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. 2011 Mar 4:8:93.
doi: 10.1186/1743-422X-8-93.

Virulence of H5N1 virus in mice attenuates after in vitro serial passages

Jing Li  1 , Bohua Liu, Guohui Chang, Yi Hu, Dawei Zhan, Yukun Xia, Yongqiang Li, Yinhui Yang, Qingyu Zhu
Affiliations

Virulence of H5N1 virus in mice attenuates after in vitro serial passages

Jing Li et al. Virol J. .

Abstract

The virulence of A/Vietnam/1194/2004 (VN1194) in mice attenuated after serial passages in MDCK cells and chicken embryos, because the enriched large-plaque variants of the virus had significantly reduced virulence. In contrast, the small-plaque variants of the virus and the variants isolated from the brain of mice that were infected with the parental virus VN1194 had much higher virulence in mice. The virulence attenuation of serially propagated virus may be caused by the reduced neurotropism in mice. Our whole genome sequence analysis revealed substitutions of a total of two amino acids in PB1, three in PB2, two in PA common for virulence attenuated variants, all or part of which may be correlated with the virulence attenuation and reduced neurotropism of the serially propagated VN1194 in mice. Our study indicates that serial passages of VN1194 in vitro lead to adaptation and selection of variants that have markedly decreased virulence and neurotropism, which emphasizes the importance of direct analysis of original or less propagated virus samples.

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Figures

Figure 1
Figure 1
Plaque forming assay of VN1194-W, VN1194-P and other variants isolated from VN1194. Confluent monolayer of MDCK cells were inoculated with 10-fold serial dilutions of H5N1 virus and overlaid with 1% hypo-Tm-solved agarose. After two d inoculation at 35°C, cells were stained with neutral red and plaque morphology was evaluated. The larger plaques were discriminated from smaller plaques when their size was two times larger than smaller ones. A: VN1194-W; B: VN1194-P; C: 1194-SP; D: 1194-LP; E: 1194-ML; F: 1194-MB.
Figure 2
Figure 2
Growth curves of 1194-LP and 1194-SP. Cells were inoculated at a 0.01 MOI (multiplicity of infection). The plaque titers of the supertant of MDCK (A) or A549 (B) cells infected with 1194-LP (◊) or 1194-SP (*) were determined at appropriate time points on MDCK cells. Each curve is the average of three independent experiments.
Figure 3
Figure 3
Survival rates of BALB/c mice infected with VN1194-W, VN1194-P and other variants. Six to 8-week-old, 15-17 g femal mice with 10 in each group were infected i.n. with 100 pfu (A-C) and i.v. with 50 pfu (D).
Figure 4
Figure 4
Replication of VN1194-W, VN1194-P and 1194-LP viruses in mouse lung, blood and brain. Mice were infected i.n. with 100 pfu (A), or i.v. with 50 pfu (B) of each virus. On days three and six p.i., three mice in each group were sacrificed and virus titers in each organs or tissues were determined by TCID50 on MDCK cells. The ratio of positive to total sample was labeled on the top of each histogram. A: Virus titers in the lung of mice infected with VN1194-W were significantly lower on three p.i. but significantly higher on six p.i. than those in the lung of mice infected with the other two viruses (P < 0.01). The differences of virus titers in mouse brain on daythree or six p.i. between the mice infected with 1194-LP and those infected with the other two viruses were also significant (P < 0.01). B: virus titers in the lung of mice infected with VN1194-W were significantly higher on both day three and six p.i. than those in the lung of mice infected with the other two viruses (P < 0.01), and the differences of virus titers in mouse brain on day three or six p.i. between the mice infected with 1194-LP and those infected with the other two viruses were also significant (P < 0.01).
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