Information Flow and Genetics

TWiM #175: Neomycin is Antiviral

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper

n/a

1.2. Main paper; discussion starts at 15:18 minutes

The Most Interesting Things (according to students)

There are state microbes!!!!! Also, although antibiotics are typically only recommended for treatment of bacterial infections, this study shows that some antibiotics may protect from viral infections as well.

The abstract for this paper cannot be copied due to licensing restrictions.  Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
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  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
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  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 22 (ASM_22):Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life. 

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • na
 S L
  • na
 S H
  • Identify the bacterial target(s) of specific antibiotics mentioned in the podcast.
  • Match the virus with its specific antibiotic treatment mentioned in the podcast.
 M L
  • Differentiate between results that indicate a systemic-drug effect and a localized drug effect.
  • Analyze phenotypic data from knock-out organisms to determine the involvement of particular host genes in pathogen virulence. 
 M H

1Papers: Snippet (S) or Main (M)
2Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

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4.2. Main Paper

  • PEGylation (22:56–23:29): When a drug is PEGylated, the drug is conjugated with polyethylene glycol (PEG), so that it will not be degraded quickly in the body.  When treating Hepatitis C with interferons, the patients were given PEGylated interferon, which is a chemically conjugated interferon that lasts longer in the body so that interferon-stimulated genes (ISG) would be expressed to help fight the viral infection.
  • Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) (30:33–30:52): CRISPR/Cas is a method for editing the genome that is bioengineered using a bacterial defense system.  Here, it was used to generate mice lacking specific proteins.  When mice lacking MAVS, a protein downstream of cytosolic receptors, were treated with neomycin, there is significant reduction of interferon-stimulated genes (ISG) production.   Next, to identify which receptor was important, they used mice lacking either Toll-like Receptor 3 (TRL3) or Toll-like Receptor 7 (TLR7).  Their results indicated that neomycin used RNA sensor pathways that involved TLR3, but not TLR7.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • n/a

5.2. Main Paper

  • Antibiotics (15:30–50:17): Aminoglycosides such as neomycin are bactericidal and applied topically.
  • Antiviral Properties (17:29–17:45): The antibiotic binds to the 30S ribosomal subunit of bacteria and inhibits protein synthesis in both bacteria and viruses.
  • Cytokines (21:14–31:33): Interferons bind receptors and turn on interferon stimulated genes (ISGs).  The ISGs promote  antiviral properties.
  • Inflammatory Response (44:44–45:30): Aggravation of cytokines will induce an inflammatory response in the host which activates the adaptive immune response to clear the pathogen.

6. Podcast Questions 

  1. Antibiotics typically function by inhibiting critical biological processes.  Which of the following antibiotic-targeted processes that are effective for bacterial pathogens would also likely be effective for a typical viral pathogen?
    1. Ribosomes translate RNA to produce protein
    2. Molecules to transfer peptidoglycan segments
    3. Enzymes synthesize cell wall polymers
    4. None of the above, as they are missing in viruses
  2. Match the antibiotic with the virus it affects. (1 =  herpes; 2 = flavivirus;  3 = Zika)
    1. ____ azithromycin
    2. ____ neomycin
    3. ____ nonchonmycin
  3. A specific hypothetical conjugated-drug induces expression of genes A, B, and C, and the drug was administered in a topical cream to the skin.  Where would you be able to find the high expression of genes A, B, and C if the drug has localized and not systemic effect?
    1. The lungs
    2. The skin
    3. The brain
    4. Throughout the body
  4. The researchers in the paper were able to identify which receptors were involved in neomycin’s effects on a viral pathogen using mice lacking specific receptors.   In a similar hypothetical experiment, a researcher knew that treatment with compound Q induces expression of immune gene A. She wants to identify compound Q’s receptor that initiates this response.  So, she treated mice lacking each of three receptors (X, Y, and Z) with compound Q or saline control and found the following results for gene A expression (see table).  Which receptor is most likely involved in compound Q’s immune gene expression response?
Mouse genotype Treatment Expression of gene A
wild-type control none
wild-type drug high
receptor X deletion control none
receptor X deletion drug high
receptor Y deletion control none
receptor Y deletion drug high
receptor Z deletion control none
receptor Z deletion drug none
    1. receptor X
    2. receptor Y
    3. receptor Z
    4. none of the receptors
    5. all of the receptors

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1 and 4bc,ef,hi).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in the line graphs and strip plots related to the experiments.
  • Identify the lag, log, and stationary phases of a growth curve or other disease measure.
  • Analyze growth and disease measurement curves to predict pathogen load.
  • Analyze growth and disease measurement curves to make conclusions about which compound within a mixture impacts the pathogen.
  • Analyze growth and disease measurement curves to make conclusions about how the microbiome impacts compound effectiveness.
  • Analyze growth and disease measurement curves to make conclusions about how treatment regime impacts compound effectiveness.
Experimental Background (Gopinath et al., Figure 1)

Antibiotics have revolutionized the treatment of bacterial infections.  However, some of their mechanisms and effects on the eukaryotic host are not yet entirely clear. Of particular interest to this group was genital herpes infection which is sexually transmitted, caused by Herpes simplex virus-2 (HSV-2), and affects 520 million people globally.  In this study, Gopinath et al (2018) wanted to investigate the effects of different antibiotics on the vaginal microbiome during HSV-2 infection.  First, they treated mice with a normal microbiome (panels a–d, g and h) or germ-free mice (panels e and f) with a hormone to synchronize their uterine cycles to eliminate that as a confounding variable.  Next, multiple antibiotic mixtures (panels a and b) or single antibiotics (panels c–f) were then applied intravaginally as a pre-treatment (for 6 days) before HSV-2 infection (panels a–f) or as a post-infection treatment (for 3 days) (panels g and h).  Mice were scored for disease each day (panels a, c, e, and g) and vaginal washes were taken to quantify infection using a plate count method for infectious virus (plaque forming units; PFU; panels b, d, f, and h).

7.1.2. Questions

  1. The researchers used germ-free mice and mice with a normal (conventional) vaginal microbiome.  Which panels show data for mice with the conventional vaginal microbiome? Pick all that apply.
    1. panels a and b
    2. panels c and d
    3. panels e and f
    4. panels g and h
  2. Here the graph showing the disease score (panel a) is similar in nature to the growth curve, and thus has similar features.  What part of the curve would be considered the stationary phase for PBS-treated mice?
    1. Day 1-3
    2. Day 4-6
    3. Day 6-7
    4. Day 8-12
  3. In this initial portion of this study normal vaginal microbiome mice were pre-treated with an antibiotic cocktail (ABX) or saline (PBS) for 6 days, infected with HSV-2, then symptoms observed for twelve days (panel a).  Disease score correlates to symptoms severity progressing from no inflammation symptoms to genital inflammation, genital lesions, limb paralysis, and near death. If disease score is a good indicator of viral load, which day and which treatment in this experiment (panel a) should have the highest amount of virus? 
    1. Day 1-3; PBS-treated
    2. Day 4-6; PBS-treated
    3. Day 6-7; PBS-treated
    4. Day 8-12; PBS-treated
    5. Day 1-3; ABX-treated
    6. Day 4-6; ABX-treated
    7. Day 6-7; ABX-treated
    8. Day 8-12; ABX-treated
  4. In this initial portion of this study normal vaginal microbiome mice were pre-treated with an antibiotic cocktail (ABX) or saline (PBS) for 6 days, infected with HSV-2, then viral counts quantified each day for three days (panel b).   They surprisingly found that the antibiotic mixture treatment impacted the HSV-2 virus.  Which day’s data indicate the antibiotic mixture has antiviral effect?  
    1. Day 1
    2. Day 2
    3. Day 3
    4. All days
  5. The next experiment was to determine which pre-treatment antibiotic was responsible for the antiviral effect observed using the antibiotic cocktail.  Based on the disease score data (panel c), which antibiotic is responsible for the reduced disease, and how do you know?
    1. Ampicillin because the disease score over time has the largest area under the curve (AUC).
    2. Vancomycin because the disease score over time is higher than the PBS control (open circles).
    3. Neomycin because the disease score over time  is nearest the antibiotic cocktail mixture (ABX).
    4. None, all are necessary for the antiviral effect because each has the effect nearest PBS alone.
  6. To investigate whether the normal mouse vaginal microbiota might contribute to the antiviral drug effect, they performed their next experiment using germ-free mice (panel e) that were treated with neomycin (neo) or saline (PBS) using the same treatment protocol (panels e and f). If the microbiome is necessary for the anti-viral effect, what results would you expect to find when you compare PBS- and drug-treated disease scores for these germ-free mice?
    1. Disease score for neo in germ-free should be higher than PBS in germ-free.
    2. Disease score for neo in germ-free should be equally as low as PBS in germ-free.
    3. Disease score for neo in germ-free should be lower than PBS in germ-free.
    4. Disease score for neo in germ-free should be equally as high as PBS in germ-free.
  7. The results for the experiment regarding the contribution of normal mouse vaginal microbiota to neomycin’s effect are shown in panel c (normal microbiota) and panel e (germ-free; no microbiota).  What do these results indicate about the impact of the vaginal microbiome on the antiviral effect of the antibiotic towards HSV infection?
    1. The normal microbiota is not required for the antibiotic to show antiviral effects.
    2. The normal microbiota is required for the antibiotic to show antiviral effects.
    3. The microbiota are destroyed by the antibiotic treatment so no effects are found.
    4. Vaginal microbiota are themselves antiviral, so no antiviral effects are observed.
  8. The authors next asked whether a post-infection antibiotic treatment could also be antiviral. To answer this question, they infected mice with a normal vaginal microbiome with HSV-2 and treated with an antibiotic or saline (PBS) at 4 hours, 24 hours, 48 hours, and 72 hours (panel g).  What do these results indicate about the post-infection effectiveness of the antibiotic against HSV-2?
    1. The antibiotic is effective as post-infection antiviral because disease score is high for both PBS and antibiotic.
    2. The antibiotic is effective as post-infection antiviral because disease score is much less for antibiotic than PBS.
    3. The antibiotic is ineffective as post-infection antiviral because disease score is high for both PBS and antibiotic.
    4. The antibiotic is ineffective as post-infection antiviral because disease score is much less for antibiotic than PBS.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify features in the data visualizations that show specific feature relevant for data analysis.
  • Identify the lag, log, and stationary phases of a growth curve or other disease measure.
  • Analyze mutant phenotypes to make conclusions about which proteins impact disease symptoms and viral load in the presence and absence of a drug.
Experimental Background (Gopinath et al., Figure 4bc,ef,hi)

Antibiotics have revolutionized the treatment of bacterial infections.  However, some of their mechanisms and effects on the eukaryotic host are not yet entirely clear. Of particular interest to this group was genital herpes infection, which is sexually transmitted, caused by Herpes simplex virus-2 (HSV-2), and affects 520 million people globally.  In this study, Gopinath et al. (2018) wanted to investigate the effects of different antibiotics on the vaginal microbiome during HSV-2 infection. In prior experiments, the researchers surprisingly found the aminoglycoside antibiotic neomycin had an anti-viral effect, reduced symptoms, and reduced pathogen load.  They further found the effect was independent of the vaginal microbiome.  Based on a transcriptome analysis, they speculated that this unusual effect might be related to nucleic acid sensing pathways in the immune response to viruses. Therefore, they investigated whether genes involved in the viral response, mainly comprised of the TLR3 signaling pathway, are required for neomycin’s effect. To investigate which genes in the TLR signaling pathway might be necessary for the antibiotic’s anti-viral activity, they quantified disease and viral counts in PBS- and neomycin-treated mice lacking specific genes in the TLR3 signaling pathway: Tlr3 (panels b-c), Trif (panels e-f), and Irf3/Irf7 (panels h-i) or wild-type mouse (panels b and c) vaginal tissue after 6 days of saline (PBS) or neomycin (Neo) treatment. 

quantified disease score (panel b) and viral counts (panel c)

7.2.2. Questions

  1. Here the graph showing the disease score in panel h is similar in nature to the growth curve and thus has similar features.  What part of the curve would be considered the log phase?
    1. Day 1-4
    2. Day 5-8
    3. Day 8-10
    4. Day 9-12
  2. What notation in the gene expression data bar graphs (panels a, d, and g) tells you the comparison being made is statistically significant, that is truly different?
    1. symbol height
    2. whiskers
    3. asterisks
    4. color
  3. To identify which host genes contribute to neomycin’s antiviral effect on disease symptoms, wild-type mice (129B6F2, panel b), mice lacking TLR3 (Tlr3 -/–, panel b), mice lacking TRIF (Trif -/-, panel d), and mice lacking IRF3/7 (Irf3/7 -/-, panel g) were pre-treated with antibiotic (neo) or PBS (control) and then infected with HSV-2.  Disease scores were taken daily for twelve days. Based on these data, which TLR3 signaling proteins contribute to neomycin’s anti-viral effect on disease symptoms?
    1.  TLR3
    2. TRIF
    3. IRF3/IRF7
    4. None contribute.
    5. All three contribute.
  4. Viral counts in the different mice were quantified for each of three days after infection (panels c, f, i).  Which comparison(s) show statistically significant differences between viral loads among these data? [wild-type is 129B6F2]
    1. Day 3; wild-type PBS and wild-type neo
    2. Day 3; tlr3-/- PBS and tlr3-/– neo
    3. Day 3; trif-/- PBS and trif-/- neo
    4. Day 3; irf3/irf7-/- PBS and irf3/irf7-/- neo

8. Paper Information and Licensing

8.1. Snippet paper

  • n/a

8.2. Main paper

License

Icon for the Creative Commons Attribution 4.0 International License

Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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