Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice

Abstract

Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouse-adapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin-Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research.

Keywords: embryonated eggs; growth system; hemagglutinin; influenza B virus; mouse-adapting; neuraminidase.

Figure 1

Schematic depicting mouse-passaging procedure. Passage 1 mice were infected i.n. with 20 µL of stock virus. In 3 d.p.i., the lungs were harvested and homogenized before pooling of the lung lysate. Passage 2 mice were then infected with 20 µL of pooled lung lysate and the procedure was repeated up to passage 10. After homogenizing passage 10 lungs, the lysate was diluted and used to infect either embryonated chicken eggs or MDCK-London cells. Infected embryonated eggs were incubated at 33 °C for 72 h before being moved to 4 °C overnight. Allantoic fluid was harvested the next day. An amount of 25 cm² infected MDCK-London cells were incubated at 33 °C for 48 h before clarified supernatant was used to infect a 75 cm² flask. The 75 cm² flask was incubated for 48 h at 33 °C before expansion into a 225 cm² flask. Finally, 225 cm² MDCK-London cells were incubated at 33 °C for 72 h before harvesting virus from supernatant. Schematic created in BioRender.

Figure 2

Live challenge with wild-type B/Florida/4/2006 and B/Phuket/3073/2013. (A) TCID₅₀ values for each strain used for initial passage infection. Values shown are an average of three independent experiments with error bars representing standard deviation between three independent replicates. Statistical significance was determined through unpaired t-test, ** p < 0.01; (B) Weight loss over 14 days shown after i.n. infection with 1:2 dilution of allantoic fluid of each parent virus. A ≥25% initial weight lost (dotted line) was used as a threshold for humane sacrifice of the animals.

Figure 3

Determination of pathogenicity of maB/Yam in mice. (A) TCID₅₀/mL titers of both strains after 10 lung-lung passages and expanded in either embryonated eggs (p10E) or MDCK-Ln cell culture (p10C). (B) Table breaking down MLD₅₀ values calculated using the Reed–Meunch method (31) for each virus strain. (C) Mice challenged with doses corresponding to logTCID₅₀ of FL/4/06 p10E or p10C were followed for 14 d.p.i. for weight loss (top) and survival after challenge with 5log TCID₅₀ units (bottom). A ≥25% initial weight lost was used as a threshold for humane sacrifice of the animals. (D) Mice challenged with doses corresponding to logTCID₅₀ of Phu/13 p10E or p10C were followed for 14 d.p.i. for weight loss (top) and survival after challenge with 5log TCID₅₀ units (bottom). A ≥25% initial weight lost was used as a threshold for humane sacrifice of the animals. Statistical differences in TCID₅₀/mL titers were calculated using unpaired t-tests, while statistical significance between group survival was calculated through log-rank Mantel–Cox test. Statistical analysis was performed in GraphPad Prism 9.0 (* p < 0.05, ** p < 0.01).

Figure 4

Pathogenicity of FL/4/06 after 20 total lung-lung passages. (A) TCID₅₀/mL titers of FL/4/06 p20E and p20C. (B) Table breaking down MLD₅₀ values calculated using the Reed–Meunch method (31) for FL/4/06 p20E and p20C. (C) Mice challenged with doses corresponding to logTCID₅₀ of FL/4/06 p20E or p20C were followed for 14 d.p.i. for weight loss (top) and survival after challenge with 6log TCID₅₀ units (bottom). A ≥25% initial weight lost was used as a threshold for humane sacrifice of the animals. (D) Comparison of survival probability of mice challenged with 5log TCID₅₀ of p10E or p20E. No statistically significant difference in survival probability was observed. Statistical differences in TCID₅₀/mL titers were calculated using unpaired t-tests, while statistical significance between group survival was calculated through log-rank Mantel–Cox test. Statistical analysis was performed in GraphPad Prism 9.0 ** p < 0.01).

Figure 5

FL/4/06 surface glycoprotein sequence analysis. The HA and NA genes were reverse transcribed into cDNA, PCR amplified, and sequenced using Sanger sequencing. (A) The major antigenic sites [35] of the HA protein are shown to be identical to the parental virus. (B) The single nucleotide mutation observed between the parental virus and the mouse-adapted HA sequences was a single silent mutation between nucleotide 241 and 320. (C) A representative amino acid alignment of the NA protein from positions 161–320 shows 100% sequence conservation between the parental and the mouse-adapted viruses.