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The evolving threat of influenza viruses of animal origin and the challenges in developing appropriate diagnostics.

Influenza is a contagious respiratory disease responsible for pandemics and seasonal epidemics, and it thereby imposes a considerable economic burden through loss of productivity and the costs of associated medical treatments. Because of the relatively frequent zoonotic transmissions of influenza viruses from avian poultry to humans, attention had been primarily focused before 2009 on avian influenza viruses (e.g., H5, H7, and H9 subtypes, which have the potential to cause pandemics) (1). Nature, however, caught us by surprise in that the first pandemic of this century was caused by a swine virus that is of the same subtype as human seasonal H1N1 virus (2). This occurrence also reflects the highly unpredictable nature of the influenza A virus. In this review, we discuss the nature of influenza viruses and the challenges of developing molecular diagnostic tests for detecting new pandemic strains.

Influenza Viruses

The influenza viruses belong to the family Orthomyxoviridae (3). There are 3 types of influenza viruses: A, B, and C. These 3 types exhibit different degrees of antigenic variations, host specificity, and pathogenicity. Type A virus undergoes much faster evolution and shows more antigenic variation than the other 2 types. Influenza B and C viruses usually infect humans and they are not discussed further in this article. By contrast, type A virus infects a wide range of avian and mammalian species. The influenza A viral genome contains 8 RNA segments encoding at least 10 viral proteins. These segments have been numbered according to length, in descending order. The fourth and sixth viral segments encode hemagglutinin [(HA).sup.2] and neuraminidase (NA) surface viral glycoproteins, respectively. Influenza A viruses are classified into different subtypes depending on the nature of the HA and NA proteins that they carry. Aquatic fowls are believed to be the natural reservoirs for all influenza A viruses. There are 16 HA and 9 NA subtypes found in avian influenza viruses. Recently, an influenza virus with novel HA and NA subtypes was detected in bats, suggesting that the diversity of animal influenza virus is larger than previously thought (4). Of these HA and NA subtypes, only H1N1, H2N2, and H3N2 viral subtypes are known to have established stable lineages in humans since the last century (5).

Influenza pandemics occur at unpredictable intervals and are associated with high infection attack-rates of variable severity. There have been 4 influenza pandemics over the last 100 years, namely, the Spanish flu in 1918, the Asian flu in 1957, the Hong Kong flu in 1968, and the recent pandemic H1N1 in 2009. All of these pandemics were initiated by the spread of an antigenically novel HA from animal sources to humans. The most catastrophic among these 4 pandemics was the 1918 pandemic, which cost more than 40 million lives and had a fatality rate of about 2.5%. The other 2 pandemics in the 20th century caused about 1 million deaths each. The recent 2009 pandemic was 3-4 orders of magnitude milder, with case fatality in Hong Kong ranging from 0.03% in older adults to as low as 0.0004% in children 5-14 years old (6). Although the 2009 pandemic is considered to have been a mild one, more than 50% of Hong Kong school-going children were found to have been infected by the pandemic H1N1 during the first wave of attack--just 5 months after the pandemic alert was issued and long before vaccines were available in adequate quantities. A pandemic of higher severity (e.g., H5N1) may cause globally catastrophic impacts to human and economic health. The World Bank has estimated a pandemic with virulence comparable to that of the 1918 Spanish flu will negatively impact the global gross domestic product by 4.8% and lead to the loss of over 70 million lives in the pandemic year alone (7).

Animal Reservoirs as a Source for Influenza Pandemics

Except for the bat influenza virus, all mammalian influenza viruses, such as equine, swine, and human influenza viruses, are believed to have evolved from avian influenza viruses. Owing to the nature of their genome, influenza viruses can exchange their gene segments in coinfected cells, thereby generating progeny viruses with new genotypes. These gene reassortment events play a key role in the evolution of influenza A virus and have direct impacts on human health (8). The huge viral gene pool in the avian population is believed to be responsible for the genesis of pandemic viruses. Sequence analysis of the 1918 pandemic H1N1 virus suggested that all 8 gene segments of this virus might be of avian origin (9). However, other investigators have suggested that this virus might be a reassortant between avian and mammalian influenza viruses (10). By contrast, it is widely accepted that the pandemic human H2N2 and H3N2 viruses were reassortants between avian and human seasonal influenza A viruses. For the 1957 pandemic, a previously circulating H1N1 human strain incorporated the avian influenza A virus genes hemagglutinin (HA) (subtype H2), neuraminidase (NA) (subtype N2), and polymerase 1 (PB1). Similarly, avian HA (subtype H3) and PB1 genes were introduced into a H2N2 human virus and caused the H3N2 pandemic in 1968. The H1N1/2009 virus is a product of multiple reassortments between avian, swine and human influenza viruses (11). The HA, nucleocapsid protein (NP), and nonstructural protein (NS) genes are in the classical swine lineage and the NA and M genes are in the avian-like Eurasian swine H1N1 lineage. The polymerase 2 (PB2), PB1, and polymerase PA (PA) genes of pandemic H1N1/2009 are from the North American swine triple H3N2 reassortant virus in which the PB1 gene originated from human seasonal H3N2. This complex genotype suggests that the ancestors of this virus might undergo multiple gene reassortments in pigs. These sequencing results stronglysuggest that a swine H1N2, closely related to the North America H3N2 triple reassortant, might be one of the precursor viruses. However, due to insufficient surveillance of swine influenza viruses, exact reassortment events are yet to be investigated (11).

There is a host barrier to prevent avian influenza from infecting humans. Many avian viruses grow poorly or even are noninfectious to humans (12). With a few exceptions like highly pathogenic avian H5N1 viruses, direct spread of influenza from birds to humans is uncommon. The control of the host restriction is a polygenic trait, but the receptor-binding specificity of influenza is one of the major determinants. In general, avian influenza HA prefers to bind to [alpha]-2,3-linked sialic acid (SA), which is prevalent in duck intestines, whereas human influenza HA prefers to bind to [alpha]-2,6-linked SA, which is highly expressed in the upper respiratory tract of humans. Hence, the SA binding preference of the virus and the SA expression pattern of the host have great influence on viral tropism and host specificity. Interestingly, the swine respiratory tract possesses receptors for both avian and human influenza A viruses. Experiments involving infections of many avian and human influenza viruses have also demonstrated that pigs are susceptible to influenza viruses of zoonotic origins (13). Pigs are, therefore, proposed to be the mixing vessels that can support coinfection, replication, and reassortment among human, avian, and swine viruses. Apart from their role in the pandemic H1N1/2009, pigs are believed to have functioned as an intermediate host for H2N2 and H3N2 pandemics. In addition to these outbreaks, zoonotic transmissions of swine influenza viruses to humans have also been reported sporadically (14). Of current particular concern are the recent human cases caused by a novel reassortant of pandemic H1N1/2009 and swine H3N2 viruses (15). Hence, reassortments of influenza virus in pigs might have direct implications for human health.

Although the last pandemic has reiterated the importance of pigs for the genesis of pandemic strains, one should not overlook the role of poultry. Direct zoonotic transmissions of influenza viruses from poultry to humans (e.g., H5N1, H7N7, and H9N2) have been detected. In some of these events, limited human-to-human H5N1 transmissions were also detected. Furthermore, some of these avian viruses might require only a few mutations to adapt to mammalian hosts in experimental settings. Thus, it is possible that a new pandemic strain may directly emerge from an avian host.

Influenza research over the last 3 decades has identified several factors to help assess whether a particular animal virus might have a pandemic potential (16). These risk factors include: the prevalence of the virus in livestock; its ability to bind to receptors in the human airway; its ability to grow in human cells/organs; its airborne transmissibility between ferrets (the best animal models for studying human transmission because they can transmit influenza viruses and develop clinical signs similar to those seen in humans including fever, rhinitis, and sneezing); and its ability to reassort with human influenza viruses. Functional hemagglutinin-neuraminidase balance has been suggested to facilitate the transmission of viruses by droplets (17). Viruses with a history of frequent zoonotic transmissions to humans and with surface glycoproteins that can escape from herd immunity in humans are also classified into the high-risk category. In addition, viruses that either carry known human-adapted mutations or associate with severe disease outcomes are also considered to be capable of causing a pandemic threat.

It can never be overemphasized that a great majority of human infections caused by animal influenza viruses are associated with viruses circulating in livestock. It is very likely that yet another pandemic strain will emerge from these populations. For better influenza pandemic preparedness, it is extremely vital to have a comprehensive picture of the animal influenza viruses circulating in both animal and human populations. For this reason, the World Organization for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO) jointly established an international network, the OFFLU (OIE/FAO Network of Expertise on Animal Influenza) ( to support and coordinate global efforts to prevent, detect, and control influenza viruses in animals. In addition, the WHO has a Global Influenza Surveillance and Response System (GISRS) ( for monitoring both human and animal influenza viruses in humans, and one of the collaborating centers in this network is particularly focusing on the ecology of animal influenza viruses. In 2010, the FAO, OIE, and WHO jointly issued a tripartite concept note to strengthen the concept of "One World, One Health" (

Detection of Influenza Viruses

Influenza surveillance requires the monitoring of circulating virus strains using diagnostic methods. It is the first stage of controlling an outbreak because efficient diagnosis can minimize spread. In a clinical or field setting, rapid detection of influenza allows physicians and scientists to initiate prompt treatment, implement infection control strategies, decrease health costs, and reduce risks to healthcare workers and the wider community. Diagnostic tests are frequently evaluated and developed for improved sensitivity and reliability, because the influenza virus is constantly evolving. For a more comprehensive review on the molecular diagnostic tests for influenza virus, readers are encouraged to read recent the excellent reviews contributed by others (18-21).

There are several methods for diagnosis of influenza and each is used depending on the available resources and context. Clinical signs are commonly used to establish a tentative diagnosis. However, this diagnosis must be confirmed with specific tests, because the manifestations of disease are variable and could be a result of other viral or bacterial infections. Clinical onset is characterized by high fever, cough, headache, malaise, and inflammation of the upper and lower respiratory tract. These symptoms can last for several days. Complications in more susceptible demographic groups such as the elderly include pneumonia, hemorrhagic bronchitis, and death. Viral replication peaks at 24 h, and after 6 days there is little viral shedding. This poses one of the most important challenges for diagnostics, because individuals rarely present themselves to a doctor in the early stages of illness.

Diagnostic tests include serological tests, virus isolation, rapid antigen tests, and molecular tests (18-21). Serological methods rely on the development of an antibody response that can take several days to develop, and in some cases the disease is so rampant that an antibody response is not even formed. Influenza virus is known to replicate in Madin-Darby canine kidney cells, and a cytopathic effect can be identified after 2-3 days. This virus isolation can take several days and must be performed in a biosafety level 2 (seasonal influenza) or above (highly pathogenic or pandemic influenza viruses) facility. This process also allows propagation of the virus for further investigation, such as sequencing and biological characterization.

Other nonnucleic acid methods detect a component of the virus. Rapid antigen assays include commercially available point-of-care tests. These tests utilize commercial antibodies against influenza antigens. They can produce a result within 20 min but are relatively expensive and have variable performance. Another drawback is that many of these tests indicate only the presence of influenza and do not provide information about subtype, although some H5N1-specific rapid diagnostic tests are becoming available. Recently, rapid antigen tests for subtyping human H1 and H3 and avian H5 have been reported. The performance of these tests is yet to be improved (22). Nonetheless, rapid antigen tests are useful in a clinical setting and can expedite the decision to administer antiviral treatment.

Among diagnostic tools available for detecting influenza, molecular tests are the most sensitive and rapid. These tests can be performed with high throughput at a moderate cost. These tests amplify target nucleic acids to allow identification. Molecular tests include reverse-transcription PCR (RT-PCR), real-time RT-PCR, nucleic acid sequence-based amplification, loop-mediated isothermal amplification (LAMP), microarray, and pyrosequencing. As a prelude to a pandemic, molecular diagnosis of influenza, in particular, should be harmonized globally. In a pandemic scenario there will be a sudden increase of samples to be tested, causing pressure for faster turnaround and the need for accurate and cost-effective results. Molecular diagnostic techniques show the most potential to meet these challenges.

RT-PCR is a technique widely used in laboratories to amplify RNA, and the necessary primer sequences are often publicly available. An extension of RT-PCR is real-time RT-PCR, for which quantitative results can be generated in real time. Real-time RT-PCR is the most sensitive, informative technique yielding rapid results, with the only drawback being that the reagents and start-up cost are high. Recent studies have indicated that viral loads might associate with disease outcomes, which suggests that the quantitative results might be a useful prognostic indicator in some clinical cases. The RT-PCR method uses fluorescent dyes or probes to detect the amplicon; several platforms are available, such as SYBR green, Taqman, and molecular beacons. The systems for RT-PCR and real-time RTPCR mainly detect the M gene, which is highly conserved, for influenza A virus detection. Primers and probes designed to detect influenza subtypes target conserved regions of the HA gene. However, these subtyping assays are normally designed for viruses with known significance to human health.

Multiplex PCR is a modification of PCR for which several primer sets are used concurrently to detect the presence of several gene targets. This method is useful in a diagnostic laboratory where multiple pathogens in a single sample can be investigated. For example, a multiplex PCR assay system to detect H5N1 and other human respiratory pathogens has been developed (23).

PCR-EIA is an alternative method that identifies PCR amplicons by use of a biotinylated RNA probe through an enzyme immunoassay (24). This method has higher sensitivity than the traditional RT-PCR methods, but it was developed only for detection of the M gene and limited HA subtypes. In addition, there is an added cost of the enzymatic reagents.

Nucleic acid sequence--based amplification also employs reverse transcriptase to generate a cDNA (25). The cDNA is synthesized by a primer containing a promoter for the T7 polymerase. The second primer attaches to the single-stranded cDNA and the reverse transcriptase synthesizes a second strand of DNA. The reaction also contains RNase H to destroy the original RNA template. The T7 polymerase binds to the double-stranded DNA to generate a complementary RNA that acts as a template as the reaction continues in a cyclic fashion. This isothermal method does not require expensive PCR instrumentation, but this approach might be limited by RNA secondary structure, which sometimes makes primer design and multiplexing difficult.

The LAMP assay is also a nucleic acid--amplification method that uses reverse transcriptase and DNA polymerase in a single-step isothermal reaction (26,27). The approach requires more extensive effort for primer designs and assay optimizations, but the method has been explored for several reasons. Specifically, it is a technique that is compliant with the challenges of influenza surveillance in places with limited resources. The LAMP assay is an alternative method that is rapid with high specificity to a target and can be used in a front-line clinical laboratory without the need for expensive, complex equipment and specialized staff.

Recently, several studies have demonstrated the feasibility of using microarrays for the detection and subtyping of influenza A viruses (28). In addition, some of these arrays could be used to differentiate highly pathogenic H5N1 from less pathogenic H5N1 viruses (29). Pyrosequencing is another new technique that provides high-throughput screening of influenza viruses (30). Assays for antiviral resistance marker detection have been developed. These approaches would be useful for conducting influenza surveillance.

All molecular assays are limited by primer and probe design and RNA quality. Target sites are subject to mutations that reduce primer specificity. Therefore, molecular assays must always be evaluated and, if necessary, revised. Sample quality is also an important factor, because tests show differential detection limits depending on the origin and quality of the sample. Despite an optimized methodology for molecular assay, there are several points at which errors can occur. Sample handling and storage, nucleic acid extraction, enzyme transcription fidelity, enzyme inhibition, and contamination are all critical points for optimal detection of influenza virus.

Presently, there is no standardized universally acceptable influenza detection technique (either in poultry or in humans) that can allow easy comparison of surveillance studies in different countries. Because the resources and technical skills available in each situation are highly variable, it is unlikely that a consensus detection method will emerge. For the time being, the WHO has recommended techniques that are available on its website.

Apart from providing guidelines and technical support for influenza diagnosis, GISRS monitors human influenza activities around the world throughout the year. Representative and/or important viruses isolated from these surveillance activities will be further characterized in various WHO collaborating centers under this network. In particular, it is recommended that novel and/or unsubtypable influenza viruses be immediately referred to appropriate reference laboratories under GISRS for further characterization (31). Information from these activities is critical for laboratory diagnostics, vaccines, antiviral susceptibility, and risk assessment.

Challenges for Pandemic Preparedness and the Next Pandemic

The 2009 pandemic might have helped further strengthen the capacity for diagnosing influenza in many diagnostic laboratories. Standard assays for influenza detection and human influenza subtyping are normally well established for use in clinical diagnostic laboratories. Although several viral subtypes are recognized as viruses of high pandemic potential, our existing knowledge is not sufficient to provide an accurate prediction of the next pandemic strain. Clinical diagnostic assays specific for HA subtypes that have a history of infecting humans have been established. These assays, however, are entirely based on known HA sequences. Some of these assays are viral-lineage specific and their usefulness is limited to regional applications. Hence, the question arises, are we really ready for the next pandemic that may emerge unpredictably? Do regional or routine diagnostic laboratories have the capacity to analyze unusual influenza viruses in humans? Is it possible for us to achieve early detection of novel human influenza viruses once they have started to emerge in humans?

PCR-based detection assays specific for each HA and NA subtype have been established to detect viral sequences in animal specimens (32, 33). These assays would be useful to analyze unsubtypable human influenza A viruses. The amplicons generated from these assays are usually large, and the products would be extremely useful for downstream characterization (e.g., sequencing). However, the ability to amplify long fragments in these assays is often compromised by the assay sensitivity. Although these assays would be useful to detect isolated virus cultures, they might not be highly suitable for clinical practice. Recently, Tsukamoto et al. (34) reported the development of SYBR green-based real-time RT-PCR for subtyping of all HA and NA genes of avian influenza viruses. These assays were evaluated with an extensive number of avian viruses of different subtypes. In addition, a great proportion of avian fecal specimens positive for influenza A viruses were correctly subtyped by these assays. However, it is not known how well these assays work with human clinical samples. In addition, there is uncertainty about whether these assays are able to detect viruses circulating in nonavian species. Nonetheless, the development of a panel of diagnostic tests that can detect all HA and NA subtypes should be encouraged. Ideally, these panels should cross-react with all animal, but not human, influenza viruses. These assays should be robust and easy to establish in basic clinical laboratories.

Because the above HA- and NA-subtype--specific primers are not guaranteed to cross-react with the corresponding HA and NA subtypes, alternative strategies that may be helpful for characterization of unsubtypable human influenza viruses should also be considered. The M and NP genes of influenza viruses are highly conserved; several universal RT-PCR assays for these 2 genes have been established. Sequencing information deduced from these amplicons can sometimes help to partly reveal the origins of some unsubtypable viruses. Although sequence data generated from these universal assays might allow the development of more specific primers for virus detection, these assays are unable to specify the HA and NA subtypes.

In practical terms, universal primers that can cross-react with all HA subtypes would provide additional advantages for developing tests that are more specific for newly identified human influenza viruses (35, 36). The identity of these amplicons can be determined by performing sequencing or microarray analysis in a timely manner. In addition, such studies also might provide useful information for developing relevant serological assays for virus detection.

Apart from the above classical PCR-based tests, many other alternative approaches might be used to deduce additional sequencing information of unsubtypable human influenza viruses. Universal primers for full-genome amplifications have been reported (37, 38). The deep-sequencing approach can also provide very comprehensive information. However, because these assays are technically more demanding or require additional diagnostic infrastructure, it would not be easy to implement these tests in general diagnostic laboratories. In addition, one should also consider the cost, time, and testing materials needed for these assays.

Determining the antiviral susceptibility of these novel human influenza viruses would be critical for prompt antiviral treatments. Currently, there are 2 classes of antiviral agents for influenza, M2 ion channel inhibitors and NA inhibitors. These drugs are experimentally effective against most animal influenza viruses. Molecular assays (e.g., real-time RT-PCR assay and pyrosequencing) for specific mutations known to confer antiviral resistance (e.g., H275Y in N1, E119V in N2, and S31N in M2) have been established (39, 40), but these assays are unlikely to be useful for detecting such mutations in a pandemic strain that is yet to emerge. Furthermore, viruses containing resistant mutations located at other positions/segments would not be picked up by these assays. Thus, molecular tests for antiviral resistance should be interpreted with great caution. Currently, phenotypic tests, such as neuraminidase inhibition assays, are still the preferred option for testing viruses that are suspected to have antiviral resistance mutations. This is particularly true for viruses that emerge from animals, because our understanding about animal influenza viruses is not as comprehensive as that for human influenza viruses. In addition, these tests can be done only on virus cultures, where some virus mutants might be selectively enriched through virus propagation in Madin-Darby canine kidney cells. Therefore, data obtained from these assays may not always represent the true nature of the original specimen.

Molecular tests that allow rapid genotyping of influenza viruses would also be useful to detect reassortants between human and animal viruses (41, 42). Reassortment events should be closely monitored by real-time surveillance, because novel reassortants may have altered virulence and/or transmissibility. Genotyping assays for influenza viruses in humans and poultry should be encouraged.

Although in this review we have focused only on the challenge of molecular testing of influenza viruses, serological tests are equally important for clinical diagnosis and public health surveillance. In addition, the role of virus isolation is also extremely important for the control of influenza viruses. Much of our understanding of human and animal viruses still relies heavily on basic virus culture techniques. On the other hand, characterization of live infectious virus isolates still plays a key role in our current production of influenza vaccine. Although there are many advantages of using molecular tests for characterizing and detecting influenza viruses, key regional laboratories are encouraged to maintain the basic virological techniques and expertise to strengthen global pandemic preparedness.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: L.L.M. Poon, National Institutes of Health (NIAID contract HHSN266200700005C), the Research Fund for the Control of Infectious Disease Commissioned Project from Food and Health Bureau, Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/06).

Expert Testimony: None declared.


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Polly W.Y. Mak, [1] Shanthi Jayawardena, [1] and Leo L.M. Poon [1] *

* Address correspondence to this author at: Centre of Influenza Research, School of Public Health, LKS Faculty of Medicine, the University of Hong Kong, 21 Sassoon Rd., Hong Kong, People's Republic of China. Fax +28551241; e-mail

[1] Centre of Influenza Research and School of Public Health, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong Special Administrative Region, People's Republic of China.

[2] Nonstandard abbreviations: HA, hemagglutinin; NA, neuraminidase; PB1, polymerase 1; NP, nucleocapsid protein; NS, nonstructural protein; PB2, polymerase 2; PA, polymerase PA; SA, sialic acid; OIE, World Organization for Animal Health; FAO, Food and Agriculture Organization of the United Nations; GISRS, Global Influenza Surveillance and Response System; RT, reverse transcription; LAMP, loop-mediated isothermal amplification.

Received July 4, 2012; accepted August 24, 2012.

Previously published online at DOI: 10.1373/clinchem.2012.182626
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Title Annotation:Mini-Review
Author:Mak, Polly W.Y.; Jayawardena, Shanthi; Poon, Leo L.M.
Publication:Clinical Chemistry
Article Type:Report
Geographic Code:9CHIN
Date:Nov 1, 2012
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