MicrobiologyBytes

Welcome to the Weekend!

World Malaria Day recognises global efforts being made in the fight against malaria. First established in 2007 by the World Health Assembly, it falls on 25 April every year.

Measles outbreak: In graphics
Great use of data by BBC News – students take note – this is how you do it.

Strain of measles possible cause of dolphin deaths [Audio]
Scientists think that more than a hundred dead dolphins found washed up on the Italian coast, may have been infected with a killer strain of “measles”. [Human measles, or another Paramyxovirus?]

Mapping the H7N9 avian flu outbreaks
Where are the 104 human cases reported so far and where might the virus go next?

Promising Treatment for New Human Coronavirus
A new report says that two antiviral drugs, ribavirin and interferon-alpha 2b, will stop nCoV from replicating in cells grown in the lab.

Radioactive Listeria cures cancer – or does it?
This report just appeared in PNAS. Let’s hope it’s a generalisable method, but for the present this is one small scale study – still a long way to go before this is a routine treatment.

The Truth About Why Microbes Make You Sick
Between fevers, congestion and diarrhea, there are numerous ways that microbes can make us feel sick. But just how do microorganisms cause these symptoms?

Gonorrhea could be untreatable by 2015 [Audio]
There’s been a sharp increase in the number of cases of the sexually transmitted infection gonorrhoea – up 25% in 2011. It is also becoming harder to find antibiotics that treat it, which has raised the prospect that it could soon become untreatable.

Holy Virus Treasure Trove, Batman!
Think about the type of animal that would make an ideal host for a virus. It would gather in large dense groups, making it easier for the virus to jump into fresh hosts. It should have a relatively long lifespan, so any single individual has many chances of becoming infected. It would certainly travel over long distances to spread the infection far and wide. Humans certainly fit the bill. So do bats.

It has been known for over 100 years that a major biogeographic barrier exists between the Australo-Papuan and Wallacean region on the one hand, and southeast Asia on the other, with different groups of both terrestrial vertebrates and invertebrates occurring on either side of the “Wallace Line“. It has even been suggested that this boundary has protected Australia from the recent H5N1 avian influenza epidemic. Of the major groups of terrestrial mammals, only rodents and bats extend across this region from southeast Asia into Australia. There are 13 species of Old World fruit bat that occur only to the west of Wallace’s Line and 67 species that are confined to the east, while 20 species have wide distributions throughout the region and occur on both sides of the line.

The aim of a new paper in PLOS ONE is to investigate the occurrence of henipaviruses in fruit bat populations in the regions of northeast Australia (Queensland), New Guinea (Papua New Guinea) and Wallacea (Indonesia and East Timor) by testing the hypothesis that Nipah virus is restricted in distribution to west of Wallace’s Line. Fruit bats were sampled from northeast Australia, Papua New Guinea, East Timor and Indonesia, and tested for the presence of anti-Hendra virus (HeV) and anti-Nipah virus (NiV) antibodies. PCR tests were also conducted to determine the presence of henipavirus RNA.

The authors found that fruit bats from regions on both sides of the line tested positive for Nipah virus and other related henipaviruses. Only certain species of fruit bats carried Nipah virus but even in their absence, other bat species could still carry these related viruses. Henipaviruses were also detected in some species not previously known to carry these viruses. Based on these results, the authors conclude that Wallace’s line is not a restricting factor for the transmission of Nipah virus.

The Distribution of Henipaviruses in Southeast Asia and Australasia: Is Wallace’s Line a Barrier to Nipah Virus? (2013) PLoS ONE 8(4): e61316. doi:10.1371/journal.pone.0061316
Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia’s closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace’s Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace’s Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.

The ongoing dance between a virus and its host shapes how the virus evolves. While human adenoviruses typically cause mild infections, recent reports have described newly characterized adenoviruses that cause severe, sometimes fatal human infections. A new paper describes a systems biology approach to show how evolution has affected the disease potential of a recently identified novel human adenovirus. A comprehensive understanding of virus evolution and pathogenicity is essential to our capacity to foretell the potential impact on human disease for new and emerging viruses.

Predicting the next eye pathogen: analysis of a novel adenovirus. (2013) MBio. 4(2): e00595-12. doi: 10.1128/mBio.00595-12
For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses.

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

Pretty damn important.

How do we know? Because humans have a gene encoding a protein which seems to be dedicated to preventing influenza virus infection. Interferon-inducible dynamin-like myxovirus resistance protein (MxA) is found in membranes of the smooth endoplasmic reticulum–Golgi intermediate compartment. On influenza virus infection of the cell, it redistributes to sites of virus replication and promotes missorting of the myxovirus nucleocapsid (N) protein into membrane-associated, large perinuclear complexes. By preventing the N protein entering the nucleus, influenza virus replication is disrupted.

Influenza A viruses of avian or swine origin sporadically enter the human population but do not readily transmit between individuals. In rare cases, however, they establish a new virus lineage in humans. The mechanisms by which invading viruses overcome the species barrier are not well understood, but multiple adaptations to the new host are required. Surprisingly little is known about adaptive mutations that overcome restriction factors of the intrinsic and innate host defense system.

A recent paper in PLOS Pathogens identifies adaptive mutations in pandemic strains of influenza that confer resistance to the interferon-induced antiviral factor MxA. The resistance-enhancing mutations changed several amino acids in the viral nucleoprotein which is the main nucleocapsid component. These mutations were sufficient to increase the pathogenicity of an avian influenza virus strain in a Mx-positive mouse model. Interestingly, the resistance-associated amino acids are counter-selected in circulating avian influenza strains, because they compromise general viral replication fitness. Innate immunity factor MxA provides a barrier against zoonotic introduction of influenza A viruses and adaptive mutations in the influenza N protein should be carefully monitored.

Why should you care about this? -> H7N9

Pandemic Influenza A Viruses Escape from Restriction by Human MxA through Adaptive Mutations in the Nucleoprotein. (2013) PLoS Pathog 9(3): e1003279. doi:10.1371/journal.ppat.1003279
The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.

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

Principles of Molecular Virology

I always tell my students that any stage of virus replication can be a target for antiviral therapy – as long as it is essential to replication and specific to the virus and therefore that inhibiting it does not damage the host cell. So far we have mostly limited ourselves to a very few stages of the replication cycle, and we only have one or two drugs (against influenza virus) that inhibit the vital uncoating step of the replication cycle. Understanding the processes involved is therefore of great importance in developing new drugs. A recent paper in PLOS Pathogens examines the uncoating of rhinovirus particles and makes some interesting findings.

Human rhinoviruses (HRV) are members of the picornavirus family, and are one of the major causative agents of the common cold. Additionally, they play important roles in the exacerbation of asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Similar to other picornaviruses, the rhinovirus particle are composed of 60 copies each of four capsid proteins, VP1, VP2, VP3 and VP4, arranged on an icosahedral lattice. The diameter of the particle is roughly 30 nm. The virus genome is a single-stranded RNA molecule of positive sense, about 7100 bases in length. It carries a covalently linked peptide (VPg) at its 5′-end and a poly-(A) tail of about 70 to 150 bases at its 3′-end. The 5′-nontranslated region is approximately 650 bases in length, highly structured, and involved in cap-independent translation initiation and RNA replication.

Minor group rhinoviruses, e.g. the prototype strain HRV2, bind members of the low-density lipoprotein receptor (LDLR) family including LDLR and LDLR-related protein for entry via clathrin-dependent endocytosis. Once in the endosome, the low pH leads to dissociation of the virus from the receptors as well as to structural changes in the viral capsid. The native virion sedimenting at 150S converts into the subviral A-particle sedimenting at 135S and devoid of the internal capsid protein VP4 and exposure of amphipathic N-terminal sequences of VP1 renders it hydrophobic, thus allowing its direct attachment to the inner endosomal membrane. These processes are accompanied by an expansion of the virus shell by about 4% along with the opening of symmetry-related channels. The largest channels are at the two-fold axes, whereas the smaller ones are located near the pseudo three-fold axes and at the base of the star-shaped vertices of the icosahedron. Finally, the RNA is released through one of these pores, most probably at a 2-fold axis. The final product of this uncoating process is the empty capsid (80S B-particle). Most enteroviruses undergo similar conformational changes; however, with the exception of minor receptor group rhinoviruses, the process appears to be triggered by receptor binding and possibly assisted by low pH.

These structural modifications can be mimicked, at least partially, in vitro. Exposure to pH <5.8 converts native HRV2 preferentially into A-particles whereas incubation at 50°C–56°C in low ionic strength buffers favours conversion into B-particles (empty capsids). In vivo, and in the presence of liposomes in vitro, both VP4 and N-terminal sequences of VP1 insert into lipid bilayers. They might contribute to formation of a pore connecting the virus interior with the cytosol of the host cell, thus allowing for the transit of RNA in its unfolded form.

The mechanism of RNA exit is poorly understood. Energy would be required for breaking the hydrogen bonds of the double-stranded regions in the encapsidated RNA genome in order to allow the RNA to thread through an opening only large enough to enable passage of a single strand. It appears likely that either the poly-(A) tail at the 3′-end or the VPg peptide linked to the 5 end of the RNA begins to emerge from the virion since other modes might be unproductive (e.g., simultaneous exit of both ends would be expected to impede complete uncoating and thus to be abortive). Directionality of this process may indicate that the RNA adopts a defined conformation inside the viral shell suggesting a well-organized process of assembly and uncoating.

The new paper shows that RNA exit does indeed occur in a specific and ordered manner, starting from the 3′-end. Ordered exit of RNA also suggests that the virus genome becomes organized during packaging or assembly, which may occur co-transcriptionally. Therefore, it is likely that the process of encapsidation begins when the 5′-end emerges from the replication complex or at least before the complete RNA has been synthesized. It is also possible that the same applies to other viruses with ssRNA of positive polarity. This would imply that in these viruses, the 3′-end becomes encapsidated last, remaining near the capsid wall presumably in close proximity to one of the holes poised to open upon uncoating, thus resulting in a ‘last-in-first-out’ process of assembly and uncoating.

So all we need now is a drug to inhibit this process, and we’ve cured the common cold. Well, some of them maybe :-)

Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End. (2013) PLoS Pathog 9(4): e1003270. doi:10.1371/journal.ppat.1003270
Viral infection requires safe transfer of the viral genome from within the protective protein shell into the host cell’s cytosol. For many viruses this happens after uptake into endosomes, where receptor-binding and/or the acidic pH trigger conformational modifications or disassembly of the shell, allowing the nucleic acids to escape. For example, common cold viruses are converted into subviral particles still containing the single-stranded positive sense RNA genome; subsequently, the RNA escapes into the cytoplasm, leaving behind empty capsids. We triggered this process by heating HRV2 to 56°C and found that 3′- and 5′-end emerged with different kinetics. Crosslinking prevented complete RNA egress and upon nuclease digestion only sequences derived from the 5′-end were protected. Part of the RNA remaining within the viral shell adopted a rod-like shape pointing towards one of the two-fold axes where the RNA is presumed to exit in single-stranded form. Egress thus commences with the poly-(A) tail and not with the genome-linked peptide VPg. This suggests that assembly and uncoating are well-coordinated to avoid tangling, kinetic traps, and/or simultaneous exit of the two RNA ends at different sites.

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.