HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

OASL suppresses infectious bursal disease virus replication by targeting VP2 for degrading through the autophagy pathway.

Infectious bursal disease (IBD) is an acute and fatal immunosuppressive disease caused by infectious bursal disease virus (IBDV). As an obligate intracellular parasite, IBDV infection is strictly regulated by host factors. Knowledge on the antiviral activity and possible mechanism of host factors might provide the theoretical basis for the prevention and control of IBD. In this study, RNA-sequencing results indicated that many host factors were induced by IBDV infection, among which the expression levels of OASL (2´,5´-oligadenylate synthetase-like protein) was significantly upregulated. OASL overexpression significantly inhibited IBDV replication, whereas OASL knockdown promoted IBDV replication. Interestingly, the antiviral ability of OASL was independent of its canonical enzymatic activity, i.e., OASL targeted viral protein VP2 for degradation, depending on the autophagy receptor p62/SQSTM1 in the autophagy pathway. Additionally, the 316 lysine (K) of VP2 was the key site for autophagy degradation, and its replacement with arginine disrupted VP2 degradation induced by OASL and enhanced IBDV replication. Importantly, our results for the first time indicate a unique and potent defense mechanism of OASL against double-stranded RNA virus by interaction with viral proteins, which leads to their degradation. IMPORTANCE: OASL (2´,5´-oligadenylate synthetase-like protein) exhibits broad-spectrum antiviral effects against single-stranded RNA viruses in mammals, potentially serving as a promising target for novel antiviral strategies. However, its role in inhibiting the replication of double-stranded RNA viruses (dsRNA viruses), such as infectious bursal disease virus (IBDV), in avian species remains unclear. Our findings indicated a unique and potent defense mechanism of OASL against dsRNA viruses. It has been previously shown in mammals that OASL inhibits virus replication through increasing interferon production. The groundbreaking aspect of our study is the finding that OASL has the ability to interact with IBDV viral protein VP2 and target it for degradation and thus exerts its antiviral effect. Our results reveal the interaction between avian natural antiviral immune response and IBDV infection. Our study not only enhances our understanding of bird defenses against viral infections but can also inform strategies for poultry disease management.

Cell Culture Adaptive Amino Acid Substitutions in FMDV Structural Proteins: A Key Mechanism for Altered Receptor Tropism.

The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology.

The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

Development of visual detection of African swine fever virus using CRISPR/LwCas13a lateral flow strip based on structural protein gene D117L.

African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.

Continuous clinicopathological and molecular recognition of very virulent infectious bursal disease virus in commercial broiler chickens.

Gumboro virus is one of the most dangerous immunosuppressant viruses that infect chickens and causes massive financial losses worldwide. The current study aims to conduct a molecular characterization of chicken farms for the infectious bursal disease virus (IBDV). Based on postmortem (PM) lesions, 125 bursal samples from 25 farms were collected from clinically diseased commercial chicken farms with increased mortality and suspected Gumboro virus infection. Pooled bursal samples from suspected IBD-vaccinated flocks were tested for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Fifteen out of 25 pooled specimens were found positive for IBDV, with a 60% detection rate, and confirmed positive for very virulent IBDV (vvIBDV) by sequence analysis. Nucleotide phylogenetic analysis of VP1 and VP2 genes was employed to compare the 5 chosen isolates with strains representing different governorates in Egypt during 2022. All strains were clustered with vvIBDV with no evidence of reassortment in the VP1 gene. The VP1 and VP2 genes are divided into groups (I, II). The strains in our study were related to group II, and it acquired a new mutation in the VP2 gene that clustered it into new subgroup B. By mutation analysis, the VP2 gene of all strains had a characteristic mutation to vvIBDV. It acquired new mutations in HVRs compared with HK46 in Y220F, A222T/V in all strains in our study, and Q221K that was found in IBD-EGY-AH5 and AH2 in the loop PBC in addition to G254S in all strains in our study and Q249k that found in IBD-EGY-AH1 and AH3 in the loop PDE. These mutations are important in the virulency and antigenicity of the virus. The VP1 had 242E, 390M, and 393D which were characteristic of vvIBDV and KpnI restriction enzyme (777GGTAC/C782) in addition to a new mutation (F243Y and N383H) in IBD-EGY-AH1 and AH4 strains. According to the current study, the strains were distinct from the vaccinal strain; they could be responsible for the most recent IBDV outbreaks observed in flocks instead of received vaccinations. The current study highlighted the importance of molecular monitoring to keep up to date on the circulating IBDV for regular evaluation of commercial vaccination programs against circulating field viruses.

Molecular epidemiology of infectious bursal disease virus in the Near East and Persian Gulf regions.

RESEARCH HIGHLIGHTS: Different field IBDVs were found to circulate in the Near and Middle East.Multiple atypical genotypes (A3B1, A4B1, A6B1) were found to circulate extensively.Traditional very virulent IBDVs (A3B2) were a minority of the detected strains.Viral exchanges can be hypothesized between the region and different continents.

Whole Genome Sequencing of Infectious Bursal Disease Viruses Isolated from a Californian Outbreak Unravels the Underlying Virulence Markers and Highlights Positive Selection Incidence.

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were not fully uncovered. Therefore, segments of both isolates were amplified, successfully cloned, whole genome sequenced by Next Generation Sequencing, genotyped, and the leading virulence factors were entirely investigated in terms of phylogenetic and amino acid analysis and protein modeling for positive selection orientation and interaction analysis. rA and rB isolates displayed the highest amino acid identity (97.84-100%) with Genotype 3 strains. Interestingly, rA and rB contained all virulence hallmarks of hypervariable (HVR), including 222A, 242I, 249Q, 256I, 284A, 286T, 294I, 299S, and 318G, as well as the serine-rich heptapeptide sequence. Moreover, we pinpointed the A3B2 genotype of rA and rB, predominant in non-reassortants, and we highlighted the absence of recombination events. Furthermore, gene-wise phylogenetic analysis showed the entire genes of rA and rB clustered with the vvIBDVs and emphasized their share in IBDV virulence. VP5 showed a virulence marker, MLSL (amino acid sequence). VP2 encountered three significant novel mutations apart from the HVR, including G163E in rA and Y173C and V178A in rB, all residing within interacting motifs. VP4 contained 168Y, 173N, 203S, and 239D characteristic for the vv phenotype. A235V mutation was detected at the dsRNA binding domain of VP3. In VP1, the TDN triplet and the mutation (V4I) were detected, characteristic of hypervirulence occurring at the N-terminus responsible for protein priming. Although selection analysis revealed seven sites, codon 222 was the only statistically significant selection site. The VP2 modeling of rA and rB highlighted great structure fitness, with 96.14% Ramachandran favored positioning including the 222A, i.e., not influencing the structure stability. The 222A was found to be non-interface surface residue, associated with no interaction with the attachment-mediated ligand motif. Our findings provide pivotal insights into the evolution and underlying virulence factors and will assist in the development of control strategies via sequence-based continuous monitoring for the early detection of novel vv strains.

Duck plague virus tegument protein vp22 plays a key role in the secondary envelopment and cell-to-cell spread.

Duck plague virus (DPV) is one of the major infectious and fatal diseases of geese, ducks, and other wild waterfowl. The DPV UL49 gene product VP22 is one of the most abundant tegument proteins. However, the role of the DPV VP22 is enigmatic to be clarified. In this study, we found deletion of the UL49 gene resulted in reduced viral growth curve and smaller plaque size in duck embryo fibroblast (DEF) cells, confirming that DPV VP22 is required for efficient viral growth in vitro. In addition, deletion of the UL49 gene inhibited the secondary envelopment of the virus, the release of viral particles, and the spread of viruses between cells. Our study signified the importance of VP22 for DPV secondary envelopment, release, cell-to-cell spread, and accumulation of viral RNA. These findings provide a basis for further study of the function of VP22 in DPV or other herpesviruses.

The evolution of chikungunya virus circulating in Indonesia: Sequence analysis of the orf2 gene encoding the viral structural proteins.

Chikungunya virus (CHIKV) is an arthropod-borne virus that has caused several major epidemics globally, including in Indonesia. Although significant progress has been achieved in understanding the epidemiology and genotype circulation of CHIKV in Indonesia, the evolution of Indonesian CHIKV isolates is poorly understood. Thus, our study aimed to perform phylogenetic and mutation analyses of the orf2 gene encoding its viral structural protein to improve our understanding of CHIKV evolution in Indonesia. Complete orf2 gene sequences encoding the viral structural proteins of Indonesian-derived CHIKV were downloaded from GenBank until August 31, 2022. Various bioinformatics tools were employed to perform phylogenetic and mutation analyses of the orf2 gene. We identified 76 complete sequences of orf2 gene of CHIKV isolates originally derived from Indonesia. Maximum likelihood trees demonstrated that the majority (69/76, 90.8%) of Indonesian-derived CHIKV isolates belonged to the Asian genotype, while seven isolates (9.2%) belonged to the East/Central/South African (ECSA) genotype. The Indonesian-derived CHIKV isolates were calculated to be originated in Indonesia around 95 years ago (1927), with 95% highest posterior density (HPD) ranging from 1910 to 1942 and a nucleotide substitution rate of 5.07 × 10-4 (95% HPD: 3.59 × 10-4 to 6.67 × 10-4). Various synonymous and non-synonymous substitutions were identified in the C, E3, E2, 6K, and E1 genes. Most importantly, the E1-A226V mutation, which has been reported to increase viral adaptation in Aedes albopictus mosquitoes, was present in all ECSA isolates. To our knowledge, our study is the first comprehensive research analyzing the mutation and evolution of Indonesian-derived CHIKV based on complete sequences of the orf2 genes encoding its viral structural proteins. Our results clearly showed a dynamic evolution of CHIKV circulating in Indonesia.

PRMT5 Facilitates Infectious Bursal Disease Virus Replication through Arginine Methylation of VP1.

The infectious bursal diseases virus (IBDV) polymerase, VP1 protein, is responsible for transcription, initial translation and viral genomic replication. Knowledge about the new kind of post-translational modification of VP1 supports identification of novel drugs against the virus. Because the arginine residue is known to be methylated by protein arginine methyltransferase (PRMT) enzyme, we investigated whether IBDV VP1 is a substrate for known PRMTs. In this study, we show that VP1 is specifically associated with and methylated by PRMT5 at the arginine 426 (R426) residue. IBDV infection causes the accumulation of PRMT5 in the cytoplasm, which colocalizes with VP1 as a punctate structure. In addition, ectopic expression of PRMT5 significantly enhances the viral replication. In the presence of PMRT5, enzyme inhibitor and knockout of PRMT5 remarkably decreased viral replication. The polymerase activity of VP1 was severely damaged when R426 mutated to alanine, resulting in impaired viral replication. Our study reports a novel form of post-translational modification of VP1, which supports its polymerase function to facilitate the viral replication. IMPORTANCE Post-translational modification of infectious bursal disease virus (IBDV) VP1 is important for the regulation of its polymerase activity. Investigation of the significance of specific modification of VP1 can lead to better understanding of viral replication and can probably also help in identifying novel targets for antiviral compounds. Our work demonstrates the molecular mechanism of VP1 methylation mediated by PRMT5, which is critical for viral polymerase activity, as well as viral replication. Our study expands a novel insight into the function of arginine methylation of VP1, which might be useful for limiting the replication of IBDV.

Detection and Analysis of West Nile Virus Structural Protein Genes in Animal or Bird Samples.

West Nile virus (WNV) is an important zoonotic pathogen, which is detected mainly by identification of its RNA using PCR. Genetic differentiation between WNV lineages is usually performed by complete genome sequencing, which is not available in many research and diagnostic laboratories. In this chapter, we describe a protocol for detection and analysis of WNV samples by sequencing the entire region of their structural genes capsid (C), preM/membrane, and envelope. The primary step is the detection of WNV RNA by quantitative PCR of the NS2A gene or the C gene regions. Next, the entire region containing the structural protein genes is amplified by PCR. The primary PCR product is then amplified again in parallel reactions, and these secondary PCR products are sequenced. Finally, bioinformatic analysis enables detection of mutations and classification of the samples of interest. This protocol is designed to be used by any laboratory equipped for endpoint and quantitative PCR. The sequencing can be performed either in-house or outsourced to a third-party service provider. This protocol may therefore be useful for rapid and affordable classification of WNV samples, obviating the need for complete genome sequencing.

Infectious bursal disease virus in Western Europe: the rise of reassortant strains as the dominant field threat.

Infectious bursal disease virus (IBDV) is a highly contagious birnavirus causing a burdensome immunosuppressive disease in chickens. IBDV features a remarkable antigenic, pathogenic and genetic heterogeneity, with significant implications on disease manifestation, control measures and diagnostic approaches. The recent proposals of comprehensive phylogenetic classification systems offered the ideal platform for large-scale molecular surveys, which are crucial to gather epidemiological data and inform control efforts. In this study, the IBDV scenario was investigated in most of Western Europe by considering the results of diagnostic activities performed internationally throughout 2021. In total, 470 bursal samples from nine different countries were analysed by RT-PCR targeting the VP2. When a field virus was identified, the VP1 was also characterized. Most of the 132 detected field viruses were highly homologous reassortants featuring a very virulent-like VP2 and a classical-like VP1 (genotype A3B1). Despite emerging recently, these reassortants were already signalled in several countries in North-Western Europe and associated with subclinical infections. Here, we report their further spread in the region, where they currently represent the dominant field threat. Two other IBDV types were found, one in Italy, where all the identified viruses clustered in a clade of the A3B1 genotype previously reported only in Russia and the Middle East, and the other in Portugal, where the recently characterized A9B1 genotype was confirmed to circulate. The obtained data suggest the recent occurrence of a major shift in the Western European epidemiological landscape of IBDV, stressing the importance of steady monitoring and sharing of information among different countries and laboratories.RESEARCH HIGHLIGHTS The IBDV scenario in Western Europe seems to have radically changed in recent years.IBDV reassortants were found to be the dominant field type in the region.Local circulation of two other IBDV types was detected in Italy and Portugal.

Cryo-EM structures of infectious bursal disease viruses with different virulences provide insights into their assembly and invasion.

Infectious bursal disease virus (IBDV) causes a highly contagious immunosuppressive disease in chickens, resulting in significant economic losses. The very virulent IBDV strain (vvIBDV) causes high mortality and cannot adapt to cell culture. In contrast, attenuated strains of IBDV are nonpathogenic to chickens and can replicate in cell culture. Although the crystal structure of T = 1 subviral particles (SVP) has been reported, the structures of intact IBDV virions with different virulences remain elusive. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the vvIBDV Gx strain and its attenuated IBDV strain Gt at resolutions of 3.3 Å and 3.2 Å, respectively. Compared with the structure of T = 1 SVP, IBDV contains several conserved structural elements unique to the T = 13 virion. Notably, the N-terminus of VP2, which is disordered in the SVP, interacts with the SF strand of VP2 from its neighboring trimer, completing the ß-sheet of the S domain. This interaction helps to form a contact network by tethering the adjacent VP2 trimers and contributes to the assembly and stability of the IBDV virion. Structural comparison of the Gx and Gt strains indicates that H253 and T284 in the VP2 P domain of Gt, in contrast to Gx, form a hydrogen bond with a positively charged surface. This suggests that the combined mutations Q253H/A284T and the associated structural electrostatic features of the attenuated Gt strain may contribute to adaptation to cell culture. Furthermore, a negatively charged groove in VP2, containing an integrin binding IDA motif that is critical for virus attachment, was speculated to play a functional role in the entry of IBDV.

Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein.

The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.

An Improved, Dual-Direction, Promoter-Driven, Reverse Genetics System for the Infectious Bursal Disease Virus (IBDV).

The infectious bursal disease virus (IBDV), one member of the Birnaviridae family, causes immunosuppression in young chickens by damaging the mature B cells of the bursa of Fabricius (BF), the central immune system of young chickens. The genome of IBDV is a bisegmented, double-strand RNA (dsRNA). Reverse genetics systems for IBDV allow the generation of genetically manipulated infectious virus via transfected plasmid DNA, encoding the two genomic viral RNA segments as well as major viral proteins. For this purpose, the minus-sense of both segment A and segment B are inserted into vectors between the polymerase I promoter and the corresponding terminator I. These plasmids facilitate the transcription of the viral minus-sense genome but copy the plus-sense genome as well viral protein translation depends on the activity of VP1 and VP3, when transfected into 293T cells. To further improve rescue efficiency, dual-direction promoters were generated based on the polymerase II promoter in the reverse direction in the backbone of the pCDNA3.0 vector. Therefore, the polymerase I promoter transcribes the viral minus-sense genome in the forward direction and the polymerase II promoter transcribes viral mRNA, translated into viral proteins that produce infectious IBDV. We also found that the rescue efficiency of transfecting two plasmids is significantly higher than that of transfecting four plasmids. In addition, this dual-direction promoter rescue system was used to generate R186A mutant IBDV since Arg186 is the arginine monomer-methylation site identified by LC-MS. Our data furtherly showed that the Arg186 monomer methylation mutant was due to a reduction in VP1 polymerase activity as well as virus replication, suggesting that the Arg186 methylation site is essential for IBDV replication.

Oligomers of hepatitis A virus (HAV) capsid protein VP1 generated in a heterologous expression system.

BACKGROUND: The quasi-enveloped picornavirus, Hepatitis A Virus (HAV), causes acute hepatitis in humans and infects approximately 1.5 million individuals a year, which does not include the asymptomatically infected population. Several severe outbreaks in developing nations in recent years have highlighted the reduction in HAV endemicity, which increases the risk of infections in the vulnerable population. The current HAV vaccines are based on growing wildtype or attenuated virus in cell culture, which raises the cost of production. For generation of cheaper, subunit vaccines or strategies for antibody-based diagnostics, production of viral structural proteins in recombinant form in easily accessible expression systems is a priority. RESULTS: We attempted several strategies for recombinant production of one of the major capsid proteins VP1, from HAV, in the E. coli expression system. Several efforts resulted in the formation of soluble aggregates or tight association of VP1 with the bacterial chaperone GroEL. Correctly folded VP1 was eventually generated in a discrete oligomeric form upon purification of the protein from inclusion bodies and refolding. The oligomers resemble oligomers of capsid proteins from other picornaviruses and appear to have the correct secondary and antigenic surface structure. CONCLUSIONS: VP1 oligomers generated in the bacterial expression system can be utilized for understanding the molecular pathway of HAV capsid assembly and may also have potential biomedical usages in prevention and diagnostics of HAV infections.

Molecular phylodynamics of infectious bursal disease viruses.

The present study was conducted to study the molecular phylodynamics of the Indian field IBDVs. A total of 13 organized commercial poultry farms and 29 village poultry flocks were recruited in the study. The broiler flocks showed 15.25-60.18% mortality, followed by 12.4% in improved native poultry varieties and 5% in indigenous birds. The 664 bp hypervariable VP2 gene fragment of Western and Central Indian vvIBDVs showed 97.14-98.79 and 94.49-96.69% identity to Pakistani and South Indian vvIBDVs, respectively. An isolate was 99.54% identical to the Ventri-Plus vaccine strain, while three IBDVs showed maximum identity with the Georgia strain. Out of 22, 19 strains showed typical vvIBDV amino acid signature, while three strains showed substitutions specific to classical IBDVs. Central Indian vvIBDVs showed conserved substitutions at N212D and E300A, which can be used as a regional marker. Phylogenetic genogrouping placed global IBDVs into seven genogroups based upon virulence and geographical distribution. Nineteen field vvIBDVs were placed in the G3 genogroup, and the other three were grouped with classical IBDVs in G1 genogroup. A nucleotide span from 584 to 1248 covering VP2 hypervariable fragment was found suitable for correct genogrouping of field IBDVs. The Bayesian evolutionary analysis showed tMRCA of the year 2009 for 8 Western Indian vvIBDVs with vvIBDV from Pakistan. Central Indian vvIBDVs were evolved in the year 1991 from BD-3 and PY12 strains of vvIBDVs from Bangladesh and Pondicherry, respectively. An isolate showed evolution in year 2010 from the Nigerian ABIC strain, while three classical strains showed tMRCA of the year 2009 with the Georgia strain as a recent common ancestor.

Soluble overexpression and purification of infectious bursal disease virus capsid protein VP2 in Escherichia coli and its nanometer structure observation.

As part of the development of an infectious bursal disease virus (IBDV) subunit vaccine, this study was designed to improve the expression of highly soluble VP2-LS3 (Haemophilus parasuis lumazine synthase 3, LS3) protein by using different tagged vectors in E. coli. IBDV VP2-LS3 gene was designed and synthesized. Fusion tags, GST, NusA, MBP, Ppi, γ-crystallin, ArsC, and Grifin were joined to the N-terminus of VP2-LS3 protein. Seven expression plasmids were constructed, and each plasmid was transformed into E. coli BL21 (DE3) competent cells. After induction by IPTG, the solubility and expression levels of the various VP2-LS3 proteins were analyzed by SDS-PAGE and Western Blot analysis. The fusion tag that significantly promoted soluble expression of the VP2-LS3 protein was selected. Recombinant proteins were purified using Ni-NTA affinity chromatography, then cleaved by using TEV protease and detected by using transmission electron microscopy. Gel electrophoresis and sequencing analysis showed that all seven recombinant vectors were successfully constructed. GST, NusA, MBP, Ppi, γ-crystallin, ArsC, and Grifin enhanced the expression and solubility of VP2 protein; however, MBP was more effective for the high-purity production of VP2-LS3. Western Blot analysis confirmed successful generation of VP2-LS3 fusion protein in E. coli. The result of transmission electron microscopy showed that VP2-LS3 formed nano-sized particles with homogeneous shape and relatively uniform size. This study established a method to generate VP2-LS3 recombinant protein, which may lay a foundation for the development and subsequent study of IBDV subunit vaccines.

Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes.

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.