MicrobiologyBytes

Virus-induced cell death is a determinant of pathogenesis. Mammalian reovirus is a versatile experimental model for identifying viral and host intermediaries that contribute to cell death and for examining how these factors influence viral disease. In this study, we identified that in addition to apoptosis, a regulated form of cell death, reovirus is capable of inducing an alternate form of controlled cell death known as necroptosis. Death by this pathway perturbs the integrity of host membranes and likely triggers inflammation. We also found that apoptosis and necroptosis following viral infection are activated by distinct mechanisms. Results suggest that host cells can detect different stages of viral infection and attempt to limit viral replication through different forms of cellular suicide. While these death responses may aid in curbing viral spread, they can also exacerbate tissue injury and disease following infection.

Reovirus activates a caspase-independent cell death pathway. MBio. 2013 May 14; 4(3). pii: e00178-13. doi: 10.1128/mBio.00178-13
Virus-induced apoptosis is thought to be the primary mechanism of cell death following reovirus infection. Induction of cell death following reovirus infection is initiated by the incoming viral capsid proteins during cell entry and occurs via NF-κB-dependent activation of classical apoptotic pathways. Prototype reovirus strain T3D displays a higher cell-killing potential than strain T1L. To investigate how signaling pathways initiated by T3D and T1L differ, we methodically analyzed cell death pathways activated by these two viruses in L929 cells. We found that T3D activates NF-κB, initiator caspases, and effector caspases to a significantly greater extent than T1L. Surprisingly, blockade of NF-κB or caspases did not affect T3D-induced cell death. Cell death following T3D infection resulted in a reduction in cellular ATP levels and was sensitive to inhibition of the kinase activity of receptor interacting protein 1 (RIP1). Furthermore, membranes of T3D-infected cells were compromised. Based on the dispensability of caspases, a requirement for RIP1 kinase function, and the physiological status of infected cells, we conclude that reovirus can also induce an alternate, necrotic form of cell death described as necroptosis. We also found that induction of necroptosis requires synthesis of viral RNA or proteins, a step distinct from that necessary for the induction of apoptosis. Thus, our studies reveal that two different events in the reovirus replication cycle can injure host cells by distinct mechanisms.

Bluetongue is a severe disease of ruminants, notably sheep and cattle. The causal agent, the dsRNA Bluetongue virus, is spread by an insect vector and occurs in its vector’s habitat in temperate climates throughout much of the world. BTV is the type member of genus Orbivirus in the family Reoviridae, with 26 known serotypes. When bluetongue first broke out in the United Kingdom in autumn of 2007, the disease was already rapidly spreading throughout continental Europe, causing high mortality rates in sheep and having a detrimental effect on the livestock trade through trade restrictions and loss of stock. The only effective weapon against the disease is control of the spread of BTV through rigorous vaccination programmes. Currently available commercial vaccines are based on both inactivated virus and live, attenuated strains and protect against a single serotype or multiple serotypes when provided as a cocktail. However, the possibility of recombination between the live vaccine strain(s) and wild-type virus in infected animals, leading to the emergence of new infectious strains has motivated efforts to develop safer vaccines.

One approach in the development of an inherently safe vaccine has been the production of Bluetongue virus-like particles (VLPs). BTV has a nonenveloped icosahedral structure, with four main structural proteins (VP3, VP7, VP5 and VP2) arranged in concentric shells around the segmented double-stranded RNA genome and minor structural and nonstructural proteins involved in virus replication. French et al. have shown that these four structural proteins, expressed in insect cells using a baculovirus expression system, assemble into virus-like particles devoid of nucleic acid.

This paper describes plant-based high-level expression of assembled subcore-, core- and virus-like particles of BTV serotype 8. Purified preparations of the VLPs, consisting of all four structural proteins, elicited an immune response in sheep and provided protective immunity against challenge with a South African BTV-8 field isolate. This demonstrates that plant expression provides an economically viable method for producing complex VLPs, such as those of BTV, with the desired biological properties. It represents a significant advance in the use of plant-based systems for the production of complex biopharmaceuticals. The methods employed could also be applied to other situations where the expression of multiple proteins is required, such as the reconstruction of metabolic pathways.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J. 06 May 2013 doi: 10.1111/pbi.12076
Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.

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

Human adenoviruses are non-enveloped icosahedral viruses with linear double stranded DNA genomes. The genome is transcribed from both strands and it is organized into several transcription units named mainly according to when they are expressed during the virus life cycle. Five early transcription units encode the E1A, E1B, E2, E3 and E4 proteins, two delayed early units encode the IVa2 and pIX proteins and there is one major late transcription unit (MLTU). The major functions of the early gene products are to force the host cell to enter the S phase in order to provide optimal conditions for viral DNA replication and for suppression of the host antiviral response. The major late gene products are the viral structural proteins which package the viral DNA into new virus particles.

Group C adenoviruses also encode two small RNAs, called virus-associated (VA) RNAI and VA RNAII. They are non-coding RNAs and transcribed by RNA polymerase III. Both VA RNAs are about 160 nucleotides long and GC rich. Expression of the VA RNAs begins during the early phase of infection and increases rapidly to a high level during the late phase. Inactivation of VA RNAI results in a 10–20 fold decrease in virus production, whereas deletion of VA RNAII alone has little impact on virus replication. The functional significance of VA RNAI is well documented whereas little is known about the function of VA RNAII. The primary function of VA RNAI appears to be to block the activity of RNA-dependent protein kinase (PKA), a double-strained RNA activated inhibitor of translation. VA RNAI also stabilizes ribosome-associated viral mRNAs resulting in enhanced levels of viral protein synthesis. In addition, VA RNAI binds efficiently to Exportin 5, interfering with the nuclear export of the cellular RNAi and miRNA precursors and Dicer processing. Finally, large amounts of VA RNA-derived small RNAs associate with RNA-induced silencing (RISC) complexes.

During the last decade, increasing numbers of small RNAs have been identified and characterized and it has become evident that the small RNAs are critical regulators of gene function (Bartel, 2004, Seto et al., 2007 and Zaratiegui et al., 2007). There are three main categories: short interfering RNAs (siRNAs which are ∼21 nt in length), microRNAs (miRNAs, ∼22 nt in length) and PIWI-interacting RNAs (piRNAs, ∼24–32 nt in length). Deep sequencing technologies combined with bioinformatic strategies have revolutionized the identification of rare small RNAs. This study examines the expression of adenovirus-encoded small RNAs at different times after infection using deep sequencing. Adenovirus-encoded small RNAs may thus constitute a front-line defense and be crucial for the survival of the virus.

Identification of adenovirus-encoded small RNAs by deep RNA sequencing. Virology. 06 May 2013. pii: S0042-6822(13)00200-6. doi: 10.1016/j.virol.2013.04.006
Using deep RNA sequencing, we have studied the expression of adenovirus-encoded small RNAs at different times after infection. Nineteen small RNAs which comprised more than 1% of the total pool of small RNAs at least one time point were identified. These small RNAs were between 25 and 35 nucleotides long and mapped in the region of the VA RNAI and RNAII genes. However, the overlap was incomplete and some contained a few extra nucleotides at the 3′ end. This finding together with the observation that some of the small RNAs were detected before VA RNA expression had started might indicate that they are derived from other precursors than VA RNAI and II. Interestingly, the small RNAs displayed different expression profiles during the course of the infection suggesting that they have different functions. An effort was made to identify their mRNA targets by using computer prediction and deep cDNA sequencing. The most significant targets for the earliest small RNAs were genes involved in signaling pathways.

Viruses have developed remarkable mechanisms to inhibit the adaptive and innate immune systems of their hosts. Clearly, viral entry glycoproteins play critical roles in these activities. However, many of these roles and biological pathways are poorly defined. With new infectious diseases emerging and classical viral diseases reemerging, closer examination of viral entry glycoproteins as targets for preventative or therapeutic strategies is warranted.

Survival of infection with Ebola virus (EBOV) depends on the ability of the host to mount early and strong immune responses. However, given that EBOV cases are associated with 40%–90% human mortality, EBOV has developed intricate solutions to human immunological defenses. Enveloped viruses, like EBOV, must deposit their genetic material within a cell to ensure their propagation. The roles of viral envelope glycoproteins in mediating virus attachment to host cells and catalyzing the subsequent fusion of the viral and host plasma membranes have been well described. Given the limited number of genes in EBOV and other viruses, it stands to reason that these conformationally labile glycoproteins are also involved in more than just the initial steps of a productive infection. There is strong evidence that viral entry glycoproteins (GP) are modulators of host antiviral defenses. This article discusses current structural understanding of the functions of envelope entry glycoproteins in immune evasion using EBOV as an example.

• How Does Glycosylation of Ebola Virus Envelope Proteins Facilitate Immune Evasion?
• What Roles Do Shed Viral Glycoproteins Play in Immune Evasion?
• How Do Viral Glycoproteins Actively Suppress Host Immunity?
• What Are the Innate Restriction Strategies Targeted toward Viral Glycoproteins?

The Secret Life of Viral Entry Glycoproteins: Moonlighting in Immune Evasion. (2013) PLoS Pathog 9(5): e1003258. doi:10.1371/journal.ppat.1003258

West Nile virus (WNV) is a small, enveloped, single-stranded RNA virus in the family Flaviviridae. In the natural transmission cycle WNV circulates between mosquitoes as vectors and birds as reservoir hosts. Most noticeably, WNV can infect a wide taxonomical range of vertebrate species but most of them do not sufficiently support virus replication for transmission. Disease symptoms rarely occur, except in humans and horses where WNV infections are frequently accompanied by a mild fever (West Nile fever), which occasionally results in the development of neurological disorders with fatal outcome.

The cellular receptors and determinants that mediate entry of WNV are unclear to date. The notable ability of WNV to infect a broad range of species (mosquitoes, reptiles, birds and mammals), and virtually every in vitro cell line is supposed to be related to cellular proteins, relevant for virus entry and replication, which are highly conserved among divergent host species.

By using integrin knock-out cell lines which lack the particular integrin subunits, this study demonstrate that the presence of αv-, β1- or β3-integrins is not required for the attachment of four different WNV strains to the cell surface. However, β1- and β3-integrin expression significantly enhances virus amplification. These findings imply that other routes are used in the absence of these integrins, or that different routes are generally used in parallel.

Integrins modulate the infection efficiency of West Nile virus into cells. J Gen Virol. 08 May 2013
The underlying mechanisms allowing West Nile virus (WNV) to replicate in a large variety of different arthropod, bird and mammal species are largely unknown but are believed to rely on highly conserved proteins relevant for viral entry and replication. Consistent with this, the integrin αvβ3 has been proposed lately to function as the cellular receptor for WNV. More recently published data, however, are not in line with this concept. Integrins are highly conserved among diverse taxa and are expressed by almost every cell type at high numbers. Our study was designed to clarify the involvement of integrins in WNV infection of cells. A cell culture model, based on wild-type and specific integrin knock-out cell lines lacking the integrin subunits αv, β1 or β3, was used to investigate the susceptibility to WNV, and to evaluate binding and replication efficiencies of four distinct strains (New York 1999, Uganda 1937, Sarafend and Dakar). Though all cell lines were permissive, clear differences in replication efficiencies were observed. Rescue of the β3-integrin subunit resulted in enhanced WNV yields of up to 90% regardless the virus strain used. Similar results were obtained for β1-expressing and non-expressing cells. Binding, however, was not affected by the expression of the integrins in question, and integrin blocking antibodies failed to have any effect. We conclude that integrins are involved in WNV infection but not at the level of binding to target cells.

Mosquitoes infected with the malaria parasite Plasmodium falciparum are significantly more attracted to human odors than uninfected mosquitoes.

Researchers investigated the response of mosquitoes infected with P. falciparum malaria parasites and uninfected to human odor collected on fabric. Mosquitoes that were infected with the parasites landed and probed significantly more than uninfected mosquitoes in response to the odor. Previous research has already shown that the malarial parasite can alter mosquito behavior in ways that increase the rate of malaria transmission. For example, malaria-infected mosquitoes also consume larger, more frequent blood meals than their uninfected counterparts.

Studies of mosquito behavior in the context of malaria transmission usually use uninfected mosquito subjects. This study suggests that such behavioral studies may not always be representative of the behavior of infected mosquitoes. They conclude that understanding the olfactory changes underlying the behavior of these infected mosquitoes may help identify new compounds that could be used to develop mosquito traps for surveillance programs.

Malaria Infected Mosquitoes Express Enhanced Attraction to Human Odor. (2013) PLoS ONE 8(5): e63602. doi:10.1371/journal.pone.0063602
There is much evidence that some pathogens manipulate the behaviour of their mosquito hosts to enhance pathogen transmission. However, it is unknown whether this phenomenon exists in the interaction of Anopheles gambiae sensu stricto with the malaria parasite, Plasmodium falciparum – one of the most important interactions in the context of humanity, with malaria causing over 200 million human cases and over 770 thousand deaths each year. Here we demonstrate, for the first time, that infection with P. falciparum causes alterations in behavioural responses to host-derived olfactory stimuli in host- seeking female An. gambiae s.s. mosquitoes. In behavioural experiments we showed that P. falciparum-infected An. gambiae mosquitoes were significantly more attracted to human odors than uninfected mosquitoes. Both P. falciparum-infected and uninfected mosquitoes landed significantly more on a substrate emanating human skin odor compared to a clean substrate. However, significantly more infected mosquitoes landed and probed on a substrate emanating human skin odor than uninfected mosquitoes. This is the first demonstration of a change of An. gambiae behaviour in response to olfactory stimuli caused by infection with P. falciparum. The results of our study provide vital information that could be used to provide better predictions of how malaria is transmitted from human being to human being by An. gambiae s.s. females. Additionally, it highlights the urgent need to investigate this interaction further to determine the olfactory mechanisms that underlie the differential behavioural responses. In doing so, new attractive compounds could be identified which could be used to develop improved mosquito traps for surveillance or trapping programmes that may even specifically target P. falciparum-infected An. gambiae s.s. females.

Remember Koch’s Postulates? These are the four criteria designed to establish a causal relationship between a causative microbe and a disease:

1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Simply finding a potentially-disease causing organism does not necessarily mean it’s up to no good!

The bacterium Mycoplasma pneumoniae is carried at high rates in the upper respiratory tracts of healthy children and usual diagnostic tests cannot differentiate between such asymptomatic carriage and actual respiratory tract infection, according to a new study. These findings are important as they suggest that clinicians may need to reconsider the clinical significance of a positive test result for the presence of this bacterium.

The researchers compared upper respiratory tract swabs and blood culture results taken from 321 children (aged 3 months to 16 years) admitted to hospital with a respiratory tract infection with those from 405 healthy children undergoing an elective surgical procedure. They found that the prevalence of M. pneumoniae (as measured using PCR tests) did not differ significantly between the asymptomatic group and the symptomatic group. There was also no difference in prevalence when diagnosed using blood tests. In addition, a high rate of other bacteria and viruses was found in both asymptomatic and symptomatic children.

This data indicates that the presence of M. pneumoniae in the upper respiratory tract is common in asymptomatic children. Current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection. Cinicians may need to readdress the clinical significance of a positive test result.

Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study. (2013) PLoS Med 10(5): e1001444. doi:10.1371/journal.pmed.1001444
Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods. This study was conducted at the Erasmus MC–Sophia Children’s Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%–25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%–20.2%) of the symptomatic children (p=0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo. Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.

Ideally, invading bacteria are detected as early as possible in critically ill patients: the strain of morbific pathogens is identified rapidly, and antimicrobial sensitivity is known well before the start of new antimicrobial therapy. Bacteria have a distinct metabolism, part of which results in the production of bacteria-specific volatile organic compounds (VOCs), which might be used for diagnostic purposes. Volatile metabolites can be investigated directly in exhaled air, allowing for noninvasive monitoring.

The aim of this review is to provide an overview of VOCs produced by the six most abundant and pathogenic bacteria in sepsis, including Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. Such VOCs could be used as biological markers in the diagnostic approach of critically ill patients. A systematic review of existing literature revealed 31 articles. All six bacteria of interest produce isopentanol, formaldehyde, methyl mercaptan, and trimethylamine. Since humans do not produce these VOCs, they could serve as biological markers for presence of these pathogens.

Targeted studies should be performed to identify potential sets of volatile biological markers and evaluate the diagnostic accuracy of these markers in critically ill patients.

Volatile Metabolites of Pathogens: A Systematic Review. (2013) PLoS Pathog 9(5): e1003311. doi:10.1371/journal.ppat.1003311