Passage
The influenza A virus is an enveloped RNA virus with potential for zoonosis (transmission from animals to humans) . It obtains its plasma membrane from its host cell, but the composition of the viral envelope and vertebrate plasma membrane are distinct. The envelope of H6-G225D consists mostly of phosphatidylethanolamine as well as some negatively charged glycerophospholipids. The outer membrane contains hemagglutinin (HA) proteins that bind to the carbohydrate known as sialic acid (SA) on host cell receptor proteins, facilitating viral entry. It also contains neuraminidase enzymes that break the bond between SA and galactose in the host membrane glycans, allowing new virions to be released.Minute changes in the genes that code for HA proteins can alter the virulence, host, and attachment mechanism. In general, influenza A infects avian species by attaching to tracheal tissue, which displays SA groups bound to receptors through an α-2,3-glycosidic linkage to galactose (Figure 1) . Avian viruses generally cannot attack human cells, in which SA is linked to galactose through an α-2,6-glycosidic linkage instead of an α-2,3 linkage.
Figure 1 Structure of the influenza A receptor in avian cell membranesIn a recent study, however, a G225D substitution in HA, caused by a GGC to GAU codon mutation, enabled avian virions to infect human cells. The stability of G225D HA proteins was compared to that of H6-P186L, which is created by a single CCC to CUC mutation and exclusively infects avian cells, and H1N1, which targets only humans. The viral RNA of H6-P186L, H6-G225D, and H1N1 was cloned using cDNA and expressed in embryonic kidney cells. The melting curve of each purified HA protein was determined using differential scanning calorimetry, with peaks representing melting temperatures (Figure 2) .
Figure 2 Hemagglutinin melting curves for H6N1 and H1N1 influenza A strains
Adapted from De vries RP, Tzarum N, Peng W, et al. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors. EMBO Mol Med. 2017;9(9) :1314-1325.
-Which viruses would provide the necessary controls for an experiment designed to determine whether H6-G225D can infect human cells but not avian cells?WT-H6N1 that attaches only to avian tracheal tissueH1N1 that attaches only to human tracheal tissueV190D and G225D double mutant, which binds neither α-2,6- nor α-2,3- sialic acids

Question

Passage
The influenza A virus is an enveloped RNA virus with potential for zoonosis (transmission from animals to humans) . It obtains its plasma membrane from its host cell, but the composition of the viral envelope and vertebrate plasma membrane are distinct. The envelope of H6-G225D consists mostly of phosphatidylethanolamine as well as some negatively charged glycerophospholipids. The outer membrane contains hemagglutinin (HA) proteins that bind to the carbohydrate known as sialic acid (SA) on host cell receptor proteins, facilitating viral entry. It also contains neuraminidase enzymes that break the bond between SA and galactose in the host membrane glycans, allowing new virions to be released.Minute changes in the genes that code for HA proteins can alter the virulence, host, and attachment mechanism. In general, influenza A infects avian species by attaching to tracheal tissue, which displays SA groups bound to receptors through an α-2,3-glycosidic linkage to galactose (Figure 1) . Avian viruses generally cannot attack human cells, in which SA is linked to galactose through an α-2,6-glycosidic linkage instead of an α-2,3 linkage.

Figure 1 Structure of the influenza A receptor in avian cell membranesIn a recent study, however, a G225D substitution in HA, caused by a GGC to GAU codon mutation, enabled avian virions to infect human cells. The stability of G225D HA proteins was compared to that of H6-P186L, which is created by a single CCC to CUC mutation and exclusively infects avian cells, and H1N1, which targets only humans. The viral RNA of H6-P186L, H6-G225D, and H1N1 was cloned using cDNA and expressed in embryonic kidney cells. The melting curve of each purified HA protein was determined using differential scanning calorimetry, with peaks representing melting temperatures (Figure 2) .

Figure 2 Hemagglutinin melting curves for H6N1 and H1N1 influenza A strains
Adapted from De vries RP, Tzarum N, Peng W, et al. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors. EMBO Mol Med. 2017;9(9) :1314-1325.
-Which viruses would provide the necessary controls for an experiment designed to determine whether H6-G225D can infect human cells but not avian cells?WT-H6N1 that attaches only to avian tracheal tissueH1N1 that attaches only to human tracheal tissueV190D and G225D double mutant, which binds neither α-2,6- nor α-2,3- sialic acids

Quizplus · Accepted Answer

11ecba2d_0eb5_c266_a3bf_91850f5d368e_MD0008_00 The passage suggests that the G225D mutation allows influenza A virions to switch their target species from birds to humans. An experiment designed to determine whether H6-G225D virions exclusively infect humans should include controls that are known to infect either human or avian species only. The effects of these control groups on cells can then be compared to the effects of H6-G225 in each type of cell. A positive control displays the response that would be expected if H6-G225D is able to infect human cells. Because H1N1 is known to infect human tissue exclusively, it works as an adequate positive control (Number II); if the G225D mutation allows virions to infect human cells, then the effects of H1N1 and H6-G225D on human tracheal cells will be similar. A negative control shows the response that would be expected if the virus did not switch target species: infection of avian cells but not human cells. WT-H6N1 serves as a negative control because it exclusively infects avian tracheal tissue (Number I). If the G225D mutation eliminates the affinity of virions for avian tissue, H6-G225D will not show a response in avian cells whereas WT-H6N1 will. In a well-designed experiment, both positive and negative controls are needed to validate the researcher's predictions and results. (Number III) The double mutant does not cause infection in either of the two target species of influenza A. It is not necessary as a control because it has the same effect as no virus at all. Educational objective: Controls are necessary experimental tools that are used to compare and validate the results of an experiment. Positive controls display an expected response; in negative controls, no response is expected.