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High morbidity and mortality in influenza are seen especially among those at the extremes of age (elderly and very young), those with underlying health conditions and pregnant women.29 Underlying health conditions especially associated with an increased risk for complicated influenza are immune-compromised individuals, either due to the underlying disease, or to immunomodulatory treatment, like organ transplant recipients and those taking medication for autoimmune conditions.30 Furthermore, chronic pulmonary disease31, diabetes mellitus, cardiovascular disease and malignancies are also considered risk factors for developing severe influenza or complications.32
Even a relatively mild, self-limiting seasonal influenza virus infection can have drastic impact on the success of a holiday or business trip. Furthermore, travel of athletes to international contests and artists to performances abroad or social gatherings like weddings can be ruined by influenza. Among the well-recognized examples spread through the media are the German national football team and coach during the 2010 World Cup in South Africa and a famous rock star of a concert that had to be cancelled as he got influenza.33–35
A large study of influenza virus infection in persons travelling to tropical and sub-tropical countries found that 1.1% of the travellers enrolled in the study seroconverted and that 40% of those who seroconverted had sought medical attention during their travel: a highly significant number.25 Influenza virus infections were acquired largely from Asia (47.5%), Africa (27.5%) and Latin America (25%).25
It is important to note that, independent of travel, seasonal influenza outbreaks have been repeatedly associated with poor outcomes even in patients without co-morbidities: including small numbers who develop severe and even lethal influenza as well as life-threatening complications. Examples include patients with severe viral pneumonia, acute respiratory distress syndrome (ARDS), post-influenza Staphylococcus aureus infection with a potential Methicillin-resistant S. aureus infection or rare examples of myocarditis and encephalitis.32,36,37 It is now generally accepted that even healthy individuals have a low but important risk of developing severe influenza-associated disease.2 Furthermore, there is a significant economic burden of seasonal influenza due to sick leave, medical care and medication. Unexpected medical events while travelling can be particularly expensive.
Anti-viral drugs are thought to be backbone of a management plan of an avian flu pandemic. Only two anti-viral drugs have shown promise in treating avian influenza: oseltamivir (Tamiflu®) and zanamivir (Relenza®). A treatment of Tamiflu® includes 10 pills taken over five days while Relenza® is administered by oral inhalation. The US Food and Drug Administration has approved both anti-viral drugs for treating influenza but only Tamiflu® has been approved to prevent influenza infection. Because antivirals can be stored without refrigeration and for longer periods than vaccines, developing a stockpile of antivirals has advantages as part of an overall strategy to control a flu epidemic. However, there are limitations to the use of antivirals: Tamiflu® needs to be taken within 2 days of initial flu symptoms for it to be effective, but many people may not be aware that they have the flu early in the disease. Some research in animals and recent experience in the use of the drug to treat human cases have also found that Tamiflu may be less effective against the recent strains for the current H5N1 virus than the 1997 strain. Improper compliance to antivirals by irresponsible individuals during an outbreak may results in the emergence of a drug-resistant strain. Lastly, there are current concerns about the safety of Tamiflu® which has been associated with increased psychiatric symptoms among Japanese adolescents.
A multitude of scientific and technological advances have occurred over the past century, allowing for a greater understanding of the dynamic relationship between the host and influenza viruses during infection. These advances, along with access to autopsy samples and the reconstitution of the 1918 pandemic virus, have facilitated a greater understanding of how the pandemic virus differs from other seasonal and pandemic influenza virus strains. Moreover, technological advancements following the 1918–1919 influenza pandemic virus have facilitated the development of preventative measures, including vaccines and antivirals, to limit widespread illness due to influenza infections.
The determination of the genomic sequence of the 1918 pandemic virus, and the subsequent reconstruction of the virus, has provided us with the opportunity to decipher the viral- and host-specific properties that contributed to the severity of the 1918–1919 pandemic. It has been demonstrated that in contrast to other influenza viruses, the 1918 pandemic virus is highly virulent and pathogenic in multiple animal species without prior adaptation [45, 50]. While obvious knowledge gaps remain, in particular with respect to the origin of the virus and the molecular mechanisms (host and/or viral) underlying differential pathogenesis as compared to other influenza viruses, there have been considerable advances in our understanding of the 1918 pandemic virus.
Since the isolation of the first human influenza virus in 1933, researchers have worked to develop an effective influenza vaccine. Current influenza vaccines are reformulated seasonally and provide protection against circulating influenza A and B viruses. The World Health Organization conducts worldwide surveillance studies throughout the year on currently circulating influenza strains, and thus recommends which strains should be included in each influenza vaccine. While the seasonal influenza vaccine is approximately 60% effective, this protection is dependent on the characteristics of the individual being vaccinated, including age and overall health, as well as the match between the strains included in the vaccine formulation and currently circulating strains. Individuals who have been vaccinated are generally protected from illness and provide a measure of protection for those who are not able to be vaccinated due to their age or other health issues through herd immunity. There has also been increasing interest in the development of “universal” influenza vaccines designed to provide protection against a wide range of antigenically-distinct influenza viruses, including those currently in circulation and those that may emerge in the future. These will not be discussed in detail as recent reviews have provided excellent discussions of this topic [51–57].
Two major classes of antivirals have emerged for therapeutic treatment of severe influenza virus infections. Adamantane antivirals target the matrix-2 (M2) surface protein, while neuraminidase (NA) inhibitors target the NA viral surface protein. Adamantane compounds were the first licensed influenza antivirals and block the M2 ion channel protein from properly functioning, thus effectively blocking membrane fusion [58, 59]. Unfortunately, adamantane antivirals are only able to target influenza A viruses limiting their application for influenza B virus infections. Further, more than 90% of influenza A viruses are resistant to this class of drugs due to the high mutation rate of the virus [58, 60]. Thus, the use of NA inhibitors is recommended. NA inhibitors block the NA surface protein and prevent the release of progeny virus and infection of additional cells. While resistance to NA inhibitors has been observed in some influenza virus strains, they are still highly effective in the majority of patients. Studies have shown that both adamantane antivirals and NA inhibitors provide protection against the 1918 virus.
Although outside the auspice of this commentary, it should be mentioned that advances in mechanical ventilation modalities, including non-invasive positive pressure ventilation, from the 1950s onwards, have provided an additional support mechanism for treatment of severely ill patients. The routine clinical use of antibiotics in the early twentieth century also heralded a new era for combating influenza viruses. As a testament to this, excess influenza mortality declined significantly from 1942 to 1951 onwards [61–63]. However, the widespread general administration of antibiotics has resulted in an escalating public health crisis due to multi-drug resistance. This has impacted the treatment of severe influenza infections, as methicillin-resistant S. aureus (MRSA) is the most frequently isolated bacteria from patients with severe influenza-bacterial co-infections in the US [64, 65] and complicated up to 55% of fatalities during the 2009 pandemic [66–69].
Classically, fatal influenza infections are primarily associated with the very young (< 5 years) and the elderly (> 65 years) resulting in a characteristic “U”-shaped mortality curve (Fig. 3). Interestingly, however, the 1918–1919 H1N1 influenza pandemic mortality curve exhibits a “W”-shape due to excess mortality in young adults 20–40 years of age due to influenza-related illness. It has been postulated that the increased disease severity in young adults was likely associated with immune status due to the lack of pre-existing immunity in this population. Further, more than 99% of fatal infections occurred in those < 65 years of age and nearly 50% of all influenza-related deaths during the 1918 pandemic were in those aged 20–40 years. Influenza and pneumonia fatality rates in those aged 15–34 years were more than 20 times higher than in previous years and absolute risk of influenza-related death was higher in those < 65 years of age than those > 65 years old. It is still not fully understood why this occurred, but it is possible that an antigenically similar influenza strain circulated prior to 1889, providing a level of protection against the novel H1N1 pandemic strain to those born prior to 1889. Additionally, archaeserological and epidemiological evidence have shown that an H3 subtype influenza virus may have been responsible for the 1889 influenza pandemic, which circulated until the emergence of the 1918 pandemic virus, leaving those individuals who had not been exposed to an H1 subtype virus highly susceptible to the pandemic virus. It has also been suggested that the generation of an excessive inflammatory response (“cytokine storm”) in healthy, young adults infected with the 1918 virus may have contributed to the excess mortality seen within this age group. Recent in vivo studies with the 1918 virus have shown a marked upregulation of inflammatory cytokines, along with the suppression of important antiviral immune responses [34, 45]. In addition, other influenza strains, such as fatal H5N1 infections in humans, have also been associated with the deleterious consequences of an excessive inflammatory response. Ultimately, the case fatality rate was so severe in young adults during the 1918–1919 pandemic that the average life expectancy rate in the US dropped by ~ 12 years.
Physiological symptoms of the 1918 pandemic virus generally lasted for 7 days and were described as feeling cold, shivering, high fever, weakness, nausea, loss of appetite, pharyngitis, cough, and bloodshot eyes. In some patients, a short “rebound” to normal health would occur that was followed by an aggressive recrudescence of disease and, ultimately, death. Similar to the 1889 pandemic, the majority of fatal infections resulted from respiratory complications. However, it has also been demonstrated that excess influenza fatalities during the 1918–1919 pandemic were associated with an acute aggressive bronchopneumonia (including epithelial and vascular necrosis, hemorrhage, edema, and bacterial-associated variant pathology within the lungs) and a severe acute respiratory distress-like syndrome associated with severe facial cyanosis.
Autopsies performed on preserved lung tissues in the modern era have revealed acute pulmonary hemorrhage and secondary bacterial infections associated with pulmonary lesions in nearly all the fatal cases examined [41, 43, 47]. Streptococcus pneumoniae was present in many cases; however, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pyogenes also appeared to complicate fatal cases [48, 49]. Neutrophilic pulmonary infiltration was seen in cases of pneumococcal pneumonia, while cases of staphylococcal pneumonia were marked by multiple microabscesses infiltrated by neutrophils. However, alveolar cell damage was seen in each case along with pulmonary repair and remodelling. Tissues from each of the fatal cases examined had similar pathologic presentation, independent of which pandemic wave they were associated with. Despite the difference in mortality rates, each wave showed similar cellular tropism, infecting both type I and type II pneumocytes, as well as the bronchiolar respiratory epithelium.
Influenza viruses from waterbirds can cross the species barrier and infect numerous other species (see Fig. 1). A recent example of bird-to-animal transmission is the mortality amongst harbour seals [Phoca vitulina] of the North-European coastal waters following infection with the LPAI H10N7 virus,,. Various outbreaks of LPAI H3, H4 and H7 viruses causing severe respiratory disease and mortality amongst harbour seals have also occurred in the past decades along the New England coast of the United States of America,,,. The exact transmission route between seals is unknown but it is likely to occur via the respiratory route, most probably whilst the seals are resting on land. It is currently unknown if adaptation of influenza viruses from waterbirds is needed to allow the virus to infect and transmit amongst seals. In addition to seals, LPAI viruses have been isolated from a long-finned pilot whale [Globicephala melas] and Balaenopterid whales (species unknown),, and serological evidence for infection was reported in various other marine mammal species (for review see). However, the available data is very limited and it is remains unclear if LPAI viruses can also cause outbreaks of disease in other marine mammals similar to harbour seals.
Waterbirds and shorebirds of the orders Anseriformes (mainly ducks, geese and swans) and Charadriiformes (mainly gulls, terns and waders) are considered the natural host reservoirs of LPAI viruses (see Fig. 1). In wild birds LPAI viruses predominantly infect epithelial cells of the intestinal tract, and are subsequently excreted in the faeces. However, infection of wild birds with LPAI viruses is typically sub-clinical and occurs in the absence of obvious lesions,,. Every year, LPAI viruses cause outbreaks amongst waterbirds. These outbreaks are most commonly associated with the increased presence of juvenile, immunologically naïve birds in the population and occur during migration when contact rates between, and within, populations are high. The relatively high virus prevalence in waterbirds may be due, in part, to virus transmission through the faecal–oral route via surface waters.
Rare cases of humans infection with avian influenza A viruses have been reported, with the exception of highly pathogenic avian influenza A (H5N1) viruses, which have caused 667 infections and 393 deaths as of June 27, 2014. In 2003, 89 people were infected with influenza A virus of H7N7 subtype that caused conjunctivitis and one fatality in Netherlands. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype A have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of preexisting immunity among humans to viruses of this subtype. Avian influenza A (H7N9) is a subtype of influenza viruses that have been detected in birds in the past. This particular H7N9 virus had not previously been seen in either animals or people until it was found in March 2013 in China. Since then, a total of 450 cases with 165 deaths have been reported, and infections in both humans and birds have been observed. The disease is of concern because most patients have become severely ill. Most of the human cases with H7N9 infection have reported recent exposure to live poultry or potentially contaminated environments, especially markets where live birds have been sold. This virus does not appear to be transmitted easily from person to person, and sustained human-to-human transmission has not been reported [32, 33].
“H7N9 influenza infection” was listed as a Category V Notifiable Infectious Disease starting from April 3, 2013, in Taiwan, and on April 24, three weeks later, Taiwan CDC announced that a 53-year-old man who had recently traveled to China is hospitalized in critical condition with a novel H7N9 infection; it is the first case of H7N9 detected outside of China [34, 35]. Taiwan CDC continued to follow up on 139 people who had had contact with the patient, and none of them was positive for the H7N9 virus. The Center for Research and Diagnostics of Taiwan CDC took another preparedness step by developing diagnostic test to detect this new virus and issued a technical briefing to the contracted virology laboratories for screening H7N9 infections. The materials are also available on the Taiwan CDC's website. So far the source of the virus appears to be birds, especially poultry and the environment contaminated by the virus, and the risk of infections seems most concentrated in live-poultry markets. Though a few family clusters have been found, experts found no evidence to conclude that person-to-person transmission is occurring and that no sustained transmission has been found. However, it is noted that limited person-to-person transmission is demonstrated in China.
Overall, 291 of the 4337 vaccinated individuals returned the questionnaire and reported adverse reactions. This is a rate of 6.7%. The majority of reported adverse reactions was found in the age between 30 and 39 years. (Figure 2)
The most frequently reported local site reactions were: local pain/pruritus or the sensacion of heat at the injection site in 3.8% out of the 4337 vaccinations, myalgia or arthralgia in 3.7%, induration or erythema at the injection site in 2.6%, lymph node swelling in 0.9%, skin rash in 0.3% and ecchymosis at the injection site in 0.1% (Figure 3).
The presence of systemic adverse reactions were reported as follows: fatigue in 3.7%, headache in 3.1%, flu-like symptoms in 2.3%, shivering in 1.9%, temperature > 38°C in 1.3%, dizziness in 1.1%, increased perspiration in 1.1%, gastrointestinal symptoms in 1.0%, drowsiness in 0.9%, , insomnia in 0.7%, formication in 0.3%, Further some severe reportable adverse reactions were observed (0.5%, Figure 3) as one case of facial nerve paralysis, one case of rheumatoid arthritic symptoms and one case of skin alteration which was reported to the local health authorities and the Paul-Ehrlich-Institute.
Currently, the H5N1 avian flu virus is limited to outbreaks among poultry and persons in direct contact to infected poultry. Avian influenza (AI) is endemic in Asia where birds often live in close proximity to humans. This increases the chance of genetic re-assortment between avian and human influenza viruses which may produce a mutant strain that is easily transmitted between humans, resulting in a pandemic. Unlike SARS, a person with influenza infection is contagious before the onset of case-defining symptoms. Researchers have shown that carefully orchestrated of public health measures could potentially limit the spread of an AI pandemic if implemented soon after the first cases appear. Both national and international strategies are needed: National strategies include source surveillance and control, adequate anti-viral agents and vaccines, and healthcare system readiness; international strategies include early integrated response, curbing disease outbreak at source, utilization of global resources, continuing research and open communication.
Whether the selection of the D222G mutation is a cause or a consequence of more severe lower respiratory tract infection is still to be resolved. It is evident, however, that its emergence is likely to exacerbate the severity of disease. The altered receptor specificity and distinctive cell tropism of the D222G mutants of influenza A(H1N1)pdm09 are hallmarks of a more dangerous pathogen, emphasizing the importance of close monitoring of the evolution of these viruses. Influenza A(H1N1)pdm09 variants with 222 G/E polymorphisms showed increased clinical virulence, and detection of such mutants in the next epidemics is mandatory for better management of ILI in individual patients as well as for surveillance purposes especially in African countries.
There were several zoonotic influenza strains that infected poultry and human during the past decade in Taiwan (Figure 2). In April 2009, a new strain of H1N1 from Mexico was found to be a novel strain of influenza for which current vaccines against seasonal flu provided little protection. This new H1N1 strain resulted from a reassortment of bird, swine, and human flu viruses. In June 2009, the WHO declared the outbreak of a pandemic which was named as pandemic H1N1/09 virus (pdmH1N1) in July 2009. It is possible that the virus has been circulating in human population since some time in the past and had not been detected.
The first case of H1N1 detected in Taiwan was confirmed on May 20, 2009, and another 9 positive cases in a row were identified within a week. The pdmH1N1 virus was isolated in late May 2009, causing a community outbreak in early July and then spreading islandwide. Taiwan CDC provided free seasonal influenza vaccine started from October; people received their vaccination at over 3,500 contracted hospitals and clinics. Then the Taiwan government approved an inactivated vaccine with influenza A/California/7/2009 (H1N1) strain known as AdimFlu-S (Adimmune Corporation, Taichung, Taiwan) and initiated mass vaccinations in November. Free AdimFlu-S vaccines were provided giving the priority to schoolchildren, the elderly, and front-line healthcare personnel. Due to the delay in vaccine development and delivery in 2010, health authorities used both vaccination and school closure to control pdmH1N1 [22–24]. Overall, the vaccine coverage rates were 76.9% for children and 24.6% for civilians in late July 2010 [22, 24, 25].
Confirmed case of pdmH1N1 influenza virus infection in Taiwan is defined as an individual with laboratory-confirmed pdmH1N1 influenza virus infection by one or more of the following tests: (1) real-time reverse transcriptase-polymerase chain reaction (RT-PCR), viral culture, or fourfold rise in pdmH1N1 influenza virus-specific neutralizing antibodies; (2) viral culture and (3) RT-PCR which can reliably identify the presence of pdmH1N1 influenza virus in specimens, especially RT-PCR, which has the highest sensitivity and specificity. With RT-PCR, pdmH1N1 influenza virus will test positive for influenza A and negative for seasonal H1 or H3. Meanwhile, commercially available rapid influenza diagnostic tests (RIDTs) detect influenza viral nucleoprotein antigen and are capable of providing results within 30 minutes. The sensitivity of RIDTs for detecting pdmH1N1 influenza varied from 10% to 70% and is directly related to the amount of virus in the specimen but inversely related to the threshold cycle value of the test. Rapid tests for influenza may detect the antigen from either influenza A or B in respiratory specimens with a high specificity (>95%), but the negative result from a rapid test does not rule out influenza infection, and most of rapid tests cannot distinguish pdmH1N1 from H3N2 influenza A viruses.
The oseltamivir-resistant pandemic influenza A (H1N1) virus strain in Taiwan was first isolated from a 20-year-old male in October 2009. The H1N1 virus isolated before the patient received oseltamivir treatment was sensitive to oseltamivir. However, three days after initiation of treatment, the virus isolated from the same patient shows a mutation involving the substitution of a histidine for tyrosine at position 275 (H275Y), which is resistant to oseltamivir. Oseltamivir treatment was not associated with statistically significant reduction in the duration of viral shedding, thereby proving ineffective in preventing viral spread in community. The role of swine in the genesis of this pandemic was again apparent. The lesson learned is that the pandemics of influenza can arise anywhere in the world and that global surveillance is merited.
The mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days.
Influenza, also known as the flu, is a respiratory illness caused by viruses belonging to the family Orthomyxoviridae. This family consists of four influenza virus genera (influenza virus A, influenza virus B, influenza virus C, and influenza virus D) that are classified based on differences in their internal glycoproteins nucleoprotein (NP) and matrix (M). Influenza type A viruses can infect humans, birds, pigs, horses, and other animals, while influenza B and C viruses are found only in humans. Influenza viruses contain a single stranded negative sense RNA genome that encodes 11 proteins. Based on the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are divided into various subtypes. There are 18 HA (H1–H18) and 11 NA (N1–N11) subtypes of influenza A viruses, that potentially form 144 HA and NA combinations. Aquatic birds including ducks, geese, and swans, are considered to be the natural reservoir of these subtypes.
Each year influenza viruses, both influenza A and influenza B are responsible for seasonal epidemics accounting for over 200,000 hospitalizations and 30,000–50,000 deaths. As per World Health Organization (WHO) estimates, influenza viruses infect between 5%–15% of the global population, annually resulting in 250,000 to 500,000 deaths, making it the leading cause of mortality after acquired immune deficiency syndrome (AIDS). In addition to annual seasonal epidemics H1N1 and H3N2 viruses have also resulted in four major influenza pandemics: The “Spanish flu” in 1918, the “Asian flu” in 1958, the “Hong Kong flu” in 1968, and the more recent 2009 H1N1 pandemic. Since 1997, human infections with a novel H5N1 subtype of highly pathogenic avian influenza (HPAI) have been reported. The first cases of human infection with H5N1 influenza were reported in 1997, when HPAI outbreaks in poultry farms and markets in Hong Kong resulted in eighteen cases and six deaths. Since then, this virus has spread to many countries in Asia, Africa, and Europe, resulting in over 424 human cases with a mortality rate greater than 60%. In addition, H9N2 and H7N7 avian influenza subtypes have also been reported to cause human infections. The most recent strain infecting humans was H7N9 in China, detected in 2011.
A number of diagnostic techniques, including virus isolation, nucleic acid amplification test (NAAT), immunochromatography-based rapid diagnostic test (RDT), etc., have been used for detection of influenza viruses in humans. Here, we review various approaches currently available or under development for diagnosis of influenza infections in humans.
The continuous spread of highly pathogenic avian influenza Type A (HPAI) H5N1 viruses in avian species across multiple continents and frequent reports of human H5N1 infection in China and Southeast Asia highlight the threat of a potential flu pandemic in the human population. At the same time, H5N1 viruses have grown into genetically and antigentically diversified viruses. Based on phylogenetic analysis of hemagglutinin (HA) protein gene sequences, at least 10 clades of H5N1 viruses (clades 0–9) have been identified,,,,. Recent studies have further assigned these viruses into four major antigenic groups (A–D). HPAI H5N1 viruses from more than one clade have caused human infection since 1997.
A key component in the global strategy to prepare for and control any pending influenza pandemic is the development of an effective vaccine. Several versions of inactivated as well as live attenuated H5N1 vaccines have been tested in humans and showed an overall good safety and immunogenicity profile mainly by using a clade 1 H5N1 virus (A/Vietnam/1203/04) as the vaccine strain per recommendations by the World Health Organization (WHO),,. Given that the majority of the world's human population is naïve to H5N1 influenza, two immunizations are needed to achieve desired levels of protective immune responses against H5N1 in contrast to the annual seasonal flu vaccine which requires only one immunization, presumably due to the priming effects by either exposure to circulating H1, H3 or Type B influenza viruses in humans or history of prior seasonal flu vaccination. The likely requirement of two immunizations in conjunction with the genetic complexity of H5N1 viruses, as evidenced by their separation into multiple subgroups, makes it difficult to prepare for the timely production of a sufficient number of doses of H5N1 vaccines in the event of an H5N1 pandemic; therefore, supplemental strategies are needed. As shown by our previously published report and confirmed by other recent studies, a DNA prime-inactivated vaccine boost is highly effective in eliciting higher protective immune responses than using either DNA or inactivated flu vaccine alone. Therefore, it may be possible to use DNA vaccines as the first dose of immunization that can be given either long before the pandemic (pre-pandemic vaccination) or shortly after the outbreak, to reduce the burden on the production of inactivated vaccines at the time of the outbreak. Furthermore, DNA vaccines can be stockpiled for a long period of time, which makes this method even more attractive.
One key issue that needs to be analyzed for the above strategy is the cross reactivity between DNA vaccines expressing H5 HA antigens from different clades. It is critical to first optimize the immunogenicity of H5 HA DNA vaccines and then to test how much cross protection can be achieved with optimized H5 HA DNA vaccines. In the current report, we constructed DNA vaccines to express wild type HA antigens without mutations at the HA1 and HA2 cleavage site from four key H5N1 strains that have caused major human infection: HK/156/97 (clade 0), VN/1203/04 (clade 1), Ind/5/05 (clade 2.1), and Anhui/1/05 (clade 2.3). Rabbit sera immunized with these HA antigens were examined for their protective antibody responses against either homologous or heterologous H5N1 viruses. Our results demonstrated an imperfect cross-reactivity profile for the protective antibody responses among these four viruses. A polyvalent formulation including three different H5 HA DNA vaccines was able to produce broad protective antibody responses with high titers against these key H5N1 isolates. Information learned from this study should facilitate the selection of candidate H5N1 vaccines to form polyvalent H5N1 DNA vaccines as part of the global strategy to prevent and control a potential avian flu pandemic.
The WHO Vaccine Composition Meeting for the 2014-2015 season held in February 2014 in Geneva approved that A/California/7/2009 (H1N1)pdm09-like virus, A/Texas/50/2012 (H3N2)-like virus, and B/Massachusetts/2/2012-like virus should be included in the trivalent vaccine formulation in the Northern Hemisphere.52 Consequently, the aforementioned novel avian strains are out of scope of the current vaccine formulation indicating the severity of the situation if the H7N9 influenza has global dissemination.
Laboratory testing in preliminary cases showed that neuraminidase inhibitors (oseltamivir, zanamivir) were effective against H7N9 infections, but the adamantanes were not. In addition, early treatment with neuraminidase inhibitors have been reported to restrict the severity of illness.34,53
When an outbreak comes to an end, it is a matter of great concern. According to the WHO, an EHF outbreak in a country is reported to be over when 42 days have passed, and new cases have not been observed. The maximum incubation period for EHF was 21 days. The 42-day period was set by the WHO (twice the maximum incubation period) to provide a strong margin of security.27 Currently, the USA, Spain, Mali, Senagal, and Nigeria are categorized as affected countries.21 Currently, EHF epidemic appears to be ending after the slowing of transmission.22
RIDTs are antigen-based tests developed for rapid diagnosis of influenza virus infections in POC settings. These tests use monoclonal antibodies that target the viral nucleoprotein and employ either enzyme immunoassay or immunochromatographic (lateral flow) techniques. Available in dipstick, cassette, or card formats, RIDTs can be completed in less than 30 min, with the results observed visually based on a color change or other optical signals. Due to simplicity in their use and the speed of obtaining assay results, RIDTs are commonly used for the diagnosis of influenza infections.
Several FDA-approved RIDTs are currently available on the market. Most of these tests can either detect or distinguish influenza A and B viruses, detect only influenza A viruses, or both influenza A and B viruses (but cannot discriminate influenza A and B). However, none of the RIDTs can distinguish between the different influenza A subtypes. Performance of RIDTs is dependent on the prevalence of circulating influenza viruses in the population. During peak influenza activity, positive predictive values are high and false positives are, therefore, likely to be observed. However, during low influenza prevalence, negative predictive values are high, with low positive predictive values. RIDTs have generally demonstrated high specificities (95%–99%) for the detection of seasonal influenza virus infections.
For diagnosis of seasonal influenza infections, RIDTs have demonstrated variable assay performance with sensitivities ranging between 10%–70%, with up to 90% specificity compared to standard RT-PCR-based assays. Performance of RIDTs have been shown to be better in children compared with adults (approximately 13% higher), potentially due to higher viral loads and longer viral shedding in children compared with adults. A meta-analysis of 159 studies involving 26 commercial RIDTs showed a sensitivity of 62.3% compared to RT-PCR approaches for diagnosis of influenza infections (both seasonal and pH1N1 virus infections). In this study, the performance of RIDTs for the detection of influenza A viruses was higher compared to influenza B viruses (percentage sensitivities were influenza A: 64.6% and influenza B: 52.2%).
During the 2009 H1N1 pandemic, RIDTs demonstrated sensitivities ranging between 10%–70% compared with RT-PCR-based assays. Using the BinaxNOW rapid antigen-based assay (Inverness Medical, Cologne, Germany), Drexler et al. reported an assay sensitivity of 11.1% while testing 144 PCR-positive clinical samples from Bonn, Germany. Early during the pandemic, a large study from New York reported 9.6% and 40% sensitivities using RIDTs BinaxNOW Influenza A&B test (BinaxNOW), 3M Rapid Detection Flu A+B test (3MA+B) compared to R-Mix culture. The low sensitivity of RIDTs during the pandemic could be attributed to poor sample quality and inexperience of the lab workers. A Centers for Disease Control and Prevention (CDC) study evaluating the performance of three different RIDTs (BinaxNOW Influenza A&B, Directigen EZ Flu A+B, and QuickVue Influenza A+B) for the detection of the pH1N1 virus had reported assay sensitivities of 40% for BinaxNOW Influenza A&B, 49% for Directigen EZ Flu A+B, and 69% for QuickVue Influenza A+B compared to a RT-PCR-based assay. Due to a high rate of false negatives, the CDC advised physicians not to discontinue antiviral therapy despite negative RIDT results. In another study, the QuickVue Influenza RIDT assay (Quidel, San Diego, CA, USA) showed an assay sensitivity of 51% in comparison with a PCR assay. Another study compared a RIDT QuickVue Influenza test with the RT-PCR-based assay and reported an assay sensitivity of 66% with 84% specificity. The positive and negative predictive values in this study were 84% and 64%, respectively. A study comparing RIDT and cell culture approaches with a multiplex respiratory viral assay (Luminex xTAG), reported a combined assay sensitivity of 17.8% for the Binax NOW Influenza A+B (Inverness, Scarborough, ME) and the 3M Rapid Detection Flu A+B (3M Medical Diagnostics, St. Paul, MN, USA). While testing nasopharyngeal aspirates from 970 young children, one study reported 84.1% sensitivity using a RIDT, QuickVue Influenza A+B ICT test (Quidel Corp., San Diego, CA, USA) compared with a viral isolation method.
For detection of avian influenza A viruses, RIDTs have demonstrated lower sensitivity compared RT-PCR-based approaches. The FDA recently approved AV Avantage A/H5N1 Flu RIDT developed by Arbor Vita Corporation, (Sunnyvale, CA, USA) for H5N1 detection. This test uses a combination of monoclonal antibodies and PDZ (Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), Zonula occludens-1 protein (zo-1)) domain containing recombinant proteins to detect NS1 protein from throat swabs and can be completed in 45 min Although RIDT have demonstrated variable sensitivity, they still remain the test of choice in most clinical virology laboratories around the world due to the speed in obtaining results, simplicity in assay procedure, and cost.
The presence of all subtypes influenza A viruses in wild aquatic birds poses serious health risks to a wide range of animal species. Influenza A viruses are enveloped, single-, negative-stranded and segmented RNA viruses belonging to the Orthomyxoviridae family; they are highly infectious respiratory pathogens in their respective natural hosts. All 16 known hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A viruses have been isolated from wild waterfowl and seabirds (Webster et al, 2006). Although some of these subtypes are non-pathogenic/non-virulent within their natural hosts and have been present in these animal reservoirs for many centuries, various subtypes are highly virulent within their natural host species and to other species (Webby et al, 2007). For example, the changing role of the highly pathogenic avian influenza virus (HPAIV) H5N1 subtype in both wild and domestic ducks has recently been documented as a potential public health hazard because they are zoonotic agents with the theoretical ability – after genetic adaptation – of a human-to-human transmission, i.e., the start of a human epidemic/pandemic (Hulse-Post et al, 2005).
The ecology of influenza A viruses is dynamic and complex involving multiple host species and viral genes. Commercial poultry farms, “wet markets,” (where live birds and other animals are sold), backyard poultry farms, commercial and family poultry slaughtering facilities, swine farms, human dietary habits and the global trade in exotic animals have all been implicated in the spread of influenza A viruses (Greger, 2006). The “wet markets” of Southeast Asia, where people, pigs, ducks, geese and chickens (and occasionally other animals) are in close proximity pose a particular danger to public health (Webster, 2004; Bush, 2005; Greenfeld, 2006; Lau et al, 2007).
Scientific opinion differs on the probability of future sustained human-to-human transmission, e.g., for H5N1 HPAIV, as well as on which viruses pose the greatest threat to humanity and to other species (Hilleman, 2002). The prevailing scientific view regarding a possible H5N1 epidemic is that sustained human-to-human transmission will occur at some unknown future date and that a prediction on the future virulence of H5N1 viruses to humans is very difficult to make. The H5N1 HPAIVs are possibly the greatest threat at the moment, although H1, H2, H3, H7 and H9 avian-derived viruses are also strong contenders as causes of potential epidemics in various species, including humans (Hilleman, 2002; Wan et al, 2008). This review will conclude that an avian influenza virus transmitted via pigs to humans poses a significant risk to cause a new influenza pandemic, possibly on the disturbing scale of the human influenza pandemic experienced during 1918-1920 (H1N1 “Spanish Flu”). Before investigating the role of swine in the influenza A viruses, it is necessary to consider the challenge which influenza A viruses pose to both human and animal health.
Genetic analysis of HA of influenza A (H1N1)pdm09 virus showed that this virus was a reassortant containing gene segments from ancestor viruses of human, swine and avian sources. Hence, Polymorphism at position 222 within the haemagglutinin (HA) molecule may have remarkable impact on viral host range, replication, and pathogenicity. It is worth noting that although the Asp222Gly mutation currently has not been associated with severe pandemic in humans.
An association between D222G and severity was initially proposed by Kilander et al. (2010) and, since then, different studies in several countries have found the D222G substitution to be more frequently associated with patients with severe pandemic influenza than in non-severe control cases. A recent study supported a role for this mutation in allowing the virus access to deeper lung tissue. Hese unusual viral attributes suggested that this new virus may possess some virulence characteristics similar to the highly pathogenic H5N1 or the 1918 pandemic influenza viruses.
This study used a conventional sequencing approach to analyze 50 H1N1pdm samples obtained from 2009 to 2011. Viruses with D222G substitution in HA protein have appeared sporadically and spontaneously in Tunisia since July 2009. Although, 7% of them, found in severe cases in the present study, had the D222G substitution. This percentage is comparable to that found in Italy (4%), in United Kingdom (6%), in France (8%) or in Spain (5%), and lower than that published in Norway (18%), and in Hong Kong (17.4%). These differences with the Norwegian and Chinese study might be due to the reduced number of severe cases analyzed in that country. In Tunisia, this mutation was observed in circulating virus obtained from severe cases and also from mild cases. The frequency of D222G substitution is higher in severe cases than mild cases. Although most of studies demonstrated the presence of D222G substitution in severe cases, it was also reported in mild cases. Therefore, the number of mild cases would need to be larger to determine whether mutant viruses are indeed circulating at a very low frequency also in non-severe cases.
The 222 G/E polymorphisms in the haemagglutinin (HA) gene of influenza A(H1N1)pdm09 virus have been associated with cases of mild to severe illness from different countries or geographical areas. Many retrospective analyses have found that cases bearing the D222G mutation were more likely to be associated with severe pneumonia, admission to intensive care facilities, and death. The majority of studies have reported that presence of D222G is sufficient to enhance virus replication and lethality in mouse models, with this effect ranging from modest to pronounced. Moreover, D222G substitution had been present in the Tunisian virus strain since pandemic season and throughout 2010–2011 season. Other groups have not observed substantial differences between wild-type and D222G viruses in mouse or ferret models, indicating the need for further investigation into the role of D222G in virulence of influenza A (H1N1)pdm09. The D222E was detected with less frequency than the D222G and only found in severe case patient. Despite the available virological, epidemiological and clinical information, the D222E substitution could confer more severity to the disease. The clinical significance of this mutation is still unclear. All above studies demonstrated that polymorphism of the HA protein, especially within the receptor binding domain, play a critical role in the binding preference and pathogenicity of influenza A(H1N1)pdm09 virus. Further study is warranted to elucidate the intriguing relationship between D222G substitution and severe disease.
Influenza viruses are known to constantly evolve and cross species barriers. The genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. The purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. In addition, we discussed the growing complexity of influenza virus–host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses.
With the aim of improving prevention and control of viral outbreaks, the Chinese government has been investing continually in the advancement of science and technology since 2003, including the appropriation of more than 12 billion RMB for research and development related to combating SARS, influenza, and other major infectious diseases. Meanwhile, China has built 11 national technology platforms, 11 national research centers, 6 national key laboratories, and 2 national engineering laboratories. In 2010, the Chinese National Influenza Centre was designated as a WHO Collaborating Centre for Reference and Research on Influenza. All these laboratories and funding contributed to application of advanced technologies in preventing and controlling infectious diseases.
Above all, quick identification of pathogens is a prerequisite to controlling emerging epidemics. To achieve it, China has developed state-of-the-art pathogen isolation and identification technologies such as high-throughput sequencing method. In contrast to the SARS-Cov debacle, H7N9, H10N8, and H5N6 were identified within China [28–30]. BGI, a Chinese company, helped Germany sequence the pathogen Escherichia coli O104:H4 within a week using high-throughput sequencing technology in 2011. Meanwhile, Chinese researchers exploring the genesis and source of emerging viruses have found that bats are natural reservoirs of SARS-like coronaviruses and have demonstrated that domestic fowl play an important vector role for H5N1 and H7N9 [4, 32, 33].
The government encourages the development of diagnostic reagents, vaccines, and medicines as well as prophylactic equipment (e.g., infrared thermometers). China's national vaccine regulatory system was confirmed to meet WHO standards in 2011. China has developed SARS, H5N1, H1N1, and H7N9 vaccines (Table 1) and became the first country to use an H1N1 vaccine. China now produces oseltamivir (like Tamiflu®) and peramivir (like Rapivab®), obviating the need to import antivirals.
China's improvements in research funding and technical capabilities have led to a series of important findings. For example, Chinese researchers have revealed the crystal structures of key viral proteins (e.g., SARS-Cov protease, H1N1 neuraminidase N1, and H5N1 polymerase PAC-PB1N complex) [36–38], which is useful for drug design, and discovered an oseltamivir-resistance mechanism in H7N9. A traditional Chinese medicine (TCM) herbal formula was confirmed to reduce H1N1 influenza-associated fever safely and with efficacy similar to that of oseltamivir in a randomized clinical trial.
There are sixteen recognized serological subtypes of type A influenza virus hemagglutinin (H1 through H16) and 9 type A neuraminidase subtypes (N1 through N9). Among the combinatorial diversity of 144 possible A/HN subtypes, relatively few subtypes have been identified as causes of human disease. Four pandemic outbreaks in the last century, one catastrophic, appear to have introduced the subsequently prevalent seasonal human influenza virus subtypes A/H1N1 (Spanish flu, 1918), A/H2N2 (Asian flu, 1957), A/H3N2 (Hong Kong flu, 1968), and A/H1N1 again (Swine flu, 1976; Russian flu, 1977). The current year 2009 has been marked by a late season pandemic-scale emergence of a novel A/H1N1 outbreak strain, raising immediate concerns for public health as well as for pork and poultry production industries worldwide.
As with the few common subtypes of human type A influenza viruses, there are similarly few subtypes of type A influenza viruses that are associated with most influenza infections of swine, horses or dogs. In distinct contrast, wildfowl species are natural hosts and a global reservoir for the majority of possible influenza A/HN subtypes. Many of these variant strains appear to be associated with endemic infections, often asymptomatic in avian hosts. Incidental infections of humans by avian influenza viruses have been documented for avian influenza subtypes A/H5N1, A/H7N2, A/H7N3, A/H7N7, A/H9N2, A/H10N7 and A/H11N9. Recent outbreaks of “bird flu” may foreshadow an eventual pandemic outbreak, in the emergence of strains and variants with enhanced pathogenicity, virulence and transmissibility in human hosts. Examples of such outbreaks include A/H5N1 Hong Kong, 1997; H9N2 Hong Kong, 1999; A/H7N7 Netherlands, 2003; A/H5N1 Southeast Asia, 2004. Some avian A/H5 and A/H7 strains of influenza virus are recognized as highly pathogenic (HP) in domestic poultry and concerns arise that this phenotype may carry over to infections of humans. Since 1997, human infections associated with the Eurasian-African lineage of A/H5N1 HP avian influenza virus have been associated with 467 documented cases in 15 countries with high mortality (282 deaths) [2; updated 30 December 2009].
Fortunately, infectious transmission of such avian influenza virus strains between humans continues to be limited. However, history suggests that further evolution of these or other type A influenza strains could emerge as a next pandemic strain. Similarly, variant type A influenza virus strains have emerged from time to time, imposing serious costs and burdens upon poultry and livestock production.
Because the natural history and the molecular biology of influenza viruses reflect such viral genome diversity, there is a critical need for rapid, sensitive, specific, and informative assays to detect and characterize any subtype of influenza virus. Benchmark standard methods that employ propagation of virus in cell culture or in embryonating chicken eggs, with assays using panels of specific serological reagents, or reverse transcriptase polymerase chain reaction (RT-PCR)-based assays, using panels of short oligonucleotide primers and probes, are either slow and time consuming, or expensive. As prevailing strains of avian influenza continue to evolve and diverge, diagnostic assays that are based only on specific recognition of short signature sequences or peptide biomarker loci will increasingly fail, through false-positive and/or false-negative results. This will adversely impact critical decision-making.
This report describes a re-sequencing pathogen microarray (RPM)-based assay for simultaneous detection, identification and characterization of any subtype of type A human or avian influenza virus, based on rapid, sensitive and specimen-specific determination of nucleotide sequences from viral hemagglutinin, neuraminidase, and other genes.
Influenza is only one of the more than 200 infectious diseases transmitted between humans and animals (McMichael, 2005; Childs et al, 2007; Ellis, 2008). Given the 30% increase of newly emerging zoonotic diseases in the final third of the twentieth century, it may well be that HIV/AIDS, Ebola/Marburg/Zaire virus, West Nile virus, Severe Acute Respiratory Syndrome virus or some other new pathogens will pose a greater threat than influenza to either the human or animal kingdoms (Greenfeld, 2006; Ellis, 2008). Yet the presence of a high prevalence avian reservoir for influenza indicates that the threat of influenza is significant, and that human and animal health must be viewed as an integrated network (Gibbs, 2005; Martinot et al, 2007; Childs et al, 2007). This is critical as public health and veterinary science is moving towards the “one world, one health” concept (Enserink, 2007).
Avian influenza infections of commercial poultry already represent the largest incidence of an animal disease ever recorded, with several hundred million wild birds, geese, chickens, turkeys and ducks having died from the virus or been culled as part of the HPAIV control program (McKenzie, 2006). Furthermore, HPAIVs will remain endemic in wild birds in Asia and other parts of the world, whatever attempts are made by the authorities in these regions of the world to mitigate them. What is in question is the extent to which HPAIV will spread to humans and if so, how lethal it will be.
To date, the major attention has focused on the capacity of H5N1 HPAIVs to infect and kill humans. In investigating the January 2004 H5N1 outbreak in Vietnam, Hien et al. (2004) noted that only humans with very close contact to infected poultry died. In addition, it has been suggested that those people who become infected with H5N1 had an immune constitution particularly susceptible to the H5N1 virus (Albright et al, 2008). Such a hypothesis is based on evidence that only a few of the people reporting extensive exposure to H5N1-diseased chickens actually become ill. In contrast, Pitzer and his colleagues (2007) could not find any evidence of human genetic susceptibility to H5N1 viruses. The crucial issue is whether a human influenza virus and an avian or animal (e.g., swine) influenza virus might reassort to create a novel reassortant virus with the capability of sustained human-to-human transmission. It appears unlikely that the wild bird/domestic duck/ chicken/human link in itself will soon create a new pandemic because after 11 years of an ongoing epidemic transmission (starting with 1997 Hong Kong outbreak), only limited human-to-human transmission has occurred. It could take decades before the “correct, i.e., human-adapted” mutations occur in the H5N1 influenza viruses (Normile, 2006; Shinya et al, 2006; van Riel et al, 2006). Swine might play a critical role as a “mixing vessel” in this evolutionary process.