Dr. Peter Palese describes how reconstructing the extinct 1918 pandemic influenza virus by reverse genetics can help us better understand molecular basis of virulence and the mechanisms by which pandemic influenza viruses are transmitted.
The UK commercial poultry industry is an important industry to the British government, the consumer and farmers alike. Worth an estimated £3.4 billion at retail value, producing over 174 million birds for consumption per year, poultry diseases are of widespread interest, both from the point-of-view of understanding different poultry farming methods, and in terms of studying the potential impact of different diseases on poultry. However, our knowledge of how poultry farms in the UK are connected to each other by the movement of people and equipment is more limited. This is essential for effective prevention and control for potential outbreaks of diseases transmitted by the movement of people and equipment between farms within the commercial poultry industry. Diseases spread in such a way include avian influenza viruses (AIV), Newcastle disease virus, Salmonella and Campylobacter species.
An epidemic of any poultry disease with high mortality or which is zoonotic, such as AIV, would result in the culling of significant numbers of birds, as seen in the Netherlands in 2003 and Italy in 2000. Such an epidemic would cost the UK government millions of pounds in compensation costs, with further economic losses through reduction of international and UK consumption of British poultry. In order to better inform policy advisers and makers on the potential for a large epidemic in the UK, we investigate the role that interactions amongst premises within the British commercial poultry industry could play in promoting an AIV epidemic, given an introduction of the virus in a specific part of poultry industry in the UK.
Poultry premises using multiple slaughterhouses lead to a large number of premises being potentially connected, with the resultant potential for large and sometimes widespread epidemics. Catching companies can also potentially link a large proportion of the poultry population. Critical to this is the maximum distance traveled by catching companies between premises and whether or not between-species transmission could occur within individual premises. Premises closely linked by proximity may result in connections being formed between different species and or sectors within the industry.
Even quite well-contained epidemics have the potential for geographically widespread dissemination, potentially resulting in severe logistical problems for epidemic control, and with economic impact on a large part of the country. Premises sending birds to multiple slaughterhouses or housing multiple species may act as a bridge between otherwise separate sectors of the industry, resulting in the potential for large epidemics. Investment into further data collection and analyses on the importance of industry structure as a determinant for spread of AIV would enable us to use the results from this study to contribute to policy on disease control.
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As this year’s flu season gets underway in the northern hemisphere, new research finds that when it comes to flu vaccination, more appears to be better. Two new studies published in the open access journal PLoS Medicine show that increasing the number of people vaccinated against influenza can decrease the burden of the disease, and not just in the individuals receiving the vaccine.
Targeted vaccination programs, in which flu vaccine is recommended for particular groups at high risk of spreading or experiencing complications of influenza, are commonly implemented. In contrast, the Canadian province of Ontario initiated a universal immunization program in 2000, in which flu vaccination is promoted and provided free of charge to everyone over the age of 6 months. The first study evaluated the effect of this universal immunization program on influenza-associated health outcomes. The researchers analyzed national and provincial data from 1997 to 2004, to compare changes in Ontario’s flu outcomes before and after introduction of universal vaccination with outcomes in other provinces, which continued targeted vaccination programs. They found that, compared with other Canadian provinces, Ontario’s universal vaccination program was associated with reductions in influenza outcomes including flu-related deaths, hospitalizations, and visits to emergency departments and doctors’ offices. The results did suggest, however, that increasing immunization rates may not be as effective in reducing mortality and health care use in older people, particularly those over 75 years of age, compared to younger people. However, even with enhanced access to free flu vaccines in Ontario, only an estimated average of 38% of the overall household population reported receiving them, suggesting that protection of older people by higher immunization rates of younger contacts who might expose them to influenza may still be of benefit.
The second study further investigated the concept of herd immunity, by which immunization of some individuals protects the overall population by reducing exposure of those who are not immunized. Using a mathematical model to simulate spread of influenza in nursing homes, researchers found that increasing the number of health care staff who are vaccinated can protect additional patients from influenza. They calculated that increasing the proportion of vaccinated health care workers from zero to 100% in a 30-bed nursing home department would reduce patient infections by about 60%, and that vaccinating seven health care workers would on average prevent one patient from getting influenza. They also found that no level of health care worker vaccination guarantees complete herd immunity, suggesting that even at high levels of immunization, increasing the number of nursing home staff who are vaccinated against flu each year will further reduce risk to patients. The authors also note that random variation, which occasionally leads to large outbreaks, limits the ability of small vaccination trials to assess the actual relationship between health-care worker vaccination and patient risk of influenza.
Patients who succumbed to influenza during the 1918 pandemic had severe lung pathology marked by extensive inflammatory infiltrate, indicating a robust immune response in the lung. Similar findings have been reported from H5N1-infected patients, raising the question as to why people expire in the presence of a strong immune response. A new paper addresses this question by characterizing the immune cell populations in the mouse lung following infection with the 1918 pandemic virus and two H5N1 viruses isolated from fatal cases. The data shows excessive immune cell infiltration in the lungs contributing to severe consolidation and tissue architecture destruction in mice infected with highly pathogenic influenza viruses, supporting the histopathological observations of lung tissue from 1918 and H5N1 fatalities. The researchers found that certain cells of the innate immune system, specifically macrophages and neutrophils, increase significantly into the mouse lung shortly following HP virus infection. Interestingly, lung macrophages and dendritic cells were shown to be susceptible to 1918 and H5N1 virus infection in vitro or ex vivo, suggesting a possible mechanism of immunopathogenesis. Identification of the precise inflammatory cells associated with lung inflammation will be important for the development of treatments that could potentially enhance or modulate host innate immune responses. While reducing virus load through anti-viral intervention remains the best treatment option for H5N1 patients, therapies that moderate immunopathology may help to reduce the high case fatality rate currently associated with this virus infection.
H5N1 and 1918 Pandemic Influenza Virus Infection Results in Early and Excessive Infiltration of Macrophages and Neutrophils in the Lungs of Mice. PLoS Pathogens, 4 (8)
Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. These results together show that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection. In addition, primary macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro and in infected mouse lung tissue.
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Viruses are released from infected cells in the form of virions, which contain all the essential factors necessary for initiating infection in a new target cell. For influenza virus, it is known that virions contain the virus genome, a lipid envelope, and at least nine virus-encoded proteins. A recent paper published in PLoS Pathogens (Cellular Proteins in Influenza Virus Particles. 2008 PLoS Pathogens 4(6): e1000085) used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. A detailed proteomic analysis of purified influenza virus particles was carried out using mass spectrometry and database searching for protein identification. In addition to the nine virus-encoded proteins, the study also identified identified 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope.
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These host proteins are present both inside the influenza virus particle and on the virus envelope. All viruses require host cell factors to complete their replication cycles, and they also have to contend with the antiviral defense mechanisms of the host. Virus–host interactions may therefore provide the key to understanding virus pathogenesis and may also present us with new targets for the design of antiviral drugs. For influenza virus, information on the requirement of cellular factors is limited, but the description of these 36 host proteins that are packaged into the virion provides a foundation for further analysis into the involvement of these cellular pathways in the influenza virus replication cycle. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes.
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To control influenza outbreaks or a pandemic, it is important to identify and characterize the different vectors that could promote influenza virus transmission between people. The respiratory tract of influenza virus-infected individuals is the main reservoir for the chain of transmission in a community. Based on experiments with animal models and observational field studies, large respiratory droplets are considered to be the most frequent vectors sustaining influenza transmission. However, experimental studies with animals with no direct contact have demonstrated that aerosols also play a significant role. In humans, the hypothesis that there is an aerosol route of transmission is supported by indirect evidence in special circumstances, such as confinement for a prolonged period of time in an airplane in the presence of a patient infected with influenza virus. In addition, it has been documented that human influenza A viruses can survive for a prolonged period on different types of surfaces once they are present in the environment. Although controversial, the possibility that contaminated surfaces and fomites could act as vectors of transmission needs to be considered in the context of overall influenza pandemic preparedness.
For any environmental contamination to be relevant, the virus should not only remain infectious but also persist at a sufficient concentration to enable it to reach the respiratory tract via finger contamination. Rhinovirus is the most common respiratory virus known to be easily transmitted by this route. Whether influenza virus is also commonly transmitted by this route remains a subject of debate. However, given that the biological properties of a potential influenza virus pandemic strain have not been established, this route of transmission has to be considered. The severe acute respiratory syndrome coronavirus SARS highlighted the ability of respiratory viruses to act in unconventional ways since environmental contamination by stools played a significant role in some population clusters. All these questions should be considered not only from a scientific standpoint. We must also take into account and provide answers to the many possible questions raised by various communities and public health authorities.
The authors of a recent paper hypothesized that banknotes may be one of various possible influenza vectors and may offer opportunities for infection. In Switzerland, a small country with a population of approximately 7 million, it is estimated that 20 to 100 million banknotes are exchanged each day, and billions of individual notes are exchanged daily worldwide. So could influenza be transmitted by money?
Survival of influenza virus on banknotes. Appl Environ Microbiol 2008 74: 3002-7
Successful control of a viral disease requires knowledge of the different vectors that could promote its transmission among hosts. We assessed the survival of human influenza viruses on banknotes given that billions of these notes are exchanged daily worldwide. Banknotes were experimentally contaminated with representative influenza virus subtypes at various concentrations, and survival was tested after different time periods. Influenza A viruses tested by cell culture survived up to 3 days when they were inoculated at high concentrations. The same inoculum in the presence of respiratory mucus showed a striking increase in survival time (up to 17 days). Similarly, B/Hong Kong/335/2001 virus was still infectious after 1 day when it was mixed with respiratory mucus. When nasopharyngeal secretions of naturally infected children were used, influenza virus survived for at least 48 h in one-third of the cases. The unexpected stability of influenza virus in this nonbiological environment suggests that unusual environmental contamination should be considered in the setting of pandemic preparedness.
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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:
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.
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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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.
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