J Vet Sci.  2017 Aug;18(S1):307-313. 10.4142/jvs.2017.18.S1.307.

Effect of the fourth nucleotide at the 3′ end of neuraminidase and matrix viral genomic RNA on the pathogenicity of influenza virus A/PR/8/34

• Lee CY
• Kwon HJ
• Nguyen TT
• Kim I
• Jang HK
• Kim JH

• KMID: 2412555
• DOI: http://doi.org/10.4142/jvs.2017.18.S1.307

Abstract

Twelve nucleotides located at the 3"² end of viral genomic RNA (vRNA) are conserved among influenza A viruses (IAV) and have a promoter function. Hoffmann's 8-plasmid reverse genetics vector system introduced mutations at position 4, C nucleotide (C4) to U nucleotide (U4), of the 3"² ends of neuraminidase (NA) and matrix (M) vRNAs of wild-type A/PR/8/34 (PR8). This resulted in a constellation of C4 and U4 vRNAs coding for low (polymerases) and relatively high (all others) copy number proteins, respectively. U4 has been reported to increase promoter activity in comparison to C4, but the constellation effect on the replication efficiency and pathogenicity of reverse genetics PR8 (rgPR8) has not been fully elucidated. In the present study, we generated 3 recombinant viruses with C4 in the NA and/or M vRNAs and rgPR8 by using reverse genetics and compared their pathobiological traits. The mutant viruses showed lower replication efficiency than rgPR8 due to the low transcription levels of NA and/or M genes. Furthermore, C4 in the NA and/or M vRNAs induced lower PR8 virus pathogenicity in BALB/c mice. The results suggest that the constellation of C4 and U4 among vRNAs may be one of the multigenic determinants of IAV pathogenicity.

Keyword

influenza A virus; pathogenicity; promoter; reverse genetics; viral replication

MeSH Terms

3' Flanking Region/genetics/physiology
Animals
Female
Influenza A virus/genetics/*pathogenicity
Mice
Mice, Inbred BALB C
Mutagenesis, Site-Directed
Neuraminidase/*genetics
Orthomyxoviridae Infections/virology
Promoter Regions, Genetic/genetics
RNA, Viral/*genetics
RNA, Viral
Neuraminidase

Figure

• 

  Fig. 1 Comparison of virus titers of recombinant viruses with different constellation of C nucleotide and U nucleotide at position 4 in the 3′-end of the noncoding region of the viral genome. Each recombinant virus (10 EID50/200 µL/ECE; EID50, 50% egg infectious dose; ECE, embryonated chicken egg) was inoculated into eighteen 10-day-old specific pathogen-free ECEs, and 3 ECEs were harvested at 8, 12, 16, 24, 32, and 48 h post-inoculation. The virus titers were measured by 50% tissue culture infection dose (TCID50) assay in Madin-Darby canine kidney cells. *Asterisks represent a significant difference of virus titers between the reverse genetics PR8 (rgPR8) and the other recombinant viruses (p < 0.05).

• 

  Fig. 2 Relative transcription levels of viral genomic RNA (vRNA) and messenger RNA (mRNA) of recombinant viruses. (A) Relative transcription levels of vRNA and mRNA of the neuraminidase (NA) genome segments. (B) Relative transcription levels of vRNA and mRNA of the matrix (M) genome segments. Madin-Darby canine kidney cells were infected by recombinant viruses at 0.001 multiplicity of infection at 37℃, and cell lysates were harvested at 6 h post-inoculation. The vRNA and mRNA transcription levels were normalized by the transcription levels of GAPDH gene of the infected cells. The relative transcription levels of vRNA and mRNA of each recombinant virus were represented by the ratio to those of rgPR8. The data presented are the average of three independent experiments. **Asterisks represent a significant difference between the rgPR8 and other recombinant viruses (p < 0.001).

• 

  Fig. 3 Comparison of mouse pathogenicity of recombinant viruses. The 105 and 104 EID50/50 µL/mouse recombinant viruses were challenged to five 5-week-old BALB/c mice. Weight loss and mortality were monitored for 12 days. (A) Weight loss and (B) mortality of mice infected by 105 EID50 of each virus. (C) Weight loss and (D) mortality of mice infected by 104 EID50 of each virus. *Asterisks indicate weight loss of recombinant viruses is significantly different from that of rgPR8 (p < 0.05). EID50, 50% egg infectious dose; PBS, phosphate-buffered saline; wtPR8, wild-type A/PR/8/34.

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