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Another potential preventive strategy for influenza is the use of antivirals either prophylactically or for early treatment. Currently, the most effective anti-influenza drugs are the neuraminidase inhibitors (NIs: oseltamivir, zanamivir and peramivir).49 M2 inhibitors (amantadine and rimantadine) are rarely used since they suffer from rapid development of virus resistance and virtually all currently circulating seasonal influenza A viruses have pre-existing resistance.50 Furthermore, M2 inhibitors do have considerable side effects and are not effective against influenza B viruses. Although influenza viruses can develop resistance to individual NIs quite rapidly, the risk of resistance development to the whole class of drugs is unlikely and lower than with M2 inhibitors, illustrated by the fact that virtually no cross resistance to oseltamivir and zanamivir has been identified.51 For travellers, the NIs may play a role in both pre- and post-exposure prophylaxis, with confirming data coming from both animal models (mouse and ferret) and human trials.52 NIs provide protective efficacy when used preventively in an outbreak situation, or soon after the first clinical symptoms, by reducing the duration and severity of symptomatic influenza.53,54 Furthermore, several 2009 pandemic period observational studies suggest that early treatment can reduce rates of hospitalisation and in-hospital mortality.55 Some consider the use of NIs controversial because almost all published studies have been industry-funded, and the reported effects are generally minor and there have been no large randomized control trials proving efficacy for post-exposure treatment.53,54 However, a recent independent meta-analysis showed that oseltamivir in adults with influenza accelerates clinical symptom alleviation, reduces risk of lower respiratory tract complications, and admission to hospital, while increasing the occurrence of nausea and vomiting.56
Although NIs have relatively mild side effects, their cost and modest efficacy suggest they should play only a limited role in routine pre-travel advice. However, elderly or other high-risk groups in which vaccine efficacy can be low, could be advised to consider bringing a NI for influenza early treatment, if access to medical care at the destination will be limited. Especially since in many countries NI requires a medical script to be purchased and may not be readily available resulting in an unnecessary delay. These drugs could also play a role in mass transportation settings like cruises or group travel. The use of NIs does lead to reduction in disease duration—if used within 48 h after first symptoms—about 1 day—and to reduction in disease severity, although this has been also a matter of debate.46 In specific cases, such reductions may be crucial: e.g. athletes, politicians, scientists and those travelling for business.57 The prophylactic use of NI decreases the chance of being infected.57 To our knowledge, and in light of the recent Olympics in Rio de Janeiro and upcoming sport events, NIs are not currently listed as prohibited substances by the World Anti-Doping Agency (WADA) [https://www.wada-ama.org/en/what-we-do/prohibited-list (26 October 2016, date last accessed)].
The CDC currently suggests that patients infected with avian influenza should be treated with oseltamivir or zanamivir. Furthermore, the curative use of NIs is recommended as early as possible, preferably within 48 h, for patients hospitalized with confirmed or suspected influenza, with severe, complicated, or progressive illness or at high risk for influenza-associated complications (e.g. children <2years, adults ≥65years, nursing home residents, individuals with major co-morbidities) according to CDC guidelines (www.cdc.gov/flu/professionals/antivirals/summary-clinicians.htm). Currently, oseltamivir is FDA registered for influenza treatment for any age and as chemoprophylaxis from 3 months of age. Age cut-offs for zanamivir use are currently 7 and 5 years, respectively. Prophylactic dosing with chloroquine—known to have some non-specific antiviral efficacy in vitro—did not translate into clinical protection in a large randomized controlled community trial during the H1N1 outbreak in 2009 and therefore does not seem to have a role in travel medicine regarding influenza prevention.58
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.
Several studies have addressed the effectiveness of non-pharmaceutical interventions (NPIs) in reducing influenza virus spread, especially in the case of a pandemic. For seasonal influenza, most attention has been focused on hand hygiene and the use of facemasks. These NPI’s may be especially important when someone in the immediate environment or a travel companion is infected.38 For instance, careful hand hygiene and the use of facemasks appear to reduce household transmission of influenza virus when implemented within 36 h of symptom onset of the index patient.39 The general utility of hand hygiene and facemasks in reducing influenza spread has been confirmed by a recent meta-analysis40 although the quality of the data in many studies, particularly when children are involved, is relatively poor.41 Furthermore, in studies that focus on scenarios in which there is active, on-going influenza transmission in the population, like during a pandemic, large variation in effectiveness of these NPIs has been observed. Despite these limitations, there seems to be sufficient evidence to conclude that facemasks, hand hygiene and reduced crowding are effective in reducing the spread of influenza.42 Hand hygiene would be relatively simple for travellers to implement and several studies suggest that the use of alcohol based sanitizers and hand washing after touching contaminated surfaces can be effective.43,44
However, these trials were not conducted in travellers. Facemask usage in travellers is particularly controversial and may only have measurable impact when a close companion (i.e. shared living quarters) is infected.45 In mass gatherings, there seems to be a (very) modest decrease in risk of infection in persons using facemasks.46 Furthermore, the overall effectiveness of masks and respirators is likely dependent on consistent and correct usage.47 In this light, it is important to note that up to 40% of influenza cases may be transmitted prior to the onset of symptoms.48
Severely ill EHF patients require intensive supportive care, which is the mainstay of therapy.19 In October 1, 2014, 2 candidates for EHF vaccines had clinical-grade vials available for phase-1 clinical trials. One of these formulations was cAd3-ZEBOV (GlaxoSmithKline, Raleigh, SC, USA) and recombinant vesicular stomatitis virus - Zaire ebolavirus (rVSV-ZEBOV) (Public Health Agency in Winnipeg, Canada). A series of coordinated phase-1 trials will be initiated in more than 10 sites in Africa, Europe, and North America.25 Although no approved specific therapy is currently available in the treatment of EHF, several drugs such as brincidofovir, favipiravir, and ZMapp are under investigation for EHF. In the current outbreak, convalescent blood and plasma therapies have been used in a few patients. The numbers are too small to draw any conclusions on their efficacy.26
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].
HA, the major surface protein of IAV, mediates the viral binding, membrane fusion and viral entry. Determination of the crystal structures of HA0 provides important information for understanding the pH-induced conformational changes of HA at the pre-fusion, intermediate and post-fusion states for development of anti-influenza drugs. Although a series of anti-influenza drugs targeting the NA and M2 ion channel are currently available, the emergency of drug-resistance viruses has raised the great concern on their ineffectiveness against the newly emerging IAVs and the HPAI viruses. Thus, it is essential to develop novel anti-IAV drugs with new targets. A number of protein-based or small molecule anti-IAV agents have been shown to interfere with the HA-mediated membrane fusion by targeting the receptor binding, blocking the cleavage of HA0, or by inhibiting the low pH-mediated conformation changes of HA. It seems not easy to find or design small compounds targeting the binding event of influenza virus, but the conformational change of HA2 which mediates membrane fusion, is a promising target for developing anti-influenza drugs. Novel influenza virus entry inhibitors may provide more selections for combination therapy with NA inhibitors and M2 ion channel blockers for treating and preventing influenza virus infection and potential pandemic outbreak.
In addition to above inhibitors, some natural molecules also possess good inhibitory activity against influenza A virus infection (Figure 6). For example, catechins isolated from green tea, like EGCG, were found to exhibit mild anti-influenza effect. Further modifications of catechins derived a set of better inhibitors. To be better anti-influenza drug candidates, catechin derivatives possess broad spectrum anti-influenza activity. Besides, curcumin, the widely used spice and coloring agent in Indian food, was proved to be a good virus entry inhibitor targeting HA with EC50 value of 0.47 μM. Modification of curcumin may produce a series of novel HA targeting inhibitors. Another kind of small molecule inhibitor is derived from andrographolide, like AL-1. AL-1 showed significant activity against avian influenza A (H9N2 and H5N1) and human influenza A H1N1 viruses in vitro. AL-1 is capable of direct interfering with viral HA to block viral binding to cellular receptors as was demonstrated by its inhibitory activity on viral adsorption to red blood cells. In 2009, Li’s group in China reported the first three small saponins molecule which inhibit HA. These three compounds can potently inhibit the entry of a H5N1 virus (A/Viet Nam/1203/2004) with IC50at low μM level. Further modifications revealed that the reduction of R1 to a disaccharide chain would abolish the inhibitory activity of these inhibitors. Therefore, anti-influenza agents from natural products, especially those from Traditional Chinese Medicine (TCM), are promising lead compounds. Some lead compounds from TCM are extensively and intensively reviewed by Xu et al. in 2010.
Personal protective equipment is important to prevent transmission of novel A/H1N1 as stated earlier by Shine et al. However, vaccination is the most effective means of preventing influenza transmission and associated morbidity and mortality. It is most important to realize that an effective measure against a pandemic is to have vaccinated and well-informed health care workers.
Unfortunately, the A/H1N1 vaccination coverage was extremely affected by an ongoing public discussion about potential side effects. Therefore, we analysed self reporting questionnaires concerning adverse reactions in 4337 HCWs and medical students.
Of course, this study - that was initiated in the acute event of a pandemic and a safety discussion - has a variety of limitations. Apart from the paucity of demographic data, self reporting questionnaires are largely limited since there may be a number of individuals who do not return the questionnaire despite adverse reaction manifestation. However, there was a need to assess potential adverse reactions since the general population and the HCWs asked for data about the new vaccine. Therefore, we decided to undertake a self reporting study despite that fact that the extent of underreporting of side effects can not be examined precisely in the chosen design. It is noteworthy that with our self reporting system we found a rate of 6.7% (291 of 4337 vaccinations). This frequency differs slightly from data from other studies but points to a safe vaccine in terms of acute adverse reactions. A prospective, randomised study with 178 participants by Vajo et al. concluded that all adverse events were rare, mild, and transient. Using the vaccine Fluval P, the most frequent reactions in this study were pain at injection site (eight cases) and fatigue for 1-2 days after vaccination (three cases). Concerning the vaccination rate we can report a rate of 4337 of about 10 000 employees of the hospital. This is a vaccination rate of over 40%. In a parallel study in Frankfurt/Main, the influenza vaccination rates of the HCWs of the University Hospital Frankfurt were measured. In this study, we were also able to show that the 2009 vaccination rate (seasonal influenza [40.5%], swine flu [36.3%]) was better than the average annual uptake of influenza vaccine in the German health care system (approximately 22% for seasonal and 15% for swine flu).
In meantime, a number of studies were published that also addressed safety issues of the H1N1 vaccination in healthcare workers. I.e. an inactivated, split-virus, unadjuvanted AH1pdm vaccine, manufactured in Japan, was given to HCWs from October 19, 2009. A retrospective cohort study was conducted and and severe adverse events were rare. A recent study using monovalent vaccination (Panenza; Sanofi Pasteur, Val de Reuil Cedex, France) among HCWs in a university hospital setting in Thailand also reported a low rate of side effects. The most common adverse reaction was fatigue/uncomfortable feeling (24%).
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
The mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days.
In the words of the late Director General of World Health Organization, Dr Lee Jong Wook, '"it is only a matter of time before an avian flu virus acquires the ability to be transmitted from human to human, sparking the outbreak of human pandemic influenza...we don't know when this will happen but we do know that it will happen". Factors that suggest that an AI pandemic would be less severe than past influenza pandemics include advances in medicine such as the availability of antiviral medications and vaccines, and international surveillance systems. However, there are also factors that suggest than an avian influenza pandemic could be worse than the 1918 pandemic, such as a more densely populated world, a larger immunocompromised population of elderly and AIDS patients, and faster air travel and interconnections between countries and continents which will accelerate the spread of disease. Nevertheless, unlike the past, we have the prior knowledge of a possible impending pandemic and the knowledge of how to contain and control it. Preparedness, vigilance and cooperation, on local, national and international levels, are our best weapons against a deadly bird flu pandemic.
Influenza A viruses (Family Orthomyxoviridae) impose a large burden on both human and animal health worldwide. Influenza A viruses can be categorised into different subtypes based on genetic and antigenic differences in the two surface glycoproteins of the virus, the haemagglutinin (HA) and neuraminidase (NA). Wild waterfowl and shorebirds are the natural reservoirs of influenza A virus and can be infected with viruses harbouring combinations of 16 different HA subtypes and nine different NA subtypes. Recently, two novel influenza A virus subtypes (H17N10 and H18N11) have been identified in rectal swabs collected from the little yellow-shouldered bat [Sturnira lilium] and the flat-faced fruit-eating bat [Artibeus jamaicensis planirostris],,. Influenza viruses of this subtype have not been isolated from any other animal order and it is unknown whether these viruses might be able to cross the species barrier. In contrast, there is significant inter-species transmission of influenza viruses from waterbirds, such that animals ranging from domestic poultry to humans can also become infected. Accordingly, infection with influenza virus has wide-reaching ramifications. For example, whilst some influenza virus strains are largely asymptomatic in chickens (and are hence referred to as low pathogenic avian influenza [LPAI] viruses) others cause severe disease in chickens that is often fatal within 48 h (and are hence referred to as highly pathogenic avian influenza [HPAI] viruses). Outbreaks of HPAI viruses can cause devastation for the poultry industry resulting in the mass slaughter of millions of birds. Similarly, outbreaks of influenza viruses amongst thoroughbred horses have disrupted numerous race meetings and resulted in the death of infected horses. In humans, seasonal influenza viruses are a significant cause of morbidity and mortality and constitute an economic burden of $10.4 billion dollars per year in the U.S.A. alone. The diversity and complexity of influenza virus infections across so many different animal species suggests that a one-health approach is the only comprehensive way to reduce the burden of disease. Here, we seek to highlight how influenza viruses spread from their natural avian hosts to mammals, and what the virus needs to overcome in order to ensure the success of these inter-species transmission events. We highlight the consequences that this inter-species transmission has, not only for human health, but also for the health of wild animals and the success of industries such as poultry farming.
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.
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.
This is an immunodiffusion-based approach used to measure antibody responses to influenza virus internal proteins NP and M, following vaccination or infection. Due to lower sensitivity, complement fixation has been replaced by HAI, VN, and EIA assays.
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.
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.
Our multiplex RT-LAMP method not only can minimize the use of expensive lab instruments and devices, but also can detect broad-spectrum human influenza viruses and avian influenza viruses infecting humans as accurately and more rapidly than conventional RT-PCR-based detection methods suitable for use in on-site testing. This method will improve and aid in the diagnosis of influenza infections and potentially increase the speed of clinicians to provide appropriate treatment.
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.
Promptly after the SARS epidemic, the Chinese government accelerated the establishment of an effectual and national unified management system for public health emergencies and enacted two laws: the Regulation on Public Health Emergency and the Measures for the Administration of Information Reporting on Monitoring Public Health Emergencies and Epidemic Situation of Infectious Diseases [18, 19]. In addition to defining the standards and grades of public health emergencies, these laws support the construction of command systems and clarify the responsibilities and the leadership role of the chief executive of central and local governments previously held by the Centers for Disease Control and Prevention (CDC). Accordingly, the executive capacity of the command systems has been much improved. Importantly, China also established an emergency information dissemination system to enable timely (within 2 hours), accurate, and comprehensive release of information. Moreover, both central and local governments are now expected to be prepared for a public health emergency response (e.g., techniques, personnel, materials, and management preparedness).
In 2004, China revised the Prevention and Treatment of Infectious Diseases Law, adding SARS and avian flu as notifiable diseases and revising the law to comply with the principal rules of infectious disease prevention and control (i.e., infection source control, interruption of route of transmission, and susceptible people protection). The law adheres to the “five early” principle of early detection, diagnosis, reporting, isolation, and treatment. Early isolation can restrain contagion. The law applied China's experience in emerging epidemics to prevent and control 37 infectious diseases.
The Chinese Ministry of Health (CMH) issued a technical guide for avian flu prevention and control in 2004. It requires that suspected and confirmed cases be handled quickly at designated hospitals with the equipment to prevent nosocomial infection. The epidemiological and etiological data of patients should be acquired to enable determination of human-to-human transmission capacity. The guide suggests that persons exposed to dead poultry infected by avian flu virus be isolated and observed for 7 days. In order to control zoonotic infectious diseases, China revised its Law on Animal Disease Prevention in 2007, adding an animal epidemic surveillance and reporting system for timely disclosure of animal epidemics and providing compensation to farmers for economic loss due to culling infected or potentially infected poultry.
Guided by the aforementioned laws, a series of social innovations enacted after the SARS epidemic have improved China's ability to combat emerging diseases. The CMH issued a swine flu prevention guide on April 29, 2009, 12 days before the first reported H1N1 case. On April 3, 2013, 4 days after the first H7N9 confirmed case, the CMH also issued a nosocomial H7N9-infection prevention guide. Administrative reforms resulted in better handling of H5N1, H1N1, and H7N9 relative to SARS. Importantly, in keeping with its move toward greater transparency, after confirmation of the first H1N1 case on May 11, 2009, China posted patient zero's travel information publicly on the same day. Likewise, after confirming the first H7N9 avian flu case on March 30, 2013, China published detailed information about the patient's medical consultation. Transparency helps to subdue rumors and maintain social stability.
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.
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.
Emerging known and unknown influenza viruses create profound threats to public health; platforms for rapid detection and characterization of influenza viruses are critically needed to prevent and respond to any potential outbreaks in Taiwan. During the past decade, quickly developing and far-reaching technology of biotechnology were wildly applied; the most commonly used methods to identify and quantify influenza infection at Taiwan CDC can be subdivided into three broader phases, phase 1, pre-SARS, and techniques measuring viral infectivity (viral plaque assay, TCID50, and immunofluorescence assay); traditionally cell culture based on virus isolation has been regarded as “golden standard” for the detection and diagnosis of virus infection, and it is the technique to which all other test methods have been compared. After SARS outbreak, the laboratory diagnostic technics were moved to the next phase, which are to examine viral nucleic acid and protein (qPCR, immunoblotting, ELISA, and hemagglutination assay). The development of molecular methods for the direct identification of specific viral genome from clinical sample is one of the greatest achievements during this period. The advance and current phase of the Center for Research, Diagnostics and Vaccine Development of Taiwan CDC is to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome (DNA microarray, liquid chip array, and SNP). Clearly nucleic acid amplification techniques including PCR, nucleic acid sequence-based amplification, and multiplex detection system are proven technologies for rapid detection and molecular identification for most known human influenza viruses. Influenza viruses are traditionally detected using specific antibody-based immunoassays or immune-fluorescence assays. On the other hand, RT-PCR and real-time RT-PCR assays using specific primers against vial nucleic acids were more advanced and specific and provide faster results than end-point assays of influenza detection and in many cases have sensitivities equal to or better than cell culture. Unfortunately, high mutation rates of influenza virus may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use irrelevant; therefore, it is highly demanded to develop rapid and universal identification and detection technologies. Furthermore, facing the growing threat of interspecies transmission of influenza viruses resulting in the emergence of new infectious pathogens, the DNA microarray was applied in Taiwan CDC for diagnosis, identification, sequencing, and subtyping of influenza virus. Recently, a new molecular biology-based microbial detection method for rapid identification of multiple virus types in one specimen has been developed. Microbial Detection Array (MDA) detects viruses using probes against genomic DNA sequence within 24 hours. Each probe tests for a particular sequence of DNA and small groups of probes can be used to check for specific viruses up to the species level, different from current PCR technologies that focus on small, prioritized sets of high-risk biological pathogens. MDA, however, can identify a broad range of organisms, including pathogens on a priority screening list, sequenced bacteria, or viruses that might not be anticipated. Such technology has great potential for improving diagnostic processes and in different applications. New technologies have been continuously added in the field of biomedical sciences, which has gradually enriched science and enormously improved the quality and quantity of research output and will provide better preparedness for the next outbreak from a new emerging influenza virus.
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.