I am currently working on some in vivo studies. I need my influenza viruses, A/PR/8/34 (H1N1) and A/HK/8/68 (H3N2) to become adapted in mice (BALB/c). I am trying to generate the mouse adapted virus by lung serial passage. My procedure is as below:
1. Infect 2 mice intranasally, 29 uL in each nostril without anesthesia.
2. Incubate for 4 days.
3. Harvest the lungs of the mouse that showed most severe weight drop in 1.5 mL PBS.
4. Homogenized the lungs tissue, get the supernatant, filterd with 0.45 um filter and proceed to next passage.
Previously, after incubation, I harvest the lungs of both of the mice and pooled them together. However I realized it may not be appropriate as the mutation of virus in each mouse is independent; one might have adapted well and one may not but pooling them together and allow them to infect next mice, gene re-assortment between the adapted virus and non-adapted virus may occur and lead to a less virulence virus in next passage. ( I am not sure about this, it is my guess, I discuss with my co-researcher and he was agreed with me, and thats why current I am only selecting the mouse with most severe weight drop. I am having this speculation was because during one of the passage, one of the H1 infected mouse showed a very significant weight drop and symptoms at day 4 post-infection (>15 %) but when I pooled the lungs of the mice and proceed to subsequent passage, weight drop and symptoms of the mice in subsequent passage were not significant.)
Currently I am trying to only harvest the lungs of one of the mouse with greatest weight drop and proceed to subsequent passage. But no promising result is obtain. As a result, I hope that Prof. Racaniello can give me some advises. Is it correct for not pooling the lungs of the mice? It was also strange that the both the H1 and H3 viruses I obtained in each passage dose not show any HAU. (I am using 0.5 % chicken RBC). Present of the viruses in lungs and oropharyngeal were confirmed by PCR.
The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to α2,3 together with decreasing binding to α2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals.
Article Adaptation of Pandemic H1N1 Influenza Viruses in Mice
You will have to passage your virus several times in mice in order to adapt the virus. See attached protocol. Let me know if you have other questions. Good luck.
Where did you get your PR8? The strain was adapted to mice shortly after it was isolated in 1934 by serial passages--10 were used, I believe. Subsequent passages in eggs or mice did not eliminate the adaptation to mice. Also, it is quite possible that by inoculating the mice without anesthesia you did not get enough virus into the nose. Without the irritation of anesthesia the mice fail to suck the virus up into the nose, pharynx and lungs. Light halothane or ether anesthesia will solve that problem. I do not have experience with A/HK/68.
Be aware that PR8 invades the brain via the olfactory bulb within hours of nasal instillation. See Majde et al., (2007) Detection of mouse-adapted human influenza virus in the olfactory bulb of mice within hours after intranasal infection. J. NeuroVirology 13:399-409.
Adaptation of pandemic H1N1 influenza viruses in mice.
Ilyushina NA1, Khalenkov AM, Seiler JP, Forrest HL, Bovin NV, Marjuki H, Barman S, Webster RG, Webby RJ.
Abstract
The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to alpha2,3 together with decreasing binding to alpha2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals.
http://www.ncbi.nlm.nih.gov/pubmed/20592084