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.

There is intense interest in developing a cure for HIV. How such a cure will be quantified and defined is not known. Researchers applied a series of measurements of HIV persistence to the study of an HIV+ adult who has exhibited evidence of cure after a stem cell transplant.

Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV was detected in blood cells, spinal fluid, lymph node, or small intestine, and no infectious virus was recovered from blood. However, HIV was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in antiretroviral treated patients. The occasional, low-level HIV signals might be due to persistent HIV or might reflect false positives. The sensitivity of the current generation of assays to detect HIV RNA, HIV DNA, and infectious virus are close to the limits of detection. Improvements in these tests will be needed for future curative studies.

The lack of rebounding virus after five years without therapy, the failure to isolate infectious virus, and the waning HIV-specific immune responses all indicate that the Berlin Patient has been effectively cured.

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient. (2013) PLoS Pathog 9(5): e1003347. doi:10.1371/journal.ppat.1003347
There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

If there is one truism about picornaviruses, it is that the entire replication cycle of these simple positive-strand RNA viruses takes place in the cytosol. This statement is usually made to directly contrast picornaviruses with retroviruses or DNA viruses that require transport to the nucleus. However, the statement is meant quite literally. Some enveloped RNA viruses enter organelles to bud from the cellular secretion pathway, while other RNA viruses replicate their genomes in tightly controlled organelle invaginations. In contrast, every step in the replication of picornaviruses, once the genome has entered the cytosol, has long been thought to take place directly in the cytoplasm or on the cytoplasmic face of membranous structures

The role of autophagosomes in poliovirus replication has long been controversial. Some believe the cytoplasmic face of these vesicles to be a site of virus RNA replication. This was primarily due to the localization of multiple virus-encoded RNA replication proteins to the autophagosome membrane. A competing hypothesis emerged observes that viral RNA replication proteins localized to single-membraned vesicles containing components of the cellular COPII machinery.

But the COPII and autophagy hypotheses might not be mutually exclusive. Single-membraned vesicles predominate in the first few hours of poliovirus infection. Later, convoluted invaginations of the single-membraned vesicles are observed. This results in structures morphologically similar to the crescent-shaped phagophore, which is the precursor to the double-membraned autophagosome. By 6 hours post-infection, double-membraned vesicles predominate. Viral proteins and active RNA replication is associated with both types of structure. However, the exponential phase of RNA replication occurs when predominantly single-membraned vesicles are present. The authors proposed a model in which single-membraned vesicles morph into double-membraned vesicles, and suggested that the single-membraned vesicles are the primary sites of viral genome replication.

Picornaviruses are among the simplest human viruses, physically consisting of a positive-sense RNA genome and a capsid. The current model for exit of picornaviruses from cells is disruption of the plasma membrane resulting in a lysis event that releases waiting cytoplasmic virions. However, if a cell full of virus-containing double-membraned vesicles lyses, releasing the vesicles, then two lipid bilayers remain between the virions and the receptors on the surface of the next cell.

This new model leaves us with a new paradigm for picornavirus replication. These viruses, for so long thought to be cytoplasmic, may in fact be more infectious if engulfed in an organelle lumen. These so-called “naked viruses,” thought to be bare in the cytoplasm, may in fact swaddle themselves in multiple layers of membranes prior to cell lysis. This work may reveal a replication strategy that can provide a mechanistic evolutionary link between the enveloped and nonenveloped viruses.

Behind Closed Membranes: The Secret Lives of Picornaviruses? (2013) PLoS Pathog 9(5): e1003262. doi:10.1371/journal.ppat.1003262

See: The Mystery of the Extra “Envelope”

The failure of poisons affecting the cytoskeleton to inhibit the replication of a diverse set of viruses strongly suggests that viruses do not require a functional cytoskeletal system for replication, either because they do not utilize it or are able to utilize alternate pathways when it is not available.

Do viruses require the cytoskeleton? Virology Journal 2013, 10: 121 doi:10.1186/1743-422X-10-121
It is generally thought that viruses require the cytoskeleton during their replication cycle. However, recent experiments in our laboratory with rubella virus, a member of the family Togaviridae (genus rubivirus), revealed that replication proceeded in the presence of drugs that inhibit microtubules. This study was done to expand on this observation. The replication of three diverse viruses, Sindbis virus (SINV; family Togaviridae family), vesicular stomatitis virus (VSV; family Rhabdoviridae), and Herpes simplex virus (family Herpesviridae), was quantified by the titer (plaque forming units/ml; pfu/ml) produced in cells treated with one of three anti-microtubule drugs (colchicine, noscapine, or paclitaxel) or the anti-actin filament drug, cytochalasin D. None of these drugs affected the replication these viruses. Specific steps in the SINV infection cycle were examined during drug treatment to determine if alterations in specific steps in the virus replication cycle in the absence of a functional cytoskeletal system could be detected, i.e. redistribution of viral proteins and replication complexes or increases/decreases in their abundance. These investigations revealed that the observable impacts were a colchicine-mediated fragmentation of the Golgi apparatus and concomitant intracellular redistribution of the virion structural proteins, along with a reduction in viral genome and sub-genome RNA levels, but not double-stranded RNA or protein levels.