Virology tidbits: April 2014

Tuesday, 22 April 2014

SARS-CoV v. MERS-CoV: differences and similarities, what do we know?

Coronaviruses are important animal and human pathogens and are the causative agent of 30-40% community acquired upper respiratory tract infections, most of them mild diseases, although severe diarrhea has been observed in children and infants. Besides relatively benign infections, the infection of infants and children has been implicated in some cases to acute asthmatic attacks and the onset of croup (whizzing cough).

Main differences between SARS-CoV and MERS-CoV

With the identi-fication of SARS-CoV in 2003 became associ-ated with more severe pulmonary disease particu-larly in immuno-compromised individuals. SARS -CoV caused se-vere lower re-spiratory disease with nearly 10% mortality and evidence of systemic spread mostly in Southeast Asian countries .

Pathology of SARS-CoV v. MERS-CoV

In contrast to SARS-CoV, the infection with MERS-CoV is leading to a higher fatality rate (50% v. 10%), although both viruses cause severe pneumonia and multiorgan dysfunction. To understand the pathogenesis, it is vital to compare various aspects of the disease, including but not limited to the receptor distribution, viral entry and affected organs as well the interference with antiviral signaling.

Genomes of SARS-CoV and MERS-CoV: whilst similar in size, SARS-CoV encodes additional genes

MERS-CoV and cytokines

Both SARS-CoV and MERS-CoV inhibit the secretion of interferon (IFN)-α and IFN-β and induce the expression of pro-inflammatory tumour necrosis factor (TNF)-α and Interleukin-6, thus inducing inflammation of surrounding tissue (and potentially necrosis). MERS-CoV also induces the expression of IL-12, IFN-γ and chemokines (e.g. RANTES/CCL-5, Il-8, IP-10/CXCL-10, MCP-1/CCL-2, MIP-1α/CCL-3) in significantly higher levels than SARS-CoV, which are required for the recruitment of T- lymphocytes to sites of inflammation. Antiviral signaling is inhibited via the inhibition of TLR mediated induction of IFN-β by the orf 4a and 4b proteins of MERS-CoV by interfering with RIG-1 and MDA5/PACT mediated signaling (whether the nucleocapsid protein is also involved has not been investigated) (see also previous blog entry).

In contrast to SARS-CoV, MES-CoV can infect and replicate in human monocyte–derived macrophages (MDM) and the aberrant induction of cytokines in these cells might contribute to disease pathogenesis. Furthermore, in MDM MERS-CoV increases the expression of MHC-class I and co-stimulatory molecules leading to an activation of the immune response.

The severity of MERS might be enhanced by the immunological status of the infected individual since symptoms are generally more severe in the elderly and immunocompromised.

SARS-CoV and MERS-CoV: receptors and cell tropism

Both SARS-CoV and MERS-CoV have been shown to infect a range of human, primate, porcine and bat derived cell lines, including but not limited to commonly used cell lines such as Vero cells and human airway epithelia cells. In the case of MERS-CoV, and in contrast to SARS-CoV, in vivo target cells include type II alveolar cells and non-ciliated cells epithelial cells (Clara cells) whereas ACE2 expressing ciliated epithelial cells (which are infected by SARS-CoV) are not susceptible to MERS-CoV infection. In addition, MERS-CoV but not SARS-CoV is capable of infecting endothelial cells as well. The receptor for MERS-CoV, first identified in Huh7 and primary human bronchial epithelial cells, was identified as dipeptidyl peptidase 4 (DDP4/CD26) and confirmed by transfecting non-permissive Cos7 cells both with bat (Pipistrellus pipistrellus) and human derived DPP4 followed by infection with MERS-CoV. The application of antibodies binding DPP4 to Huh7 and primary human bronchial epithelial cells prior to infection successfully prevented cells from MERS-CoV infection, thus further validating DPP4 being the receptor for MERS-CoV. DPP4 also specifically co-purified with the S1 subunit of MERS-CoV Spike protein. Besides being the receptor for MERS-CoV, DPP 4 has many diverse functions in glucose homeostasis, T-cell activation, neurotransmitter function, and modulation of cardiac signaling, but the enzymatic function of DPP4 is not required for viral entry (similarly the function of ACE2 is not required for SARS-CoV entry). The susceptibility for MERS-CoV of Vero cells is increased by the presence of a cell surface lung protease, TMPRSS2, as well as the presence of low-affinity receptors. During the entry of Coronaviruses into the host cell, the type II transmembrane protease TMPRSS2 activates the spike (S) protein by cleaving the mature S protein into two subunits (S1 and S2) thereby increasing the fusogenicity with the host cell receptor. In the absence of TMPRSS2, Coronavirus particles enter the cell via the endosomal pathway, which is dependent on Cathepsin L. Both SARS-CoV and HCoV-NL63 have been shown to enter the host cell via both pathways, thus suggesting that MERS-CoV might be similar and -if this is the case offer some potential options for successful treatment or prevention. Vero cells expressing TMPRSS2 show larger syncytia at 18 hrs p.i. compared to control cells, which can be blocked by the application of Camostat, a Serine protease inhibitor - Camostat however only partially blocks viral entry. This indicates that MERS-CoV, as other Coronaviruses, enters Vero cells via two independent pathways; indeed the application of both Camostat and a Cathepsin L inhibitor ((23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester or EST) not only blocks MERS-COV but also HCOV-NL63 and SARS-CoV entry into Vero-TMRSS cells. In MERS-CoV infected human bronchial submucosal gland-derived cells (Calu-3) cells , treatment with both EST and Camostat nor in combination with leupeptin is more efficacious than treatment with Camostat alone (in contrast to HCoV-Nl63 and SARS-CoV). These inhibitors were also not efficacious against MERS-CoV infection of lung derived MRC-5 and WI-38 cell lines (both are however different from mature pneumocytes, suggesting that a single treatment with Camostat is sufficient to block MERS-CoV entry into differentiated lung-derived cell lines. In the context of the infection of humans with MERS-CoV, the presence of low-affinity receptors as well as the presence of TMPRSS2 (or another S-cleaving protease) on the cell surface might sensitize cells to MERS-CoV infection. In addition, the presence of both a receptor for MERS-CoV and a S cleaving protease in a variety of animals present in the Middle East might determine potential animal reservoirs and sources of recurring transmission to humans. Since the MERS-CoV receptors in human, horse and camel are equally effective -with goat and bat receptors less effective- it might be worthwhile to extent screening beyond camels, especially in the light of the increase interest in racehorses among wealthy Arabians.

Acute renal failure

The pathology of patients infected with MERS-CoV include not only respiratory disease but also acute renal failure. Camels infected with MERS-CoV might shed viral particles in the urine, thus (potentially!) contributing to viral transmission. Infection and replication of kidneys with MERS-CoV might therefore not only lead to acute renal failure but also to shedding and transmission of MERS-CoV in urine - thus leading to new cases not only via airborne transmission but also under favorable conditions via contaminated drinking water. Indeed, DPP4 is present on the surface of both cells derived from a healthy human kidney and in primary kidney cell lines (as is ACE2, the receptor for SARS-CoV but not receptors for other HCoV). In addition to primary human kidney cells, MErS-CoV also replicates with high titers in kidney epithelial cells derived from bats, pigs, and monkey (such as LLC-MK2, Vero, and 769-P cell lines).

Although acute renal failure is a relatively late complication well after the onset of first symptoms- shedding of viral particles might partially explain familial clusters of infections. In contrast to SARS-CoV, the infection of primary kidney cells with MERS-CoV induces a more severe cytopathic effect and in higher viral titers, not only when compared with SARS-CoV but also when compared to human bronchial epithelial cells. Acute renal failure however is absent in rhesus macaques. Unfortunately, to my knowledge no post-mortem data are available from diseased patients, so at present it is not clear if the infection of kidneys causes tissue necrosis. In the case of SARS-CoV, histopathological findings revealed mainly acute tubular necrosis without abnormal pathology of the glomeruli, being the result of a systemic inflammatory response rather than a specific effect of viral infection of the kidney.

Transmission

Based on the experience from SARS-CoV related outbreaks and epidemiological data, MERS-CoV is thought to be transmitted by

Large particle respiratory droplets (by air; requires close contact).
Contact with contaminated surfaces.
Oral-fecal route.
Hospital procedures associated the generation of aerosol.

As a disclaimer, it should be noted that the precise mechanism of transmission has not been established.

Finally, the question remains how to treat patients infected with MERS-CoV? So far no specific treatment in the form of replication inhibitors exist. Treatment relies mainly on supportive care, alleviating the symptoms. Experimental treatment includes treatment of patients with Interferon-α2b and ribavirin, thus limiting viral replication. A future vaccine will most likely be based on the Spike protein and be a DNA vaccine rather than an live or an inactivated (attenuated) vaccine, similar to the experimental SARS-CoV vaccine. It should be noted that any vaccine developed might be used to vaccine animals rather than humans simply because animal vaccines are easier to be approved of.

In the meantime it is important to identify the reservoir and use this information to prevent further cases.

Wednesday, 16 April 2014

Polio in the Middle East - a tinderbox

Apart from MERS another viral disease is spreading in the Middle East: Poliomyelitis.

Poliomyelitis -or Polio for short- is recognised as a disease affecting the spinal cord since 1789 and known to have appeared in epidemics on a global scale until the vaccination became available in the 1950s. Following mass vaccination campaigns the disease was eliminated from the United States and Europe in the mid1970s and from the Middle East in the 1980s, leaving some African countries and swaths of Afghanistan and Pakistan as pockets. In the year 2000, annual cases (worldwide) were less than 1000 -down from an estimated 600000 prior to the arrival of the Polio vaccine. Wild type II Poliovirus was eradicated in 1999, but both Poliovirus type I and III continue to circulate in four countries in Asia and Africa, causing sporadic outbreaks and transmission not only in endemic areas but also those which have been declared free of Polio.

One of these previously Polio free area is Syria, which introduced mass vaccination campaigns in the 1980s (mirrored by similar campaigns in Iraq and Lebanon).

Polio vaccination rates and cases 1980-2012 (Source: WHO)

As it is well known Syria is ravaged by a civil war, which not killed an estimated 140000 people but also displaced 8 to 9 million people. These refugees often flee to the neighboring countries of Lebanon, Jordan and Iraq where they are confined to cramped refugee camps. These camps often do not have running water and only rudimentary sewage disposal system, which often overflow with the rain. It is not surprising therefore that diarrhea, respiratory diseases, Hepatitis A, scabies and lice are common diseases are particular common among the children which are not only traumatized but also often malnourished. These conditions are also a breeding ground for a resurgence of Polio, a disease transmitted via the oral-fecal route. Indeed, first cases of Polio from Syria were reported in October 2013 from the Deir Al Zour province region bordering Iraq and the region around Aleppo/Edleb in the east (bordering Lebanon). As for the refugee camps, a first unconfirmed case has been reported from a refugee camp in Taanayel/Lebanon. Furthermore, Israel reported in 2013 25 cases of Polio being identified in samples of sewage - so far however no human case has been reported and no link to the outbreak in Syria has been established. The lack of human cases might be attributed to widespread of inactivated Polio vaccine (Salk vaccine) and the high herd immunity in Israel.

Given that mass vaccinations campaigns were conducted in Syria until 2012 it is possible that a larger epidemic among Syrian refugees might have been prevented so far thanks to past immunization. Inside Syria, a mass vaccination campaign in 2012 excluded the rebel stronghold of Deir Al Zou a region that reported the first cases of Polio ten months later. This is mirrored by the resurgence of Polio in the region of Aleppo, another heavily contested area in the conflict. In the past, mass vaccination campaigns in other conflict areas were only possible due to the willingness of the parties involved to allow vaccinators into contested areas. Let’s hope that this time this is the case as well. Until then mass vaccination campaigns are imperative in refugee camps to maintain high herd immunity. Highlighting the need of vaccination campaigns is the identification of a case of Polio in Iraq, which has been linked to the outbreak in Syria.

Thursday, 10 April 2014

Tamiflu and Zanamivir: are they effective in treating Influenza or not ?

A recently published report from the Cochrane Collaboration suggested that two drugs which are used in the treatment of human Influenza are not as effective as reported in clinical studies, so it is worth to pause a moment and recapitulate how these drugs work and take a closer look at the report before rushing to any judgment.

Currently four drugs are approved for the treatment or prophylaxis of Influenza virus infection (apart from vaccinations), two belonging to the class of the adamantanes (Amantadine and Rimantadine) and two belonging to the class of neuraminidase inhibitors (zanamivir -more commonly known as Relenza- and oseltamivir -commonly known as Tamiflu). Although both are targeting processes within the replication cycle, adamantanes are only effective against Influenza A -the most severe and common form of Influenza - whereas the latter is effective against both Influenza A and B. Furthermore, adamantanes are associated with severe toxic side effects and rapid emergence of drug resistance variants, a feature shared with oseltamivir. These drug resistant virus isolates are genetically stable and can be transmitted to non-infected individuals and shed for prolonged periods by immunocompromised patients taking the drug. In the case of Relenza, no stable resistant virus isolates have been reported so far. The potential side effects limit the use of both adamantanes and oseltamivir, although both drugs have their place within the context of an epidemic or a pandemic. It is commonly accepted however that the use as a prophylactic is contraindicated in most cases, excluding immunocompromised patients perhaps (certainly in a non-pandemic); indeed, otherwise healthy individuals should be vaccinated instead. This does not mean that they can not be used to prevent the spread of Influenza within a community of otherwise healthy individuals, but one should make a careful calculation of balancing the cost and side effects with the gain - and vaccination is not only cheaper but also has less side effects. The situation is obviously different in a situation of a pandemic where mass vaccination campaigns only help to control the spread of Influenza in the long term (it takes about two weeks to develop an immunity against Influenza) or in a situation where a new virus emerges to which there is no vaccine available.

In addition, most times people claim “to have the flu” they actually do not have Influenza but are infected with other common viruses - and neither the adamantanes nor the neuraminidase inhibitors would interfere with those since they are specific for Influenza.

In order to understand how these drugs we have to take a look at the replication cycle of Influenza virus.

Replication of Influenza virus (abridged)

The surface of all Influenza viruses contains two glycoproteins -hemagglutinin (HA) and neuraminidase (NA) - the combination of these which defines the particular Influenza strain (e.g. H1N1, H5N1 or H3N7) and the variation of these molecules determines if the virus can enter human cells or not. This variation however also necessitates a new seasonal vaccine each year since the composition of the surface molecules changes each year - often these changes are small enough that antibodies against a previous strain might offer a partial protection, sometimes however a completely new strain emerges and thus having the potential to cause a pandemic. The former process is referred as antigen drift while the latter is referred to as antigenic shift.

Hemagglutinin binds the receptor on the cell surface, sialic acid, and facilitates the entry of the viral particle into the cell and the release of the viral genome into the cytoplasm of the cell in a process akin to other RNA viruses such as the Coronaviridae or Filoviridae.

Following the synthesis and assembly of nascent viral particles, the viral particle needs to be released from the cell surface in a process called budding - a process that involves cleavage of the cellular receptor (sialic acid; SA) by the viral neuraminidase. Neuraminidase deficient viral particles are attached to the cell surface and cannot be released from the cell surface and are limited to one round of replication. Furthermore, the neuraminidase is also required for infection of cells of the upper respiratory system where it cleaves the sialic acid moiety of the mucin thus allowing the virus to infect airway epithelia cells.

Apart from the hemagglutinin and neuraminidase the viral envelope of Influenza A contains other proteins required for viral replication. Of particular interest in this context is the viral M2 protein, which in infected cells it colocalizes with sites of virus budding as well as cholesterol containing lipid raft domains on the cell surface and incorporated into the virion during the budding process. Together with M1, M2 is responsible for the formation of filamentous Influenza virus particles - those that are commonly found during an infection but are largely absent in virus grown in eggs (on a side note, filamentous Influenza virus particles enter cells with macropinocytosis, a receptor independent entry pathway and are the prevalent morphology in patients - spherical particles are common in viruses grown in eggs). The M2 protein not only assists in forming filamentous viral particles, it is more importantly a proton selective ion channel which is activated by the low pH (4.9) of the endosome (concurrently with HA) thus allowing -following structural changes- the viral genome being released into the cytoplasm and imported into the nucleus of the infected cell.

The adamantanes inhibit these processes, both the formation of spherical and filamentous viral particles as well as the release of the viral genome of the cell, whilst the neuraminidase inhibitors inhibit the cleavage of SA - and thus viral entry as well as the release of newly synthesized viral particles from the cell surface.

The Tamiflu and Relenza debate

The debate about the effectiveness started as soon as both drugs were approved by the FDA for the use to treat and prevent Influenza and intensified during the “swine flu”, A/H1N1/2009, epidemic when governments worldwide began to store Tamiflu and Relenza. Early clinical trials of both zanamivir and oseltamivir suggested that both drugs can prevent the onset of Influenza if taken early and reduced symptoms 24 hrs following treatment of infected patients. More importantly, both drugs reduced the onset and complications of secondary bacterial infections - the very same which often might have lead to the death of patients during the Influenza pandemic in 1918/1919 and were the reason that scientist initially suspected bacteria to be the causative agent of the disease in the first place.

These clinical studies also emphasized the importance that both drugs need to be administrated early in the infection in order to reduce symptoms. So what is all the fuzz about? The authors of the recent report reanalyzed the 107 published and unpublished clinical study reports available from European Medicines Agency (EMA) and the manufactures of zanamivir and oseltamivir, GlaxoSmithKline and Roche respectively, as well as comments published by the FDA, EMA and the Japanese regulator. The main objective was to identify the potential benefits and harms associated with these drugs in placebo and non-placebo treated groups by reanalyzing the data from non-published and published studies from both manufactures and regulatory bodies. Criteria for effectiveness included (1) the time from taking the drug to alleviation of the first symptoms (2) hospitalizations (3) secondary infections as a result of Influenza virus infection (3) side effects of the drug (such as nausea) and (4) prophylaxis - certainly an important factor in an epidemic. Contrary to what is reported in the media, the report acknowledges that in cases of symptomatic Influenza oseltamivir and zanamivir do protect household members of infected individuals - but fail to do so in cases of asymptomatic/non -Influenza disease (the attentive reader might have realize that both drugs were specifically designed against Influenza - so who would expected that they work against other viruses anyway?). Moreover, zanamivir significantly reduces the risk of bronchitis in adults (not in children though). Even the authors concede that both drugs may have benefits. Interestingly the authors published a report in 2009 stating that oseltamivir does not prevent infections of the lower respiratory tract.
In this context one might speculate that the receptor specificity of Influenza might determine the sensitivity to neuroaminidase inhibitors.
Airway epithelia cells of in lower respiratory tract are known to express α2-3 linked sialic acid -the receptor for avian and avian-like high risk influenza virus (such as H5N1). Cells in the upper respiratory tract however preferentially express α2-6 linked sialic acid - with the exception of children, which preferentially express α2-3 linked sialic acid receptors. Recent results however indicate that the infectivity of recombined (reverse genetically modified) H5N1 which is specific for SAα2-6 is not altered by oseltamivir nor zanamivir, thus implying that other factors might play role in conferring drug resistance (apart from the HA and NA) - however it can not be ruled out that the mutations induced in the HA gene contribute to mutations of the NA. Some data derived from studies using Influenza virus infecting wild aquatic birds suggest that additional N-linked glycans at the top of HA are required for effective binding of Influenza Virus to the host cell receptor (this applies to both H5 and H7 viruses).

So what is the conclusion? Both oseltamivir and zanamivir seem to offer some protection and alleviation of symptoms. The core of the problem seems however that pharmaceutical companies often withhold data from clinical studies - this however is not unique to antiviral drugs. In the end, people may realize that getting vaccinated in the first place might be the best prophylactic. Finally, more antiviral drugs need to be developed. Two recently identified promising candidates include resveratrol and epigallocatechin gallate, both plant derived polyphenols and can be found in green tea as well as red wine. Isoquercetin has been shown to prevent the formation of oseltamivir and amantadine resistant virus particles, thus being another potential antiviral drug.