Virology Journal Club 07.02.2013

HFMD This term students taking my virology course at the University of Leicester are doing a series of tutorials involving reading and explaining research papers in virology. This is the sort of exercise which is common in graduate schools and regularly performed by researchers, and is known as a “journal club”. We cannot give students access to the facilities or equipment to study dangerous human viruses at the forefront of research, so gaining an deep understanding of the way into which research is currently being conducted in this area is the closest we can come to allowing them to be “real virologists”. Today, we are looking at the following paper:

The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates. (2012) Virology 432(2): 417-426

 

How do you read a research paper? Start with a quick scan: what’s this all about?

From the Abstract:

  • “Enterovirus 71 (EV71)” – a picornavirus.
  • “Previous studies with laboratory-infected animals”
  • “Neuropathogenesis and protein expression profiles in the thalamic tissues of EV71-infected animals”
  • “Changes in protein expression profiles following immunization with inactivated EV71 vaccine evaluated”

To process this information, copying and pasting chunks from the paper is not sufficient – you need to rewrite it in your own words. So:

  • Enterovirus 71 (EV71) is a picornavirus [single stranded RNA genome, Baltimore group IV] which causes hand–foot–mouth disease (HFMD).
  • This group of researchers from China has carried out previous work on EV71 involving laboratory-infected animals, so this is a follow-up study. [Need to find out: What did the previous results show?]
  • In this study they looked at neuropathogenesis [brain injury] and protein expression profiles [Need to find out: Just virus proteins, or overall protein expression?] in the thalamus of EV71-infected animals. [Need to find out: Why the thalamus?]
  • They also examined changes in protein expression after animals had been vaccinated with an inactivate EV71 vaccine [so no actual EV71 replication] and then infected with EV71.

At this stage it is useful to scan though the figures and explanatory figure legends in the paper to get an overview of the data:

Figure 1: Pathological [histological] changes in the thalamus, pons and spinal cord of immunized and unimmunized neonatal [does the age of the animal make a difference?] monkeys infected with EV71, versus controls [uninfected]. In vaccinated animals “a few cases of inflammatory cell infiltration in some parts of the thalamus, pons and spinal cord” were observed, but in unimmunized animals, “inflammatory cell aggregation, vascular cuffing and a small amount of neuronal cell degeneration and necrosis” were seen, i.e. more severe tissue damage.

Figure 2: Amount of virus in CNS of immunized and unimmunized monkeys infected with EV71, determined by:

a) titration of virus – no infectious virus found in immunized animals.
b) RT-PCR amplification of virus genome from tissues.

Only small amounts of vRNA found in immunized animals in comparison to unimmunized.

Figure 3: Inflammatory cytokines (IL-2, IL-4, IL-5, IL-6, TNF-α and IFN-γ) in cerebrospinal fluid of immunized and unimmunized neonatal monkeys measured by ELISA. Open circles = vaccinated, closed squares = unvaccinated. Two animals in each group, sampled over a two week period. Error bars show variability of sampling. Authors state that “⁎p<0.05 [1 in 20 chance of error] indicates a significant difference between the UI group and VI group at the same time point. ⁎⁎p<0.01 [1 in 100 chance of error] indicates remarkable significance.”

Figure 4: Protein expression profiles in the thalami of immunized and unimmunized monkeys. “A matrix of 47 proteins at days 10 and 14 p.i, [post-infection]. [Protein levels analyzed using 2D electrophoresis, mass spectrometry and Western blotting.] Large changes were observed in Cluster 6, ubiquitin-proteasome pathway-related proteins and Cluster 7, stress response proteins.

Figure 5: Gene expression of 18 selected genes in the thalami of immunized [shaded bars] or unimmunized [open bars] monkeys, measured using real-time RT-PCR analysis. Error bars indicate variation. A number of genes were upregulated, one was downregulated 14 days p.i.

[Note heading: “Quantification of protein transcript levels by RT-PCR” Protein transcript? If a student wrote that in an essay they would lose marks.]

Analysis checklist:

1. What did the authors want to find out or prove? Why? (Introduction section of the paper)

Enterovirus 71 (EV71) is a picornavirus which causes hand–foot–mouth disease (HFMD) in humans. In recent years this virus has cause large outbreaks with associated death in Asia. EV71-infected infants are especially at risk for developing CNS pathology that causes neurogenic pulmonary edema [swelling of the lungs due to brain injury], which is the major cause of death associated with HFMD. CNS damage similar to that in human disease has been observed in animals such as cynomolgus monkeys and infant rhesus monkeys. The authors chose to study changes in the brains of vaccinated and non-vaccinated infant rhesus monkeys challenged [deliberately infected with a known amount of] EV71. They note that “Based on the data obtained in these primate studies, it might be possible to use these established animal models to evaluate EV71 vaccines or therapeutic drugs with respect to histopathology, immunology and pharmacology”.

 

2. What exactly did they do? (Methods section)

EV71 virus strain FY-23 was used in this study; it was isolated from a child with clinical symptoms of severe cardiopulmonary collapse during an EV71 epidemic in Fuyang, China, in May 2008.

Inactivated vaccine was prepared from the FY-23KB virus clone [biological clone, purified by 5 sequential passages in and 2 plaques purifications in human diploid cells] by treatment with formalin.

A total of 10 1.5-month-old rhesus monkeys, 5 males and 5 females, were used in this study, divided into 3 groups:

  • 4 monkeys were given the vaccine followed by a virus challenge (immunised and infected, VI)
  • 4 monkeys were directly challenged with the virus without prior vaccination (unimmunised and infected, UI)
  • 2 monkeys were used as normal control group (unimmunised and uninfected, UU).

Two monkeys in each group were euthanised on days 10 and 14 for histopathological, biochemical and etiological examinations. The authors state that “the majority of the pathological lesions in the CNS induced by EV71 infection are found in the thalamus, pons and spinal cord (Liu et al., 2011, Wong et al., 2008 and Zhang et al., 2011)”. [So that’s why they looked these regions.]

Testing of proinflammatory cytokines (IL-2, IL-4, IL-5, IL-6, TNF-α and IFN-γ) in CSF at days 10 and 14 was performed using an ELISA assay.

Histopathological and immunohistochemical analysis of brain tissue samples was carried out on each animal.

Protein levels in brain tissue samples were analyzed and identified using 2D electrophoresis, mass spectrometry and Western blotting.

mRNA expression was measured in brain tissue was measured by real-time RT-PCR analysis.

Statistical differences between the treatment groups was calculated using two-way ANOVA and a P value of <0.05 was considered to be significant.

 

3. What were the results? (Results section)

Pathological [histological] changes were observed in the thalamus, pons and spinal cord of immunized and unimmunized neonatal monkeys infected with EV71, versus controls [uninfected] – Figure 1. In vaccinated animals “a few cases of inflammatory cell infiltration in some parts of the thalamus, pons and spinal cord” were observed, but in unimmunized animals, “inflammatory cell aggregation, vascular cuffing and a small amount of neuronal cell degeneration and necrosis” were seen, i.e. more severe tissue damage.

The amount of virus in the CNS of immunized and unimmunized monkeys infected with EV71 was determined [Figure 2] by:

a) titration of virus – no infectious virus found in immunized animals.
b) RT-PCR amplification of virus genome from tissues.

Only small amounts of vRNA found in immunized animals in comparison to unimmunized.

Inflammatory cytokines (IL-2, IL-4, IL-5, IL-6, TNF-α and IFN-γ) in cerebrospinal fluid of immunized and unimmunized neonatal monkeys were measured by ELISA [Figure 3]. Most of the inflammatory cytokines examined were significantly higher in unvaccinated animals 7-14 days post infection [as would have been expected].

Protein expression profiles in the thalami of immunized and unimmunized monkeys showed large [significant?] changes in a number of proteins 10-14 days p.i., notably in Cluster 6, ubiquitin-proteasome pathway-related proteins and Cluster 7, stress response proteins [Figure 4].

Gene expression of 18 selected genes in the thalami shows that a number of genes were upregulated, one was downregulated 14 days p.i. in unimmunized monkeys [Figure 5]. [Is this a surprise?]

 

4. What do these results mean? (Discussion section)

It has been known for some time that fatal human EV71 infections show the features typical of an inflammatory reaction. Elevated cytokines, such as IL-6, IFN-γ and TNF-α, seen in infected animals are consistent with clinical reports on EV71 infection in humans (Lin et al., 2002). The evidence presented in this paper strongly suggests that the candidate inactivated EV71 vaccine protected neonatal moneys against the effects of the virus on brain tissue, and thus presumably would have prevented them from significant injury.

Pathogenesis in the experimental animals is associated with altered expression profiles of at least 47 proteins. Although this study has identified candidate proteins which might be the subject of further research into EV71 pathogenesis, it does not in itself reveal precise mechanisms by which the virus causes brain injury, beyond the general observation of inflammation and tissue damage. How much of the inflammation is cause directly by the virus and how much by the immune system is not clear, although inflammation is clearly reduced in vaccinated animals.

 

5. What else could the authors have done? What should they do next? What are the strengths and weaknesses of this paper? Why does this research matter? (Synthesis)

Did the researchers need to use living animals, specifically monkeys, to do this research? Distasteful though this is, it is not possible to study this sort of brain injury and immune response using in vitro models such as cultured cells. Primates were required because rodents are not susceptible to EV71 infection. In addition to validating an animal model to test possible future drugs and vaccines, understanding the molecular mechanisms by which EV71 causes damage to the brain might enable the production of in vitro models which do away with the need for live animals (e.g. or large scale drug testing). Although the relatively small number of animals used in this study (only 2 monkeys per group) will not lead to highly reproducible results (nowhere near the level required in clinical trials for example), the researchers need to balance the need to get the data against causing harm to the minimum number of animals.

EV71 is a virus which first popped up in the 1960s (unknown before then) and has gone on to cause large outbreaks of human disease with neurological involvement and hundreds of child deaths, notably across Asia in recent years. For example, Vietnam recorded 63,780 cases of hand, foot and mouth disease in the first seven months of 2012 (WHO, Hand, Foot and Mouth Disease Situation Updates (22 January 2013)), about 50-60% of which were believed to be caused by EV71 (meaning other viruses have a similar pathology). Although experimental humans vaccines and antiviral drugs are being worked on, there is presently no way of preventing or treating this infection.

 

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