MicrobiologyBytes

Latest News | W.H.O. Global Alert and Response

1. Coronaviruses are a family of viruses that includes viruses that may cause a range of illnesses in humans, from common cold-type respiratory infections to SARS. Viruses of this family also cause a number of animal diseases.

2. What’s it called again?
Currently being referred to as nCoV or nCoV-2012, this virus has also been called Human Coronavirus-Erasmus Medical Center (hCoV-EMC), or Middle East respiratory syndrome coronavirus (MERS-CoV), and even “Saudi SARS” (it’s not – SARS is a related but different Coronavirus).

3. The first known case of nCoV infection was in a Saudi Arabian man who died in early 2012. This particular strain of coronavirus had not been previously identified in humans. The second confirmed case appeared in early September 2012, involving a 49-year old man in Doha, Qatar who had traveled to Saudi Arabia around the same time that the first case was identified. Currently, at least 40 cases have been confirmed, and 20 of those affected have died. The virus has also been found in Tunisia.

4. Where did it come from?
Bats. (It’s [nearly] always bats.) Bat coronaviruses carried by the genus Pipistrellus that differ from nCoV by as little as 1.8%. The existence of over 50 species of Pipistrellus bats in the Arabian Peninsula suggests that they may be the animal reservoir.

5. Symptoms of nCoV infection include renal failure and severe acute pneumonia, which often result in a fatal outcome. In humans, the virus has a strong tropism for nonciliated bronchial epithelial cells because it uses dipeptidyl peptidase 4 (DPP4, also known as CD26) as a receptor.

6. nCoV can penetrate the bronchial epithelium and evade the innate immune system, signs that it is well-equipped for infecting human cells. This suggests that although nCoV may have jumped from animals to humans very recently, it is as well adapted to infecting the human respiratory tract as other, more familiar human coronaviruses, including the SARS virus and the common cold Coronavirus HCoV-229E.

7. The virus is susceptible to treatment with interferons, immune proteins that have been used successfully to treat other viral diseases, offering a possible method of treatment in the event of a large-scale outbreak.

8. How is it transmitted?
Almost certainly like other respiratory viruses, via aerosol droplets from coughs and sneezes, but possibly also by unwashed hands contaminated with respiratory secretions.

9. Is there a vaccine?
Not yet. It is possible to make vaccines agains Coronaviruses and several SARS vaccines were developed but never put into use because the SARS outbreak died away. It should be possible to make a nCoV vaccine if we need one.

10. Is there any travel advice?
At the moment the World Health Organization says there is no reason to impose any travel restrictions. Travel advice will be kept under review if additional cases occur or when the patterns of transmission become clearer.

11. Are we all going to die?
Probably not. Most of the people who have been infected so far have been older men, often with other medical conditions. The outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003 infected over 8000 people and killed nearly 800 before burning itself out. But SARS didn’t kill us all and it’s unlikely that nCoV will either.

Other things you should know:

• 10 things you should know about H1N1 (swineflu)
• 10 things you should know about E. coli
• 10 things you didn’t know about Schmallenberg virus
• 10 things about Foot and Mouth Disease

“Scholars have argued that in risk communication a dilemma exists between the media functions of informing the media audience about rational risk behavior, and providing an arena for public deliberation about risk. Optimizing the information function would suggest that media provide clear, unanimous advice without creating confusion by reporting uncertainty and controversy. Optimizing the deliberative function, in contrast, would require media to include different (even contradictory) voices. A similar dilemma exists between incompatible expectations of different fractions of the audience. Part of the audience may trust the media to provide the best available advice. These audience members may be prepared to take the mediated advice at face value, not wanting to be unsettled by controversy and uncertainty. But another part of the audience may prefer to learn about the full spectrum of opinions, including outsider views, and may want to develop their own conclusions on whom to trust and whose advice to follow. Presenting only the mainstream view may motivate members of that part of the audience to seek information in alternative channels – such as blogs or rumors.”

Similar challenges but different responses: Media coverage of measles vaccination in the UK and China. (2012) Public Understanding of Science. doi: 10.1177/0963662512445012
For several decades scholars have studied media reporting on scientific issues that involve controversy. Most studies so far have focused on the western world. This article tries to broaden the perspective by considering China and comparing it to a western country. A content analysis of newspaper coverage of vaccination issues in the UK and China shows, first, that the government-supported ‘mainstream position’ dominates the Chinese coverage while the British media frequently refer to criticism and controversy. Second, scientific expertise in the British coverage is represented by experts from the health and science sector but by experts from health agencies in the Chinese coverage. These results are discussed with respect to implications for risk communication and scientists’ involvement in public communication.

With increasing concerns over antibiotic resistance in bacterial and other pathogens, the search for novel approaches to infection control other than stimulation of classic adaptive immunity (vaccination) are increasingly being sought. The idea of controlling a pathogen with another microorganism is not new and was initiated originally by Felix d’Herelle, one of the co-discoverers of bacteriophages. d’Herelle had earlier explored the idea of using pathogenic bacteria to control grasshoppers.

Because of a lack of understanding of both phage and bacterial pathogenesis at that time, initial experimental work in phage biocontrol by d’Herelle and those of his colleagues and followers used treatments with phage preparations of uncertain content and quality and not infrequently involving poorly conceived and uncontrolled experiments. Benefiting from our increasing understanding of pathogenesis and using more appropriate models, more recent and more rigorous experimental work has shown the value of using phages in bacterial control. Despite this, the large scale application of phages for infection control remains to be fully exploited.

In highly complex ecosystems, whether on the macro- or micro-scale, a wide variety of interactions involving competition, parasitism, and symbiosis can be found, sometimes with pathogens occupying different roles dependent on the nature of the host. Viruses have been identified for almost every species of higher animal and for most prokaryotes for which they have been sought. For any interaction of this sort, whether it be microbial pathogen and host or parasitic nucleic acid and host genome, the parasitizing entity will drive evolution of the host and vice versa. Such genetic and ecological interactions must be taken into account when considering the biological control of one organism by another.

Fleas and smaller fleas: virotherapy for parasite infections. Trends in Microbiology 26 Mar 2013 doi: 10.1016/j.tim.2013.02.006
Bacteriophages are viruses of bacteria that are used for controlling bacterial food-borne pathogens and have been proposed for more extensive usage in infection control. Protists are now recognised to harbour viruses and virus-like particles. We propose that investigation of their prevalence in parasites be intensified. We also propose that such viruses might be considered for virotherapy to control certain parasite infections of man and animals.

HIV may not always want to go unnoticed. It is known that HIV replicates more efficiently in TH cells that have been activated, and this presents the virus with a bit of a dilemma – it certainly doesn’t pay to be recognized by CTLs, the hired assassins of the immune system. But there’s also a possible benefit in triggering the activation of the TH cells that they’ve infected. Where does the balance lie? Is it conceivable that there are some conditions where it is better for HIV epitopes to be recognized than to be ignored?

To answer this question, scientists have built a complex mathematical model of interactions between TH cells, CTLs, antigen-presenting cells and viruses. They set up two different versions of the model – one with a virus that infects non-immune cells (like HCV infecting hepatocytes), and one with a virus that infects T cells (like HIV). The results are quite striking – in the case of HCV, evasion always pays off. But in the case of HIV, the dependence on TH activation seems to sometimes favor virus epitopes that are strongly recognized by cellular immune system. The authors propose that recognition of HIV epitopes by TH cells can be beneficial for the long-term survival of the virus within the body. Another key variable is how many TH cells are activated in the body by pathogens other than HIV. If this “background activation” is strong, HIV obtains no payoffs from being recognized. Studying the sequence data confirms that in the capsid genes (Gag) it is TH epitopes, rather than CTL epitopes, that are constrained and that epitopes stay the same within patient viral populations rather than between patients, again implicating chronic internal transmission between cells.

There is an important practical implication of these findings. If this model is correct, then HIV vaccines based on TH epitopes might prove counterproductive, playing into the hands of the virus. Instead, vaccines should target only CTL-specific viral epitopes.

HIV Plays (and Wins) a Game of T Cell Brinkmanship. (2013) PLoS Biol 11(4): e1001521. doi:10.1371/journal.pbio.1001521

The family Arenaviridae contains four important species that cause severe hemorrhagic zoonoses in humans. Together, they have an important impact on public health in endemic regions. Lassa virus (LASV) is endemic to Africa. The other three species (Machupo, Junin, and Guanarito viruses [MACV, JUNV, and GTOV]) are from South America. The prototypic arenavirus is lymphocytic choriomeningitis virus (LCMV), which can also cause disease in humans, especially in immunocompromised patients.

Arenaviruses carry two RNA genome segments (small, S, and large, L), which encode two genes each. The S-segment encodes the glycoprotein precursor (GPC) and, in ambisense, the nucleoprotein (NP). Similarly, the L-segment encodes the matrix protein Z and, in negative sense, the multifunctional protein L. Natural reservoirs include different species of rodents, depending on the arenavirus. The exact mode of transmission to humans is unknown but probably occurs through direct contact with the infected host or food contaminated with excrement. Direct human-to-human transmission is possible and regularly occurs in clinical settings in endemic areas.

Little is known about the pathogenesis of the diseases caused by arenaviruses. A putative explanation for the severe symptoms is an immunopathology caused by an imbalanced host–pathogen interaction with a perpetuated excessive reaction of host immune cells combined with delayed viral clearance. Early immune evasion may participate in the disease through delayed virus clearance. Treatment options for the patients are limited. In addition to intensive care, the broad-band antiviral drug ribavirin has proven to be effective if administered early in the course of the disease. The caveat is the need for early diagnosis, and this is a genuine problem, since infections with arenaviruses are initially often mistaken for malaria, typhoid fever, or other common tropical diseases due to the nonspecific nature of the symptoms. The only currently available vaccine is Candid #1. This attenuated JUNV strain was generated through multiple passaging and provided good protection in clinical trials against argentine hemorrhagic fever (AHF) with an excellent safety profile. The historical development and biological properties of this vaccine were recently reviewed in a concise overview.

Although there has been much effort to develop vaccines against LASV, none have been effective enough to warrant clinical trials. This short review summarizes the work that has been done toward the development of vaccines against hemorrhagic fever caused by arenaviruses and discusses the obstacles toward a licensed vaccine.

Vaccination Strategies against Highly Pathogenic Arenaviruses: The Next Steps toward Clinical Trials. (2013) PLoS Pathog 9(4): e1003212. doi:10.1371/journal.ppat.1003212

A vaccine to prevent shingles may reduce by half the occurrence of this painful skin and nerve infection in older people (aged over 65 years) and may also reduce the rate of a painful complication of shingles, but has a very low uptake (only 4%) in older adults in the United States.

Herpes zoster (“a.k.a. “shingles”) is a significant public health problem affecting 1 million individuals in the USA each year and associated with important illness. Herpes zoster occurs following reactivation of latent varicella zoster virus (VZV) infection and presents with a painful vesicular rash, which frequently in older individuals leads to prolonged pain, post-herpetic neuralgia (PHN), with a major impact on quality of life. Vaccine efficacy has been shown in trials; in a selected insured population; and among people with any of five specific immune-mediated diseases but not among an unselected population in a clinical setting.

Despite Advisory Committee for Immunization Practices (ACIP) recommendations, individuals with immunosuppression received the live herpes zoster vaccine in clinical practice. The lack of adherence to ACIP recommendations on vaccination is not entirely surprising given that individuals with immunosuppression are not only at increased risk of incident herpes zoster but also at significantly increased risk of herpes zoster complications, in particular prolonged, severe PHN. Previous research has suggested that the varicella vaccine may be efficacious and safe in people with immunosuppressive disorders. Similar evidence about vaccine effectiveness (VE) is lacking in relation to the zoster vaccine in individuals with serious immune suppression, beyond effectiveness among those with the selected immune-mediated disorders examined to date.

Important outstanding research questions with great relevance to policy include VE in unselected population-based elderly US populations; this includes effectiveness against PHN, which has not been assessed in routine practice. This is the first study to the best of our knowledge to assess the effectiveness of herpes zoster vaccine against both incident herpes zoster and PHN in an unselected older population including those with immunosuppression.

Herpes Zoster Vaccine Effectiveness against Incident Herpes Zoster and Post-herpetic Neuralgia in an Older US Population: A Cohort Study. (2013) PLoS Med 10(4): e1001420. doi:10.1371/journal.pmed.1001420
Herpes zoster is common and has serious consequences, notably post-herpetic neuralgia (PHN). Vaccine efficacy against incident zoster and PHN has been demonstrated in clinical trials, but effectiveness has not been studied in unselected general populations unrestricted by region, full health insurance coverage, or immune status. Our objective was to assess zoster vaccine effectiveness (VE) against incident zoster and PHN in a general population-based setting. A cohort study of 766,330 fully eligible individuals aged over 65 years was undertaken in a 5% random sample of Medicare who received and did not receive zoster vaccination between 1st January 2007 and 31st December 2009. Incidence rates and hazard ratios for zoster and PHN were determined in vaccinated and unvaccinated individuals. Analyses were adjusted for age, gender, race, low income, immunosuppression, and important comorbidities associated with zoster, and then stratified by immunosuppression status. Adjusted hazard ratios were estimated using time-updated Cox proportional hazards models. Vaccine uptake was low (3.9%) particularly among black people (0.3%) and those with evidence of low income (0.6%). 13,112 US Medicare beneficiaries developed incident zoster; the overall zoster incidence rate was 10.0 (9.8–10.2) per 1,000 person-years in the unvaccinated group and 5.4 (95% CI 4.6–6.4) per 1,000 person-years in vaccinees, giving an adjusted VE against incident zoster of 0.48 (95% CI 0.39–0.56). In immunosuppressed individuals, VE against zoster was 0.37 (95% CI 0.06–0.58). VE against PHN was 0.59 (95% CI 0.21–0.79). Vaccine uptake was low with variation in specific patient groups. In a general population cohort of older individuals, zoster vaccination was associated with reduction in incident zoster, including among those with immunosuppression. Importantly, this study demonstrates that zoster vaccination is associated with a reduction in PHN.

A new tuberculosis vaccine, MVA85A, has recently been found to be less effective than initially thought, prompting widespread consternation in the press. The excellent NHS Choices website reports that “although the stories are based on solid science, the news is actually less worrying than the headlines suggest”. MVA85A is a booster vaccine that researchers hope might help improve the effectiveness of the existing BCG vaccine. Although the BCG is effective in the UK, new vaccines and boosters are needed as it is less effective in other countries. The effectiveness of BCG against tuberculosis is variable and has been found to be less effective in countries such as South Africa, where as many as 1% of the population has TB. An effective booster vaccine would therefore be useful. Although a recent study found the new vaccine is safe, it does not appear to have performed better than the placebo in children who had already had the BCG vaccine. Despite this setback, several further lines of investigation are being pursued by researchers, who now want to look at whether the MVA85A vaccine might work better in other sub-populations, and whether it might improve protection against pulmonary tuberculosis (lung infection) in people who have HIV, for example.

NHS Choices: Disappointing results for new TB vaccine

Another paper just published has found that whole genome sequencing of Mycobacterium tuberculosis provides more accurate information on clustering and longitudinal spread of the pathogen than the standard test (classical genotyping). Importantly, whole genome sequencing revealed that first outbreak isolates were falsely clustered by classical genotyping and do not belong to one recent transmission chain. By using whole genome sequencing, the authors estimated that the genetic material of M. tuberculosis evolved at a rate at 0.4 mutations per genome per year, suggesting that the bacterium grows in its natural host (infected people) with a doubling time of 22 hours, or 400 generations per year. This finding about the evolution of M. tuberculosis indicates how information from whole genome sequencing can be used to help trace future outbreaks. Importantly, as the costs of whole genome sequencing are declining, this test could soon become the standard method for identifying transmission patterns and rates of infectious disease outbreaks.

Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study. (2013) PLoS Med 10(2): e1001387. doi:10.1371/journal.pmed.1001387

2,016 confirmed cases of measles in England and Wales were reported to the Health Protection Agency (HPA) in 2012, the highest annual total since 1994. Dr Mary Ramsay, head of immunisation at the HPA, said: “Coverage of MMR is now at historically high levels but measles is highly infectious and can spread easily among communities that are poorly vaccinated, and can affect anyone who is susceptible, including toddlers in whom vaccination has been delayed. Older children who were not vaccinated at the routine age, who may now be teenagers, are at particular risk of becoming exposed, while at school for example. Measles continues to circulate in several European countries that are popular with holidaymakers. Measles is a highly infectious disease so the only way to prevent outbreaks is to make sure the UK has good uptake of the MMR vaccine, and that when cases are reported, immediate public health action is taken to target unvaccinated individuals in the vicinity as soon as possible.”
UK HPA: http://goo.gl/2Abgf
The Guardian: http://goo.gl/rnfRh

#viaGoogle+

Google+: Reshared 5 times
Google+: View post on Google+

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.