Swine flu virus - Early and definitive confirmatory detection

Dr Ranil Dassanayake

Swine influenza refers to influenza caused by any strain of the influenza virus endemic in pigs (swine). Strains endemic in swine are called swine influenza virus. Swine flu is a highly contagious respiratory disease of pigs, caused by one of several swine influenza A viruses.

Three genera of influenza viruses, Influenza virus A, Influenza virus B and Influenza virus C are known to cause human flu. Off these, in swine, Influenza virus A is common and Influenza virus C is rare; and Influenza virus B has not been reported in swine. However, within Influenza virus A and Influenza virus C, the strains endemic to swine and humans are largely distinct. Swine influenza virus comes under the genera of Influenza virus A, which is the common cause of flu in human.

Genetic materials

Influenza viruses are made up of an outer envelop and inner viral core (nucleocapsid/ a region encapsulated by viral envelop), where the genetic materials of viruses are located. Viral envelop is made up of proteins and in influenza A, viral envelops are made up of two proteins namely, hemagglutinin (Ha) and neuraminidase (NA). Influenza A is a RNA virus and has a segmented genome/ genetic material consisting eight different linear RNA strands (PB1, PB2, PA, NS (NS1 & NS2), NP, and M (M1& M2)genes, in addition to HA and NA genes).

RNA viruses have a very high rate of mutation, a short generation time and yield a high production of virions after replication in the host’s cells. Influenza A’s most advantageous feature is its segmented genome and these strands can combine with each other and create novel genotypes, an important mechanism for extremely rapid adaptation to new hosts.

All influenza A viruses contain hemagglutinin and neuraminidase, but the structure (amino acid sequence) of these proteins differs from strain to strain due to rapid genetic mutation in the viral genome.

The rapid mutation to the virus is brought about by viral enzyme called reverse transcriptase, which synthesizes genetic materials in the production of viral progeny in the host cells. Influenza A virus strains are assigned an HA number and an NA number based on which forms of these two proteins the strain contains. Number assigned to HA and NA depends on the structure (amino acid sequence) of hemagglutinin and neuraminidase proteins.

Swine influenza

Therefore, Influenza A virus strains could be categorized according to the structure of above mentioned viral coat proteins. Swine influenza is known to be caused by subtypes H1N1, H1N2, H3N1, H3N2 and H2N3. In swine, three influenza A virus subtypes (H1N1, H3N2, and H1N2) are circulating throughout the world. In the United States, the H1N1 subtype was exclusively prevalent among swine populations before 1998; however, since late August 1998, H3N2 subtypes have been isolated from pigs.

The 2009 flu outbreak in humans that is widely known as “swine flu” is believed to be triple reassortants, containing genes from human, swine and avian viruses [It is reported Mexican swine flu virus is a mixture of four different strains, North American swine flu virus, North American avian flu, human influenza A (H1N1) flu virus (first identified 1998) and a swine flue strain found in Asia and Europe].

Emergence of reassortants is a frequent phenomena in influenza viruses and is driven by the segmented genome and takes place during coinfection (when a cell becomes infected by two/three different viral strains at the same time). During coinfection, different segments of each virion can recombine, thus creating a new viral strain emergence that is sometimes even more detrimental than the original parent viral strains.

The formation of reassortants can take place recombining genetic materials of viral strains residing in three different species, such as human, swine and avian and also two or more viral strains residing in the same species. It is believe that the pig is the mixing vessels of influenza viral strains living in different species of hosts in the formation of reassortants as pigs have receptors for avian and mammalian viruses.

Protective mechanisms

The influenza virus can constantly change their genetic materials and subsequently the structure of their proteins, thereby virus can evade the protective mechanisms (antibodies) that have developed in humans in response to previous exposures to influenza or to influenza vaccines.

Every two or three years the virus undergoes minor changes. However, every 10 to 15 years, after the bulk of the world’s population has developed some level of resistance to these minor genetic changes, influenza undergoes a dramatic genetic change (antigenic shift), often infecting hundreds of millions of people whose antibody defenses are unable to resist for new genetic changes.

This antigenic shift which causes mass infections is responsible for epidemics/pandemics. The influenza virus has also been known to change form over a much shorter period of time.

For instance, during the Spanish flu pandemic, the initial wave of the disease was relatively mild, while the second wave of the disease a year later was highly lethal.

HINI virus, which caused the 1918 flu pandemic had reportedly killed 50-100 million people worldwide. In 1957, an Asian influenza flu pandemic infected some 45 million Americans and killed 70,000. It caused about 2 million deaths globally. Eleven years later, lasting from 1968 to 1969, the Hong Kong flu pandemic afflicted 50 million Americans and caused 33,000 deaths, costing approximately $3.9 billion. In 1976, about 500 soldiers became infected with swine flu over a period of few weeks.

Immunity

Scientists believe the swine flue virus is enormously transmissible and genetics of the virus are so novel that humans are unlikely to have much immunity to it, other than some cell-mediated immunity that acquired by the previous exposure to influenza viruses.

The current seasonal flu vaccine, which targets a different H1N1 strain is also unlikely to offer any protection. WHO is currently considering the production of a new vaccine and according to them it may take at least a six months.

Currently, virus is treatable with the influenza drugs oseltamivir (Tamiflu) and zanamivir (Relenza).

However, the most important questions yet to be answered; such as will the genetic make up of the swine flu virus mutate rapidly to be untreatable by the antiviral drugs currently available and also how soon can an effective vaccine be produced against the virus. These and many more mysteries of the swine flu requiring rapid and definitive answers will be unraveled with the use of high-tech Molecular Biology tools.

Since vaccines are still under development, early and reliable screening is a key factor for efficient surveillance and effective response strategies, especially with the availability of antiviral influenza treatment.

The molecular analyses of swine flu virus offer tremendous information about the biology and genetic make up of the virus. For instance genetics of the virus, i.e. triple reassortment came into light just by unraveling the nucleotide sequence of viral genome. The virulence of the virus could also be predicted to a certain extent using the information obtained from the nucleotide sequence.

Molecular diagnose

In addition, this information can also be used to develop an efficient and reliable molecular diagnostic assay for the virus. Influenza diagnostics using Reverse Transcription followed by viral quantification with Real Time Polymerase Chain Reaction are considered to be faster and more reliable than other methods such as immunodiagnostics due to the high sensitivity and specificity and also rapid turnaround time.

The question on many minds these days would be whether Sri Lanka has such facilities to carry out influenza screening and if so where?

The molecular diagnostic lab of the Durdans Hospital of Sri Lanka is equipped with equipments required for molecular analyses of the virus and has been analyzing other human viruses for the last two years and therefore, if the necessity arises Durdans Hospital will be able to offer the services of a Molecular Detection and Quantification assay to screen the causative agent of swine flu.

However, since the virus is highly contagious, in order to prevent the threat of infection, the processing of viral samples (Nasal washings, aspirates, nasopharyngeal swabs) need to be carried out at a P4 level facility.

(The writer is a Senior Lecturer in Biochemistry and Molecular Biology, Department of Chemistry, University of Colombo and Visiting Consultant Molecular Biologist, Durdans Hospital, Colombo)