Lengthy, but good article on bird flu

Health
Bird flu: A threat to humanity
By Mehdi Pervez
Sat, 23 Sep 2006, 10:07:00



Over centuries natural calamities have hit this earth in many shapes. Sometimes in the form of floods sometimes in the form of volcanic eruptions and sometimes in the form of epidemic of deadly diseases. Every instance has caused the lives of hundreds and thousands of human lives. Even at the prime time of medical science an old disease is re-emerging with its deadliest threats. We are thinking about none other than bird flu. Bird flu is nothing new or uncommon. It first pandemically occurred in 1918 immediately after World War I and killed more than 40 million people. The later outbreaks were in 1957 and 1968. All of the attacks from bird flu killed about 100 million people around the world. Though this is not actually a human disease but its scourge of killing 10 crore people in only 87 years is a fearful matter. For more than a century, bird flu has circulated among birds, particularly domesticated fowl, but recent attention has been called to avian influenza since some strains infected humans. No longer is bird flu relegated to pigs and birds, as the virus has strengthened and mutated, resulting in a contagion that can move from bird to human. Human cases of bird flu have caused infections and death across the globe as scientists struggle to identify the dangerous strains and prevent a fatal pandemic.

Now let us discuss some of the microbiological aspects of Flu Virus. "Flu" is short for "influenza". The name goes back hundreds of years when the disease was thought to be caused by supernatural "influences". Many describe any nasty lung infection as flu, but only specific lab tests can give a proper diagnosis. There are several different viruses (and bacteria) which may infect the lung, but true flu is caused by orthomyxoviruses, of which there are three types, designated A, B, and C. An influenza virion has about 500 "spikes" sticking out from its lipid envelope. About 80% of the spikes are a glycoprotein peplomers-rod shaped viral protein called hemagglutinin (or simply, HA) which are homotrimers of class I membrane glycoprotein's. This was first identified by its ability to cause red blood cells, which carry a molecule called "heme", to agglutinate (stick together). We now know that HA is influenza's receptor-binding protein. It plays the critical role of attaching the virus to the host cell. The other 20% of the spikes are a mushroom shaped viral protein called neuraminidase (NA), which is tetramer of a class II membrane protein. This protein is an enzyme that destroys a host cell molecule called neuraminic (or sialic) acid. NA might play a part in getting the virus into the cell, but its most important function is that it helps the newly made influenza virions to easily escape from the host cell so they can infect others[1]. The virulent avian influenza H5N1 strains differ from other avian strains in that, there lies a link between HA cleavage and degree of virulence. In virulent strains the HAs contain multiple basic amino acids at the cleavage site, which are cleaved intracellularly by endogenous proteases. In contrast, in case of avirulent avian strains as well as non-avian influenza A viruses, the HAs lack the basic amino acid residues, hence not subjected to cleavage by such proteases. Moreover, all types of influenza A viruses are antigenically labile, well adapted to evade host defenses and lack mechanisms for "proof reading"; hence constant, permanent and small changes in antigenic composition are very common, which is known as antigenic drift. Another important characteristic of great public health concern is antigenic shift which results from reassortment of genetic material from different species resulting in variability of HA spikes, keeping the basic structure of the virus constant [2].

Influenza viruses that infect birds are called avian influenza viruses. Only influenza A viruses infect birds, and all known subtypes of influenza A viruses can infect birds. However, there are substantial genetic differences between the subtypes that typically infect both people and birds. Within subtypes of avian influenza A viruses there also are different strains. 13 different kinds of HA and 9 different kinds of NA genes in type A influenza is known[3].

They evolve! Molecular evolution (the evolution of molecules) is a fascinating area of evolution and of prime concern to any scientist wanting to understand viruses and how they spread. All genetic material can mutate, that is change its nucleic acids. The mutations are random, but their selection is not. "Selection" is another word for how well they survive and reproduce. Selection ensures that the mutations that increase a virus' ability to survive and reproduce will be represented in even greater numbers in the next generation. Mutations are the "fuel" for evolution because they provide the genetic variation on which selection acts. This is simply Darwin's old theory of evolution by means of natural selection, but on a microscopic scale.

All influenza viruses (all orthomyxoviruses) have RNA as their genetic material. When RNA is replicated it tends to have more errors than when DNA is replicated. These extra errors provide extra mutations upon which selection may act. That means RNA viruses (not just influenza viruses but all RNA viruses) have a high mutation rate and can evolve quickly - faster than a DNA virus or even a DNA human! Over time these mutations accumulate and eventually the virus evolves into a new strain. This progressive accumulation of individual mutations is called antigenic drift, because the shape of the antigen (the viral protein) slowly drifts into a different shape with each generation of virus. Eventually they drift so much that the original antibody can no longer bind to it.. All viruses show antigenic drift, but RNA viruses mutate faster so they drift faster. Antigenic drift is responsible for many of the localized outbreaks of different strains of influenza, especially influenza B.

Importantly, type A - but not B or C - undergo a kind of gene swapping or genetic reassortment to give it its proper name. If a cell is simultaneously infected by two different strains of type A influenza, the offspring virions may contain mixtures of each parents' genes! This really complicates things and makes it very easy for influenza A to quickly evolve new combinations of HA and NA genes. To better understand what I mean you need to learn a little bit about how we keep track of all this reassortment. We know of 13 different kinds of HA and 9 different kinds of NA genes in type A influenza. All these different kinds have evolved by antigenic drift as described earlier. Any one virion can contain only one HA and one NA. For example then an influenza A strain designated H1N1 can be produced. (We drop the "A"s at the end to make it clearer.) Along comes another virus with different kinds of HA and NA genes, let's say it is H3N7. If these two different virions infect the same cell at the same time they may produce offspring not only like themselves (H1N1 and H3N7) but also with a mixed combination (H1N7 and H3N1).

This is only a small sample of the many possible new combinations that might be made. All eight segments may take part in the reassortment. These newly created mixed genomes are very different from their parents and (probably) have never been "seen" by your immune system - or for that matter, anyone else's. This form of viral evolution is called antigenic shift, to differentiate it from antigenic drift (which occurs slowly and without a change in the gene associations). These new combinations present us with such a unique strain of virus that our immune system has to start all over to make new antibodies to combat it[1].

Since now we have seen that there are many strains of flu virus. But the strain that is mostly infecting people since 1997 is the H5N1 strain. This strain, in many ways, different and dangerous from other flu strains which we will try to explain below.

A report by a World Health Organization (WHO) committee says avian flu may have a longer incubation period and is more likely to cause diarrhea than typical flu viruses are, among other differences.

Published in the Sep 29 New England Journal of Medicine, the review was written by experts from several countries, including Vietnam, Cambodia, Thailand, the United States, the United Kingdom, Hong Kong, and Myanmar. They reviewed 71 published studies and reports, including details on 41 confirmed human cases from Vietnam, Thailand, Cambodia, and Hong Kong.

Researchers from Hong Kong report that lung cells growing in a laboratory responded much more intensely to the H5N1 virus than to an ordinary flu virus, even though the viruses reproduced at about the same rate, according to the report published online by Respiratory Research.

The H5N1 viruses were "more potent inducers" of cytokines and chemokines-chemical messengers that trigger inflammation-than H1N1 viruses were, says the report by a team led by J.S.M. Peiris of the University of Hong Kong. A flood of inflammation-triggering chemicals released by the immune systems has been referred to as a "cytokine storm."

They found that all the H5N1 viruses caused cells to secrete significantly higher levels of IP-10 (interferon-gamma-inducible protein 10), interferon beta, a type of T cell known as RANTES, and interleukin-6 than the H1N1 virus did. In addition, the 2004 versions of H5N1 caused cells to release more IP-10 at 6 hours than the 1997 version did.

"We have found that infection with H5N1 viruses led to the production of 10 times higher levels of cytokines from human cells than normal human flu viruses," said Peiris, as quoted Nov 12 in The Standard, a Chinese business newspaper.

The most alarming news about this H5N1 virus is that, Scientists reported findings which may help explain what made the 1918 pandemic influenza virus so deadly and that reveal similarities between that virus and the H5N1 avian influenza virus now circulating in Asia. The 1918 flu pandemic, regarded as the worst in history, killed as many as 100 million people

"The new studies could have an immediate impact by helping scientists focus on detecting changes in the evolving H5N1 virus that might make widespread transmission among humans more likely," the statement said[4].

Avian influenza viruses circulate among birds worldwide. Certain birds, particularly water birds, act as hosts for influenza viruses by carrying the virus in their intestines and shedding it. Infected birds shed virus in saliva, nasal secretions, and feces. Susceptible birds can become infected with avian influenza virus when they have contact with contaminated nasal, respiratory, or fecal material from infected birds. Fecal-to-oral transmission is the most common mode of spread between birds.

Most often, the wild birds that are host to the virus do not get sick, but they can spread influenza to other birds. Infection with certain avian influenza A viruses (for example, some H5 and H7 strains) can cause widespread disease and death among some species of domesticated birds[3].

Domesticated birds may become infected with avian influenza virus through direct contact with infected waterfowl or other infected poultry, or through contact with surfaces (such as dirt or cages) or materials (such as water or feed) that have been contaminated with virus[3]. Avian influenza A viruses may be transmitted from animals to humans in two main ways:

Ø Directly from birds or from avian virus-contaminated environments to people. Almost all these casualties were directly exposed to infected fowl, making contact with the virus through the birds' saliva, nasal secretions and feces, which become dry, pulverized and are then inhaled.

Ø Through an intermediate host, such as a pig[3].

A new study indicates that H5N1 avian influenza viruses are becoming less deadly to ducks, permitting them to carry the viruses for days or weeks and spread them to more susceptible birds and potentially to humans.

The findings "suggest that the duck has become the 'Trojan horse' of Asian H5N1 influenza viruses," says the report by an international team led by researchers from St. Jude Children's Research Hospital in Memphis. "The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat."

The researchers experimentally infected ducks with various H5N1 viruses, most of them dating to 2003 and 2004. About half of the infected ducks survived while shedding the virus for as long as 17 days, according to the report, published online today by the Proceedings of the National Academy of Sciences[4].

Avian influenza virus lacks the ability to 'hop' easily between people, which have probably helped to contain the problem. However, in the future, it is possible that the process of genetic reassortment could occur in a human who is co-infected with avian influenza A virus and a human strain of influenza A virus. The genetic information in these viruses could reassort to create a new virus with a hemagglutinin from the avian virus and other genes from the human virus. Theoretically, influenza A viruses with a hemagglutinin against which humans have little or no immunity that have reassorted with a human influenza virus are more likely to result in sustained human-to-human transmission and pandemic influenza. Therefore, careful evaluation of influenza viruses recovered from humans who are infected with avian influenza is very important to identify reassortment if it occurs[3].

Symptoms of Avian Influenza

Infected bird will get fever with rigor; diarrhea, paralysis then the bird will be unable to stand and keep the head up and ultimately die in 1-2 days[5].

The reported symptoms of avian influenza in humans have ranged from typical influenza-like symptoms:

1. fever (usually high)

2. headache

3. extreme tiredness

4. dry cough

5. sore throat

6. runny or stuffy nose

7. muscle aches

8. Stomach symptoms, such as nausea, vomiting, and diarrhea, also can occur but are more common in children than adults

9. Conjunctivitis is seen in some patients

Life threatening complications like viral pneumonia, respiratory distress syndrome, worsening of chronic medical conditions, such as congestive heart failure, asthma, or diabetes and multi organ failure may result in the death of the patient[3].

Laboratory Testing Procedures

Rapid antigen detection by immunofluorescence assay and enzyme immunoassay, virus isolation by culture in HeP-2, RD cells or MDCK cell lines and identification by immunofluorescence assay using specific monoclonal antibody and haemagglutination inhibition assay have been used for diagnosis. Detection of influenza- specific RNA by reverse transcriptase-polymerase chain reaction, by using primer sets specific for HA sequence of influenza A/H5 and of N1 are some of the other tests that have been developed. Serological identification by measuring the specific antibodies by haemagglutination inhibition test, enzyme immuno assay and the virus neutralisation test, more specifically the micro neutralisation test, have also been developed. Following kits are presently available:

1. Immunoflourescence assay- WHO influenza reagent kit for the identification of Influenza A/H5 virus (1997-1998, 2003 or 2004 version) which includes influenza type A/H5- specific monoclonal antibody pool along with influenza B, A/H1 and A/H3 subtype specific monoclonal antibodies.

2. Virus culture - Madin-Darby Canine Kidney cells (MDCK). ATCC CCL34.

· Inactivated virus, goat serum to A/Term/South Africa/61/H5, and chicken pooled serum to A/Goose/Hong Kong/437-4/99.

· WHO influenza reagent kit: reference antigens and reference antisera.

· Receptor destroying enzyme (RDE)[3].

Highly pathogenic avian influenza A (H5N1) is classified as a select agent, and culturing of clinical specimens for influenza A (H5N1) virus must be conducted under laboratory conditions that meet the requirements for Biosafety Level (BSL) 3 with enhancements. These enhancements include controlled access double-door entry with change room and shower, use of respirators, decontamination of all wastes, and showering out of all personnel. Laboratories working on these viruses must be certified by the U.S. Department of Agriculture. 4 recommends that virus isolation studies be conducted on respiratory specimens from patients who meet the above criteria only if requirements for BSL 3 with enhancements can be met[3].

3. Polymerase chain reaction - Gene primers from Hong Kong, Government Virus Unit.

All laboratory results for influenza A/H5N1 should be confirmed by a WHO collaborating center for influenza or by another WHO- recommended reference laboratory. The WHO reference laboratories are as below:

1. Queen Mary Hospital, University of Hong Kong.

2. National Influenza Center, Kowloon, Hong Kong.

3. National Institute of Infectious disease, Tokyo, Japan.

4. National Institute of Medical Research, UK.

5. Department of Infectious disease, Memphis, USA.

6. Centers for Disease Control and Prevention, Atlanta, USA[2].

Clinical specimens from suspect influenza A (H5N1) cases may be tested by PCR assays under standard BSL 2 conditions in a Class II biological safety cabinet. In addition, commercial antigen detection testing can be conducted under standard BSL 2 conditions used to test for influenza[3].

The range of antiviral drugs is small, but especially so when it comes to bird flu. Four different influenza antiviral drugs (amantadine, rimantadine, oseltamivir, and zanamivir) are approved by the U.S. Food and Drug Administration (FDA) for the treatment of influenza; three are approved for prophylaxis. All four have activity against influenza A viruses. (4). Two of them, amantadine and rimantadine, are ineffective against H5N1. The other two are zanamavir (commercialized as Relenza) and the widely-stockpiled oseltamivir, commercialized as Tamiflu. These medications are called neuraminidase inhibitors, which block the virus from replicating. If taken within a couple of days of the onset of illness, they can ease the severity of some symptoms and reduce the duration of sickness[6].

No definitive vaccine against the viral threat is available, because no-one knows the precise shape that it will take after mutating to the feared highly contagious form. Several prototypes are being explored, but the risk is that they could be only partially effective or even useless because the virus' genetic shape will have changed and will not be recognized by antibodies. If a pandemic does occur, one worry is about manufacturing capacity and distribution: making enough of the vaccine and getting it on time and to the right people, without causing panic or a black market or leaving poor countries helpless[6].