Recombinant Bovine parainfluenza 3 virus Protein C (P/V/C)

Basic Research Questions

• What is Bovine Parainfluenza 3 Virus and what role does the P gene play in its genome?

  Bovine Parainfluenza Virus type 3 (BPIV3) is an enveloped, nonsegmented negative-strand RNA virus within the Respirovirus genus of the family Paramyxoviridae . BPIV3 is a significant respiratory pathogen in cattle, contributing to syndromes in the bovine respiratory disease complex (BRDC) . The virus encodes six structural proteins and three accessory proteins . The P gene of BPIV3 expresses all three reading frames from a single mRNA editing site, encoding the phosphoprotein (P) as the primary product, while also containing open reading frames for the accessory C and V proteins . These proteins play crucial roles in viral replication and host immune evasion mechanisms.

• How is BPIV3 classified and what is its prevalence in cattle populations?

  Based on genetic and phylogenetic analyses, BPIV3 is classified into three genotypes (BPI3Va-c) . A meta-analysis of global BPIV3 prevalence revealed significant variations depending on detection methods. In the general cattle population, antibody detection methods showed a prevalence of 0.64, while in cattle with BRDC, a prevalence of 0.75 was observed . For antigen detection methods, a prevalence of 0.15 was observed exclusively in cattle with BRDC. Nucleic acid detection methods showed prevalence rates of 0.05 and 0.10 in general and BRDC cattle populations, respectively, while virus isolation methods revealed prevalence rates of 0.05 and 0.04 . This data suggests BPIV3 is widely distributed but detection varies significantly based on methodology.

• What are the current applications of recombinant BPIV3 vectors in vaccine development?

  Recombinant BPIV3 serves as a promising vaccine vector against various respiratory virus infections. A primary application is the development of bivalent vaccines targeting both RSV and HPIV3 infections . The chimeric bovine/human PIV3 (rB/HPIV3), where the BPIV3 F and HN genes are replaced with HPIV3 counterparts, has been used to express major protective antigens of RSV . This approach provides a vaccine strategy that overcomes technical challenges associated with live attenuated RSV vaccines . The attenuation of rB/HPIV3 is provided by the host range restriction of the BPIV3 backbone in primates, making it particularly suitable for human applications . Recent developments include using BPIV3 vectors to target SARS-CoV-2 infections .

Advanced Research Methodologies

• How can researchers establish a reverse genetics system for BPIV3 to study P/V/C protein functions?

  A robust reverse genetics system for BPIV3 can be established using a 5-plasmid approach. The methodology involves:

  • Construction of a full-length cDNA clone encoding the complete BPIV3 antigenomic RNA (approximately 15,456 nucleotides)

  • Development of three helper plasmids encoding the viral NP, P, and L proteins

  • Inclusion of a plasmid containing T7 RNA polymerase

  • Assembly of viral genome fragments using unique restriction enzyme sites

  For BPIV3 genotype C specifically, researchers have successfully developed this system by amplifying the viral RNA genome using RT-PCR and assembling separate fragments (A to F) using unique restriction enzyme sites . The T7 promoter is inserted upstream of fragment A with three G residues preceding the sequence, and a partial HDV ribozyme is included in the final fragment . This system allows for precise genetic manipulation of the P gene to study the functions of P, V, and C proteins.

• What experimental approaches can identify how P/V/C proteins modulate host immune responses?

  Research has demonstrated that PIV-3 modulates signaling pathways downstream of the type III IFN receptor to block production of several specific molecules critical for antiviral responses . To study these mechanisms:

  • Real-time PCR can be employed to measure expression levels of interferon-stimulated genes in cells infected with wild-type versus P/V/C-modified viruses

  • Time course experiments from 0-72 hours post-infection can reveal temporal aspects of immune modulation

  • Comparison of relative gene expression using the 2^(-ΔΔCT) method with GAPDH as a reference gene

  • Analysis of specific immune mediators including IL-29, IL-28, and various IFN subtypes

  The following primer sets can be utilized for quantitative analysis of relevant immune mediators:

  Target	Forward Primer	Reverse Primer
  IL-29	GAA GCC TCA GGT CCC AAT TC	GAA GCC TCA GGT CCC AAT TC
  IL-28	ACT GCA GCC ACT CCC	CTC CAG AAC CTT CAG CGT CAG
  IFN-α1	CAG AGT CAC CCA TCT CAG CA	CAC CAC CAG GAC CAT CAG TA
  IFN-α2	CTG GCA CAA ATG GGA AGA AT	CTT GAG CCT TCT

  These approaches can effectively characterize how P/V/C proteins interfere with antiviral signaling pathways .

• How can researchers generate and characterize chimeric viruses to determine the role of P/V/C proteins in host range restriction?

  The contribution of viral proteins to host range restriction can be systematically studied by generating chimeric viruses. To investigate P/V/C proteins:

  1. Construct chimeric BPIV3/HPIV3 recombinants by replacing specific BPIV3 genes with their HPIV3 counterparts

  2. Introduce unique restriction enzyme recognition sites into both BPIV3 and HPIV3 antigenomic cDNAs to facilitate gene exchanges

  3. Use site-directed mutagenesis protocols to create the necessary restriction sites

  4. Assess growth characteristics in both bovine and primate cell lines (e.g., LLC-MK2 monkey kidney and MDBK cells)

  5. Evaluate replication efficiency in animal models (e.g., rhesus monkeys) to determine attenuation phenotypes

  This approach has successfully identified the N protein as a determinant of host range restriction in primates , and similar methods can be applied to study P/V/C proteins. Complementary approaches include Western blotting and immunofluorescence analysis to confirm protein expression in infected cells .

• What strategies can be used to develop photocontrollable BPIV3 for studying P/V/C protein dynamics?

  Innovative approaches combining optogenetic systems with reverse genetics have enabled the development of photocontrollable BPIV3. The methodology involves:

  1. Insertion of an optically controllable Magnet gene into specific regions of the BPIV3 genome

  2. For P/V/C protein studies, the Magnet gene can be incorporated into the P gene region

  3. Blue light illumination can then control viral replication in a spatiotemporal manner

  This approach allows researchers to achieve precise control over viral protein expression and function, facilitating detailed studies of P/V/C protein dynamics during infection. The technology has been successfully demonstrated with insertions into the large protein gene, which encodes the RNA-dependent RNA polymerase , and can be adapted for P/V/C protein studies.