of sorafenib to the active site of FMDV 3Dpol, interacting with crucial catalytic residues, including D245, D338, S298, and N307. Additionally, sorafenib exhibited significant binding affinity to the active site motifs of cellular kinases, namely c-RAF, AKT, and PI3K, which play critical roles in the viral life cycle. The findings suggest that sorafenib holds promise as a therapeutic agent against FMDV infection. Its mechanism of action may involve inhibiting FMDV replication by reducing 3Dpol activity and regulating cellular kinases. This study provides insights for the development of novel therapeutic strategies to combat FMDV infections.

The identification and genetic characteristics of Quang Binh virus from field-captured Culex tritaeniorhynchus (Diptera: Culicidae) from Guizhou Province, China

BackgroundMosquitoes carry a variety of viruses that can cause disease in humans, animals and livestock. Surveys for viruses carried by wild mosquitoes can significantly contribute to surveillance efforts and early detection systems. In addition to mosquito-borne viruses, mosquitoes harbor many insect-specific viruses (ISVs). Quang Binh virus (QBV) is one such example, categorized as an ISV within the Flavivirus genus (family Flaviviridae). QBV has been specifically documented in Vietnam and China, with reports limited to several mosquito species.MethodsThe homogenate obtained from female mosquitoes was cultured on C6/36 (Aedes albopictus) and BHK-21 (baby hamster kidney) cell lines. Positive cultures were identified by reverse transcription-polymerase chain reaction (RT‒PCR) with taxon- or species-specific primers. Next-generation sequencing was employed to sequence the complete genomes of the identified positive samples. Subsequently, phylogenetic, gene homology, molecular evolutionary and genetic variation analyses were conducted.ResultIn 2021, a total of 32,177 adult female mosquitoes were collected from 15 counties in Guizhou Province, China. The predominant mosquito species identified were Culex tritaeniorhynchus, Armigeres subalbatus and Anopheles sinensis. Among the collected mosquitoes, three positive cultures were obtained from Cx. tritaeniorhynchus pools, revealing the presence of Quang Binh virus (QBV) RNA sequences. Phylogenetic analysis indicated that the three Guizhou isolates, along with the prototype isolate from Vietnam, formed distinct branches. These branches were primarily closely related to other QBV isolates reported in China. Comparative analysis revealed a high degree of nucleotide and amino acid homology between the Guizhou isolates and both Vietnamese and other indigenous Chinese isolates. Additionally, nonsynonymous single-nucleotide variants (SNVs) were observed in these strains compared to the QBV prototype strain.ConclusionThis study represents the first report of QBV presences in Cx. tritaeniorhynchus mosquitoes in Guizhou Province, China. Phylogenetic tree analysis showed that the three Guizhou isolates were most closely related to the QBV genes found in China. In addition, the study of the genetic characteristics and variation of this virus provided a deeper understanding of QBV and enriched the baseline data of these insect-specific flaviviruses (ISFVs).Graphical

Antimalarial dihydrochalcone isolated from Artocarpus sericicarpus Jarret leaves and in silico investigation against falcipain-2 protein

Aims: To investigate the potential antimalarial plant belonging to the Artocarpus genus, Artocarpus sericicarpus, and isolate the antimalarial active compound from leaves extract. Methods: The isolation of the antimalarial compound was based on bioassay-guided isolation. The compound isolation was conducted by chromatography and identified using spectroscopic techniques, including 1H, 13C, and 2D NMR. The compound was tested for its antimalarial activity against Plasmodium falciparum and evaluated for cytotoxicity using several cell lines, including Huh7, HepG2, BHK-21, and Vero cells. In silico investigation of the compound against falcipain-2 protein was conducted as well. Results: Fractionation and purification of dichloromethane leaves extract led to the isolation of a dihydrochalcone compound identified as 1-(2,4-dihydroxyphenyl)-3-[4-hydroxy-3-(3-methylbut-2-en-1-yl)phenyl]propan-1-one. The dihydrochalcone compound exhibited antimalarial activity with an IC50 value of 34.80 µM. Cytotoxicity test revealed CC50 values of the compound were more than 20 µg/mL, and the selectivity indexes (SI) were 4.50, 5.52, 3.02, and 6.68 on Huh7, HepG2, BHK-21, and Vero cells, respectively. The CC50 and SI indicated the nontoxic criteria of the compound. In silico investigation showed that the compound could bind to the falcipain-2 active sites. Conclusions: The dihydrochalcone compound identified as 1-(2,4-dihydroxyphenyl)-3-[4-hydroxy-3-(3-methylbut-2-en-1-yl)phenyl]propan-1-one was isolated from Artocarpus sericicarpus leaves extract showed antimalarial activity and the ability to bind to the falcipain-2 active sites on in silico investigation.

Antiviral evaluation of 1,4-disubstituted-1,2,3-triazole derivatives against Chikungunya virus.

This work aimed to investigate the antiviral activity of two 1,4-disubstituted-1,2,3-triazole derivatives (1 and 2) against Chikungunya virus (CHIKV) replication. Cytotoxicity was analyzed using colorimetric assays and the antiviral potential was evaluated using plaque assays and computational tools. Compound 2 showed antiviral activity against CHIKV 181-25 in BHK-21 and Vero cells. Also, this compound presented a higher activity against CHIKV BRA/RJ/18 in Vero cells, like compound 1. Compound 2 exhibited virucidal activity and inhibited virus entry while compound 1 inhibited virus release. Molecular docking suggested that these derivatives inhibit nsP1 protein while compound 1 may also target capsid protein. Both compounds exhibit promising antiviral activity against CHIKV by blocking different steps of virus replication.

Metavirome Analysis and Identification of Midge-Borne Viruses from Yunnan Province, China, in 2021.

Midges are widely distributed globally and can transmit various human and animal diseases through blood-sucking. As part of this study, 259,300 midges were collected from four districts in Yunnan province, China, to detect the viral richness and diversity using metavirome analysis techniques. As many as 26 virus families were detected, and the partial sequences of bluetongue virus (BTV), dengue virus (DENV), and Getah virus (GETV) were identified by phylogenetic analysis and PCR amplification. Two BTV gene fragments, 866 bps for the VP2 gene of BTV type 16 and 655 bps for the VP5 gene of BTV type 21, were amplified. The nucleotide sequence identities of the two amplified BTV fragments were 94.46% and 98.81%, respectively, with two classical BTV-16 (GenBank: JN671907) and BTV-21 strains (GenBank: MK250961) isolated in Yunnan province. Furthermore, the BTV-16 DH2021 strain was successfully isolated in C6/36 cells, and the peak value of the copy number reached 3.13 × 107 copies/μL after five consecutive BHK-21 cell passages. Moreover, two 2054 bps fragments including the E gene of DENV genotype Asia II were amplified and shared the highest identity with the DENV strain isolated in New Guinea in 1944. A length of 656 bps GETV gene sequence encoded the partial capsid protein, and it shared the highest identity of 99.68% with the GETV isolated from Shandong province, China, in 2017. Overall, this study emphasizes the importance of implementing prevention and control strategies for viral diseases transmitted by midges in China.

Grp94 Inhibitor HCP1 Suppressed the Replication of SVA in BHK-21 Cells and PK-15 Cells.

Glucoregulatory protein 94 (Grp94) is necessary for the post-viral life cycle and plays a quality control role in viral proteins, but the role of Grp94 in regulating viral replication in host cells is not well known. Therefore, finding a compound that can regulate Grp94 will help us to study the mechanism of viral replication. Previously, we synthesized a coumarin pyrazoline derivative HCP1 that is an effective inhibitor of Grp94. We suppose that HCP1 may inhibit viral replication. This study aimed to investigate the effect of HCP1 on the replication ability of Senecavirus A (SVA), so as to provide a target and a leading compound for revealing the pathogenic mechanism of the virus and developing antiviral drugs. Rat cell lines BHK-21 and porcine cell lines PK-15 were infected with SVA, and the infected cells were treated with different concentrations of HCP1. The cell viability (CCK-8), virus titer (TCID50), autophagy level, and Grp94 expression were measured. The results showed that a low concentration of HCP1 decreased viral titer and viral load in BHK-21 and PK-15 cells, and 5μM HCP1 significantly decreased the expression of SVA VP2 protein. In addition, SVA infection can lead to an increased level of autophagy, and HCP1 can inhibit host cell autophagy caused by SVA infection, thereby inhibiting viral replication and infection. These findings reveal that Grp94 is a key factor in controlling SVA replication, and its inhibitor HCP1 suppresses SVA replication by inhibiting the increase of Grp94 protein level and autophagy induced by SVA. This study will contribute to the development of a new class of small-molecule antiviral drugs.

Generation of Stable Cell Lines Expressing Akabane Virus N Protein and Insight into Its Function in Viral Replication.

Akabane virus (AKAV) is a world wide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. The nucleocapsid (N) protein of AKAV possesses multiple important functions in the virus life cycle, and it is an ideal choice for AKAV detection. In this study, we successfully constructed two stable BHK-21 cell lines (C8H2 and F7E5) that constitutively express the AKAV N protein using a lentivirus system combined with puromycin selection. RT-PCR analysis confirmed that the AKAV N gene was integrated into the BHK-21 cell genome and consistently transcribed. Indirect immunofluorescence (IFA) and Western blot (WB) assays proved that both C8H2 and F7E5 cells could react with the AKAV N protein mAb specifically, indicating potential applications in AKAV detection. Furthermore, we analyzed the growth kinetics of AKAV in the C8H2 and F7E5 cell lines and observed temporary inhibition of viral replication at 12, 24 and 36 h postinfection (hpi) compared to BHK-21 cells. Subsequent investigations suggested that the reduced viral replication was linked to the down-regulation of the viral mRNAs (Gc and RdRp). In summary, we have established materials for detecting AKAV and gained new insights into the function of the AKAV N protein.

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3–86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26–506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G.

Dengue virus serotypic replacement of NS3 protease or helicase domain causes chimeric viral attenuation but can be recovered by a compensated mutation at helicase domain or NS2B, respectively.

Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%-78% amino acid identities, resulting in incomplete immunity, where one serotype's infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5' RNA triphosphatase (5'RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations' frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells. IMPORTANCE Enzyme activities for replicating DENV 5' cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5' cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)-NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.

In vitro biological activity of cobalt(II) complexes with salicylaldimine ligands in microbial and cancer cells.

Background: More studiesusing cobalt complexes as drugs areneeded. Results: The drug action oftwo cobalt salicylaldimines was determined. The complexes and amphotericin B (20mg/ml) inhibited Candida albicans at 9-15 and 21mm. This concentration of both ligands inhibited Staphylococcus aureus at 10mm and one ligand inhibited Escherichia coli at 9mm, but the complexes and ampicillin inhibited four bacteria at 9-20 and 21-26mm. The ligands were inactive against cancer and normal cells, but the complexes and doxorubicin provided IC50valuesof 28.18-54.19 and 9.66μM against MCF-7 cells and 15.76-20.49 and 36.42μM against BHK cells. Conclusion: The ligands' activity was much improved by complexation, although they remained substandard.

A Newly Emerging Serotype A Strain in Foot-and-Mouth Disease Virus with Higher Severity and Mortality in Buffalo than in Cattle Calves in North Egypt.

A severe foot-and-mouth disease (FMD) epidemic struck several Egyptian provinces recently, causing significant losses among animals even in vaccinated farms. This study indicated the existence of the newly emerging foot-and-mouth disease virus (FMDV) and first investigated its effect on the Egyptian water buffalo (Bubalus bubalis) and cattle calves in the Beheira province, north Egypt. Twenty tongue epithelial samples from diseased calves in five infected farms were randomly collected, prepared, and propagated using baby hamster kidney-21 (BHK-21) cells. Whole genomic RNA was extracted from the cells of the third passage. A FMDV genome was detected and serotyped using one-step reverse transcription polymerase chain reactions (RT-PCRs). Nucleotide sequencing of the purified serotype-specific PCR bands was performed, and a maximum likelihood phylogenetic tree based on 600 base pairs of VP1 was constructed. The results identified FMDV, serotype A in all infected samples, whereas the serotypes O and SAT2 were negative. The obtained 20 sequences were identical to each other and similar to the newly reported strain in Egypt that belongs to the Europe-South America (Euro-SA) topotype. The epidemiological and clinical parameters associated with such a strain were fully recorded by veterinarians and analyzed in a single infected farm including 70 cattle and buffalo calves. It caused higher peracute mortalities in buffalo (25.7%; 95% CI: 13-43) than in cattle (8.6%; 95% CI: 2-24) calves. Severe clinical signs such as dullness, hypothermia, bradycardia, and cardiac arrhythmia were common to both except in fatal cases, whereas hyperthermia and respiratory signs were prevalent in cattle calves. In conclusion, we first characterized the newly emerging FMDV in the calves of Beheira as more fatal and severe in buffalo than in cattle calves.

Amino acids 1811–1960 of myosin heavy chain 9 is involved in murine gammaherpesvirus 68 infection

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 μg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811–1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.

Effects of Zinc Oxide Nanoparticles Supplementation on Growth Performance, Meat Quality and Serum Biochemical Parameters in Broiler Chicks.

The zinc oxide nanoparticles (ZnONPs) have attracted exhilarating research interest due to their novel distinguishing characteristics such as size, shape, high surface activity, large surface area and biocompatibility. Being highly bioavailable and exerting a superior efficacy than conventional zinc sources, ZnONPs is emerging as an alternative feed supplement for poultry. The present study involves the synthesis of ZnONPs through a cost effective and eco-friendly method using planetary ball milling technique and characterized for its size, shape, optical property, functional group and elemental concentration using particle size analyzer, Transmission Electron Microscopy, X-Ray Diffraction analysis, Fourier Transform Infra-Red spectroscopy, UV-Vis spectroscopy and Inductively Coupled Plasma-Mass Spectroscopy. In vitro cytotoxicity study using Baby Hamster kidney (BHK-21) cells, Vero cells and primary chick liver culture cells revealed that ZnONPs can be safely incorporated in the broiler chick's feed up to the concentration of 100mg/kg. To investigate the effects of ZnONPs on production performances in broiler chicks, a feeding trial was carried out using 150-day-old broiler chicks randomly allotted in five treatment groups. The dietary treatment groups were: T1 (80mg/kg of zinc oxide), T2 (60mg/kg of zinc methionine) and T3, T4 and T5 received 60, 40 and 20mg/kg of ZnONPs respectively. The results showed a significant improvement (p < 0.05) in the body weight gain and feed conversion ratio of broiler chicks supplemented with 20 and 40mg/kg of ZnONPs. The ZnONPs supplementation significantly (p < 0.05) increased the dressing percentage in addition to significant (p < 0.05) reduction in the meat pH compared to inorganic and organic zinc supplementation. Overall, an eco-friendly method for ZnONPs synthesis was demonstrated and the optimum dietary level (20mg/kg) of ZnONPs could enhance the growth, the meat quality and Zn uptake without any negative effects on selected serum biochemical parameters in the broiler chicks.

Circulating foot-and-mouth disease virus serotype A African-genotype IV in Egypt during 2022.

Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation. Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection. Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35-75) and the immunogenic determinants of the G-H loop of VP1 (residues 100-146 and 161-175). The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes.

A C-type lectin in saliva of Aedes albopictus (Diptera: Culicidae) bind and agglutinate microorganisms with broad spectrum.

Via complex salivary mixture, mosquitos can intervene immune response and be helpful to transmit several viruses causing deadly human diseases. Some C-type lectins (CTLs) of mosquito have been reported to be pattern recognition receptor to either resist or promote pathogen invading. Here, we investigated the expression profile and agglutination function of an Aedes albopictus CTL (Aalb_CTL2) carrying a single carbohydrate-recognition domain (CRD) and WND/KPD motifs. The results showed that Aalb_CTL2 was found to be specifically expressed in mosquito saliva gland and its expression was not induced by blood-feeding. The recombinant Aalb_CTL2 (rAalb_CTL2) could agglutinate mouse erythrocytes in the presence of calcium and the agglutinating activity could be inhibited by EDTA. rAalb_CTL2 also displayed the sugar binding ability to D-mannose, D-galactose, D-glucose, and maltose. Furthermore, it was demonstrated that rAalb_CTL2 could bind and agglutinate Gram positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as fungus Candida albicans in vitro in a calcium dependent manner. However, rAalb_CTL2 could not promote type 2 dengue virus (DENV-2) replication in THP-1 and BHK-21 cell lines. These findings uncover that Aalb_CTL2 might be involved in the innate immunity of mosquito to resist microorganism multiplication in sugar and blood meals to help mosquito survive in the varied natural environment.

In English

Today, the antiviral activity of oxide nanomaterials can be used in the fight against the viral disease COVID-19. It is thought that Ag nanoparticles may bind to the surface glycoprotein of the virus and interfere with the virus’s interaction with epithelial cells, and inhibit virus reproduction by releasing silver ions in the cell. The viruses’ inhibition with RNA (ribonucleic acid) and DNA (deoxyribonucleic acid) genomes by oxide nanocomposites action was presented. In this research, the surface structure of doped CeO2 (La2O3) was studied by nitrogen adsorption-desorption based on BET method. The silver atom’s existence in CeO2 - Ag0 can facilitate the transport of more holes to the surface and can enhance the optical, antivirus activity. The primary particle size of pure cerium dioxide is 7 nm, for CeO2 - Ag composite at 2 and 4 wt. % of silver is 6.5 and 6.9 nm; for La2O3 - Ag 27 and 35 nm, respectively. Cell viability was assessed using an MTT (3-(4,5-Dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide) assay after NPs (nanoparticles) exposure, since only viable cells have functional mitochondrial dehydrogenase enzymes that can reduce MTT to formazan. Nanoparticles were non-toxic for BHK-21(Syrian hamster kidney), Hep-2 (Human larynx carcinoma), and MDCK (Canine kidney) cells in concentrations of 10 and 100 μg/ml, while cell viability was within 76÷100 %. La2O3 and CeO2, which contained 4 wt. % of Ag at a concentration of 1000 μg/ml had a lower toxic effect: for BHK-21 cells 68 and 76 % of viable cells, respectively; for Hep-2 - 40 and 36 %, for MDCK - 42 and 48 %; La2O3 and CeO2 with 2 and 5 wt. % of Ag at a concentration of 1000 µg/ml were highly toxic. The level of ВНК-21, Нер-2, and MDCK cells viability was in a range of 7 to 37 %. It has been stated that oxides of cerium and lanthanum have a pronounced virucidal action against the Herpes simplex virus and Influenza A virus by completely inhibiting the development of its cytopathic action. The lanthanum and cerium oxides with 2 and 5 wt. % of silver inhibited the development of the virus’s CPE by more than 5.0 log10 compared to the virus control. The results show that lanthanum and cerium oxides with 2 and 5 wt. % silver have a high virucidal effect against herpes simplex virus type 1. A 1.0÷4.0 log10 reduction in the infectious titer of the Herpes virus synthesized “de novo” in the presence of lanthanum and cerium oxide nanocomposites has been shown.

A high throughput antiviral screening platform for alphaviruses based on Semliki Forest virus expressing eGFP reporter gene

Alphaviruses, which contain a variety of mosquito-borne pathogens, are important pathogens of emerging/re-emerging infectious diseases and potential biological weapons. Currently, no specific antiviral drugs are available for the treatment of alphaviruses infection. For most highly pathogenic alphaviruses are classified as risk group-3 agents, the requirement of biosafety level 3 (BSL-3) facilities limits the live virus-based antiviral study. To facilitate the antiviral development of alphaviruses, we developed a high throughput screening (HTS) platform based on a recombinant Semliki Forest virus (SFV) which can be manipulated in BSL-2 laboratory. Using the reverse genetics approach, the recombinant SFV and SFV reporter virus expressing eGFP (SFV-eGFP) were successfully rescued. The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 ​cells. Using a broad-spectrum alphavirus inhibitor ribavirin, we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study. The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z′ score. A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses. This assay provides a safe and convenient platform for antiviral study of alphaviruses.