Negative sense RNA viruses

The genomes of all cellular organisms, from bacteria to humans, consist of double-stranded DNA. But in viruses, there is tremendous diversity of virus genomes: double stranded or single-stranded, DNA or RNA, positive- or negative-sense, but only viruses have RNA genomes.

In terms of virus genomes, “negative sense” means that a single-stranded nucleic acid molecule has the opposite sequence to messenger RNA (mRNA) and so cannot be translated into protein until it has been copied. This has important biological implications for viruses with negative-sense RNA genomes. Since cells have no biochemical mechanism to copy RNA, every negative-sense RNA virus must carry within the virus particle an RNA-dependent RNA polymerase (or “replicase” as it is frequently called), or the virus genome will be biologically meaningless once in a host cell.

There are seven virus families and 31 genera of viruses with negative-stranded RNA genomes, and these groups contain some very important pathogens. Based on their similar genetic structure, four of these virus families are believed to have arisen from a common ancestor, and are grouped into a taxonomic order, the Mononegvirales (unsegmented negative-strand viruses).

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The Bornaviruses are a relatively little-studied group, giving rise to Borna disease, a neurological syndrome of warm-blooded animals. The Filoviruses have pleiomorphic (variably shaped), elongated particles approximately 80 nm in diameter and between 130-14,000 nm long - hence their name, which means “thread-like” viruses. The Filovirus genome encodes seven proteins on monocistronic mRNAs which are complementary to vRNA (the virus genome). Until recently, relatively little work has been performed on these viruses because of the difficulties of working with them, but their replication is known to be similar to that of rhabdoviruses and paramyxoviruses (also members of the Mononegvirales).

The Paramyxoviruses have enveloped particles which are 125-250nm in diameter. Their genome contains a linear arrangement of six genes, separated by repeated sequences. Paramyxoviruses include:

• Parainfluenzaviruses: These cause acute respiratory infections ranging from relatively mild influenza-like illness to bronchitis, croup and pneumonia.
• Respiratory Syncytial Virus (RSV): A major cause of lower respiratory tract disease in infants.
• Measles: A highly infectious virus spread by aerosols, which causes a systemic infection with complications including ear infections (1 in 20 cases), pneumonia (1 in 25), convulsions (1 in 200), meningitis/encephalitis (1 in 1,000), subacute sclerosing panencephalitis (SSPE) http://en.wikipedia.org/wiki/SSPE (1 in 1,000,000), and even death (1 in 2,500-5,000 cases).

The Rhaboviruses have unique bullet-shaped particles with prominent protein spikes on the surface of their lipid envelope. Rhabdovirus genomes are around 11 kilobases long and contain five genes. Diseases caused by Rhabdoviruses include vesicular stomatitis in cattle, pigs, horses and wildlife, and rabies, which causes a fatal encephalitis.

In addition to the Mononegvirales, other negative-sense RNA viruses have segmented genomes, i.e. their genomes comprise a number of separate molecules, all of which must be packaged into a particle in order to give rise to an infectious virus.

The best known of these are the Orthomyxoviruses, which include influenza virus. Influenza viruses can infect a wide variety of mammals, including humans, horses, pigs, ferrets and birds, and are a major human pathogen. Unlike other negative-sense RNA viruses, Orthomyxovirus genomes are replicated in the nucleus of the host cell, rather than in the cytoplasm.

The Arenaviruses and Bunyaviruses have another twist when it comes to genome structure: they have ambisense genomes which contain both positive-sense and negative-sense coding regions.

In summary, this is possibly the most biologically diverse class of viruses.

• Mononegvirales (replication in cytoplasm)
• Orthomyxoviruses (replication in nucleus)
• Arenaviruses, Bunyaviruses (ambisense genome)
• Different strategies of gene expression to cope with genome coding patterns.
• They include some of the most important virus pathogens.

Maybe Not Quite The End

This post is from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

Given the current scare over H5N1 influenza virus in swans in the UK, it is possibly timely to recall that I wrote a little while ago in MicrobiologyBytes about how easy it appeared to be for the highly pathogenic H5N1 avian influenza virus to change receptor and therefore host specificity: all it apparently needed was substitutions at position 129 and 134 in the HA protein to change from binding avian-type sialic acid (SA) α2,3Gal(actose) receptors to the human-type SA α2,6Gal receptor. And the outlook was gloomy, and panic was close at hand.

Fortunately for us, it turns out that things are not so simple. According to a letter in the January 2008 issue of Nature Biotechnology, it is a characteristic structural topology, and not just the α2,6 linkage, that enables specific binding of HA to α2,6 sialylated glycans. The authors state:

…recognition of this topology may be critical for adaptation of HA to bind glycans in the upper respiratory tract of humans. An integrated biochemical, analytical and data mining approach demonstrates that HAs from the human-adapted H1N1 and H3N2 viruses, but not H5N1 (bird flu) viruses, specifically bind to long α2-6 sialylated glycans with this topology. This could explain why H5N1 viruses have not yet gained a foothold in the human population.

Apparently the critical shape in humans is umbrella-like, whereas the avian receptor is characteristically cone-like. Again from the paper:

The topology of α2-3 and α2-6 is governed by the glycosidic torsion angles of the trisaccharide motifs-Neu5Aca2-3Galb1-3/4GlcNAc and Neu5Aca2-6Galb1-4GlcNAc, respectively (Supplementary Fig. 3 online).

Ram Sasisekharan and colleagues showed that human-adapted viruses with mixed α2,3/α2,6 binding ability that bound the umbrella-type receptor were efficiently transmitted, whereas viruses with the same basic specificity that did not have HA binding specificity to “long” α2,6, were not.

This means that the perceived threat of H5N1 human adaptation and rapid spread has receded somewhat, as the virus HA needs considerably more adaptation than the simple mutations that were previously assumed to change the specificity. Furthermore, these findings also allow the possibility of using glycan arrays with long α2,6 molecules for the screening of H5N1 and other avian virus isolates for possible evolutionary adaptation to the appropriate receptor binding form. They close their paper with these encouraging words:

A sufficient understanding of the avian H5N1 HA mutations leading to long α2-6 binding specificity offers an opportunity for intervention through vaccine development to negate the eventuality of a H5N1 pandemic.

Now while I am happy that the threat may not be as imminent as I thought it was, I must point out that H5N1 flu is still one of the nastiest pandemic prospects facing humanity. The virus is established as an endemic pathogen worldwide, meaning it could break into the human population just about anywhere people keep domestic poultry. While the threat of mutation leading to rapid adaptation may be a lot less severe than we thought, there is still the possibility of
recombination / reassortment leading to a virulent, human-adapted virus - and we should recall that the flu pandemics of the 1950s and 1960s were due to reassortment between the prevailing H1N1 virus and a H2N2 type in 1957, and between a H3-containing virus and the prevailing H2N2 in 1968. And the contributing avian viruses were nowhere near as well distributed as H5N1 is now, nor as virulent …

So the apocalypse is still nigh - but possibly less nigh than we may have thought. However, as the inimitable Gregory House has observed, “It’s not paranoia if they’re really after you”. And I think they still are.

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Related:

• Influenza H5N1 in Dorset, UK
• This is the End
• H5N1 influenza is no longer “bird flu”
• Human Infections with Avian Influenza H5N1 Viruses

News: Influenza H5N1 in Dorset, UK

Three mute swans have been found dead with the virulent H5N1 strain of bird flu. The three infected swans were found at the Abbotsbury Swannery, an open reserve near Chesil Beach in Dorset on December 27, 31 and January 4.

Efforts have begun to test other birds at Abbotsbury Swannery, nine miles from Weymouth.

Latest News

Related:

• H5N1 influenza is no longer “bird flu”
• Human Infections with Avian Influenza H5N1 Viruses
• This is the End
• UK national framework for responding to an influenza pandemic

UK national framework for responding to an influenza pandemic

Pandemic flu: A national framework for responding to an influenza pandemic

Influenza pandemics are natural phenomena which occurred three times in the last century. Their severity has ranged from something similar to seasonal influenza to a major threat, with many millions of people worldwide becoming ill and a proportion of these dying. No country can expect to escape the impact of a pandemic entirely, and when it arrives most people are likely to be exposed to an increased risk of catching the virus at some point. Influenza pandemics therefore pose a unique international and national challenge. As well as their potential to cause serious harm to human health, they threaten wider social and economic damage and disruption. Measures to prevent, detect and control them require coordinated international effort and cooperation, with one countrys action or inaction potentially affecting many others.
Although it is highly likely that another influenza pandemic will occur at some time, it is impossible to forecast its exact timing or the precise nature of its impact. This uncertainty is one of the main challenges for policy makers and planners. Even if as seems likely a pandemic originates abroad, it will probably affect the UK within two to four weeks of becoming an epidemic in its country of origin, and could then take only one or two more weeks to spread to all major population centres here. In addition to collaborating actively in multi-national prevention, detection and research, the Governments aims at a national level are to ensure that the UK is prepared to limit the internal spread of a pandemic and to minimise health, economic and social harm as far as possible.
This framework sets out the Governments strategic approach to achieving these aims and is intended for use by all those involved in planning for and responding to an influenza pandemic. It builds upon and supersedes the most recent version of the UK Health Departments UK Influenza Pandemic Contingency Plan (published in October 2005), expanding it to cover a more comprehensive range of impacts and responses. The framework will also inform the development of community and organisational arrangements that are appropriate to local circumstances and are sufficiently consistent to ensure an equitable and sustainable national response. It includes information to support planning and, where necessary, provides signposts to additional sources of technical information and guidance.

Related:

• Newsflash: H5N1 Influenza in Suffolk again
• H5N1 influenza is no longer “bird flu”
• Global migration of influenza viruses
• The Biology of Influenza
• Influenza pandemic - when?

Newsflash: H5N1 Influenza in Suffolk again

Defra has confirmed that the type of bird flu found in turkeys on a Suffolk farm is the virulent H5N1 strain.

Dicer is involved in protection against influenza A virus infection

RNA interference, RNAi, is a natural antiviral mechanism in plants and invertebrates. Based on the results obtained in plants, Drosophila and worms and because the RNAi machinery is present in all animals from nematodes to mammals, RNAi has often been proposed to be involved in the response to viral infection in vertebrates. In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-alpha and IFN-beta genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

Dicer is involved in protection against influenza A virus infection
J Gen Virol. 2007 88: 2627-2635

Influenza virus transmission is dependent on humidity and temperature

In temperate regions influenza epidemics recur with marked seasonality: in the northern hemisphere the influenza season spans November to March, while in the southern hemisphere epidemics last from May until September. Although seasonality is one of the most familiar features of influenza, it is also one of the least understood. Indoor crowding during cold weather, seasonal fluctuations in host immune responses, and environmental factors, including relative humidity, temperature, and UV radiation have all been suggested to account for this phenomenon, but none of these hypotheses has been tested directly. Using the guinea pig model, these authors evaluated the effects of temperature and relative humidity on influenza virus spread. By housing infected and nave guinea pigs together in an environmental chamber, they carried out transmission experiments under conditions of controlled temperature and humidity. They found that low relative humidities of 20%-35% were most favorable, while transmission was completely blocked at a high relative humidity of 80%. Furthermore, when guinea pigs were kept at 5°C, transmission occurred with greater frequency than at 20°C, while at 30°C, no transmission was detected. The data implicate low relative humidities produced by indoor heating and cold temperatures as features of winter that favor influenza virus spread.
To investigate the mechanism permitting prolonged viral growth, expression levels in the upper respiratory tract of several innate immune mediators were determined. Innate responses proved to be comparable between animals housed at 5°C and 20°C, suggesting that cold temperature (5°C) does not impair the innate immune response in this system. Although the seasonal epidemiology of influenza is well characterized, the underlying reasons for predominant wintertime spread are not clear. This provides direct experimental evidence to support the role of weather conditions in the dynamics of influenza and thereby address a long-standing question fundamental to the understanding of influenza epidemiology and evolution.

Influenza virus transmission is dependent on relative humidity and temperature
PLoS Pathog 3(10): e151

Related:

• Global migration of influenza viruses
• The Biology of Influenza

H5N1 influenza is no longer “bird flu”

Highly pathogenic avian H5N1 influenza viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33°C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2–627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human receptors.

Growth of H5N1 Influenza A Viruses in the Upper Respiratory Tracts of Mice
PLoS Pathogens 2007 3 (10) e133

What does this all mean?
The H5N1 influenza virus (formerly known as “bird flu”) has mutated to infect people more easily, although it still has not transformed into a pandemic strain. In order to acquire the capacity for efficient human-to-human transmission, H5N1 avian influenza viruses must undergo a series of genetic changes resulting in the ability to replicate at lower temperatures, in a wider range of cell types, to recognize human receptors, and other unknown phenotypic changes controlled by virus proteins. Birds usually have a body temperature of 41°C, and humans 37°C. The human nose and throat, where flu viruses usually enter, is usually around 33°C, so the virus doesn’t grow well in the nose or throat of humans. This research identifes a mutation which allows H5N1 to replicate in the cooler temperatures of the human upper respiratory tract. The H5N1 viruses circulating in Europe and Africa all have this mutation. What we don’t know at present is how many additional mutations are needed for these humanized viruses to become a pandemic strain, or how long that process will take.

Global migration of influenza viruses

Influenza A virus is able to persistently re-infect human populations by continually evading host immunity through the continuous and rapid evolution of surface antigens. This process is known as antigenic drift. Influenza virus epidemics affect temperate latitudes of the world each winter, from November to March in the northern hemisphere and from May to September in the southern hemisphere. In the United States alone, these influenza epidemics are associated with an annual average of 36,000 human deaths and 226,000 hospitalizations. Globally, the virus is associated with as many as half a million annual deaths. While rapid antigenic change is characteristic of influenza, recent studies have failed to detect antigenic drift over a single epidemic season, suggesting that important evolutionary processes may occur during non-epidemic periods, either locally or perhaps elsewhere. However, surveillance during non-epidemic periods is not conducted routinely by the network of World Health Organization influenza reference centers and, consequently, little is known about how and where the virus persists in the human population in between winter epidemics at low levels. A key question is therefore whether the virus remains locally within its host population in between epidemics, perhaps persisting within hosts in a latent state, or whether the virus migrates to other reservoirs, such as the tropics, and is later reintroduced.

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Although influenza virus has long been regarded a “cold-weather” pathogen due to its marked winter epidemics in temperate zones, recent studies show that tropical regions experience significant year-round influenza virus activity. In theory, such a “tropical belt” could serve as a year-round reservoir that harbors endemic populations of influenza virus that seasonally reintroduce viral isolates into temperate zones to trigger new epidemics. Whereas population crashes at the end of seasonal epidemics create severe evolutionary bottlenecks that limit genetic diversity, tropical zones may function as permanent mixing pools for viruses from around the world. Historically, Southeast Asia has been considered a potential epicenter for emergence of pandemic viruses due to the proximity with which humans live with their domestic animals. However, abundant data from these regions is currently unavailable, so the origins of influenza pandemics and epidemics remain unclear.

Given the ease and speed with which the influenza virus is thought to spread between humans, it is generally accepted that global chains of direct person-to-person transmission are sufficient to maintain influenza virus in the human population. However, a complete understanding of how the influenza virus transmits between humans is lacking, and whether human-to-human spread alone accounts for the seasonal emergence of epidemics has been questioned. The simultaneous appearance of influenza outbreaks separated by large distances, as well as sporadic influenza cases during summer months, suggests that the virus may instead already be “seeded” and somehow reactivated by environmental stimuli. The alternating pattern of northern and southern hemisphere epidemics could, in principle, also result from opposite climatic forces independently reactivating viral activity in these two hemispheres at alternating six-month intervals. Hence instead of continually migrating across the equator, separate virus populations could persist locally in an asymptomatic latent state over the summer months until climatic stimuli sufficiently increase host susceptibility and/or viral transmissibility to induce another epidemic. However, hypotheses of how climatic change may directly or indirectly influence viral activity and/or host susceptibility remain largely untested.

Some theories for influenza seasonality produce testable hypotheses. On one hand, if influenza virus persists locally over the summer in a latent state, then isolates sampled over multiple seasons from a single locality would cluster together on a phylogenetic tree, separate from isolates from other geographic regions. Alternatively, if the virus did not evolve locally between epidemic seasons, but rather traveled globally between epidemics, then the resulting phylogeny would show extensive intermixing of isolates from different localities. To determine whether influenza virus migrates between the northern and southern hemispheres during non-epidemic summer months or remains localized, a recent paper in PLoS Pathogens reports on an extensive phylogenetic analysis of 399 influenza A viruses sampled from New Zealand from 2000 to 2005, 88 virus sequences from Australia from 1999 to 2005, and a carefully selected sample of 52 isolates which are representative of the types present in a larger sample of 413 viruses in the USA from 1998 and 2005 (Phylogenetic Analysis Reveals the Global Migration of Seasonal Influenza A Viruses. PLoS Pathog 3(9): e131).

The results show that even in areas as relatively geographically isolated as New Zealand’s South Island and in Western Australia, global virus migration contributes significantly to the seasonal emergence of influenza A epidemics, and that this migration has no clear directional pattern. These observations run counter to suggestions that local epidemics are triggered by the climate-driven reactivation of influenza viruses that remain latent within hosts between seasons or transmit at low efficiency between seasons. However, a complete understanding of the seasonal movements of influenza A virus will require greatly expanded global surveillance, particularly of tropical regions where the virus circulates year-round, and during non-epidemic periods in temperate climate areas.

Related:

• This is the End
• Influenza: H5N1 Updates
• The Biology of Influenza
• Influenza pandemic - when?

Human Infections with Avian Influenza H5N1 Viruses

During the period 20 May to 15 Sep 2007, a total of 21 human cases of avian influenza A (H5N1) infection was reported to WHO from four countries (China, Egypt, Indonesia, and Vietnam). Fourteen (67%) of these cases were fatal.

Since 1 Dec 2003, a total of 328 human avian influenza A (H5N1) infection have been reported to WHO. Of these, 200 (61%) were fatal. All the human cases were reported from Asia (Azerbaijan, Cambodia, China, Indonesia, Iraq, Laos, Thailand, Turkey, and Vietnam) and Africa (Djibouti, Egypt, and Nigeria).

MMWR Weekly 28 Sep 2007 56 (3)

H5N1 influenza virus can also pass through a pregnant woman’s placenta to infect the foetus. Researchers also found evidence that the virus not only affects the lungs, but passes throughout the body into the gastrointestinal tract, the brain, liver and blood cells.

Related:

• The Biology of Influenza
• Influenza pandemic - when?
• This is the End