We examined whether ILI symptoms segregated with the flu vaccination records. 58.0% of study participants who were vaccinated also reported the experience of ILI symptoms. 65 study participants who received the pdm flu vaccine and filed their ILI symptoms report later during the flu season (i.e. after December 2009, and after they had received the pdm flu vaccine) experienced significantly more symptoms than the 170 study participants who did not receive the pdm flu vaccine (and also reported their ILI symptoms after December 2009). Vaccinated study participants reported 4 ILI symptoms rather than 3 symptoms on average, i.e. nasal discharge (p = 0.02), fatigue (p = 0.04), cough (p = 0.01), sneezing (p = 0.04) and chills (p = 0.05) (Table 3). The 45 study participants who received the pdm flu vaccine and mailed a swab later showed less frequent detection (6/45) of coronavirus RNA (p = 0.03), but showed a higher percentage (14/45) of rhinovirus RNA (p = 0.01) in their nasal swabs as compared to the 139 study participants who chose not to get vaccinated but mailed a swab due to symptoms of ILI (44/139).

In Argentina, trivalent inactivated influenza vaccine (IIV3) was introduced into the National Immunization Program (NIP) in 2011 for children 6 months to 2 years old and other target groups including 1) high-risk individuals (e.g., those with chronic respiratory and cardiovascular conditions, diabetes, immunocompromising conditions, etc.); 2) pregnant or postpartum women; 3) health care workers; and 4) adults 65 years old and older (9). From 2011 to 2015, vaccine coverage in young children (6 months to 2 years old) ranged between 72% (for the first dose) and 50% (for the second) (9).

IIV3 effectiveness was assessed through a case-control study carried out at three pediatric hospitals (10). Although the total number of cases was low (38 cases and 92 controls), preliminary effectiveness was 73% in children 6 months to 2 years old. Results from the Pan American Health Organization (PAHO) Network for Evaluation of Influenza Vaccine Effectiveness in the Latin America and Caribbean Region (Red para la Evaluación de Vacunas En Latino América y el Caribe–influenza, REVELAC-i) assessing influenza vaccine effectiveness showed a lower protection rate (48%) for the prevention of severe infections in children under 5 years old (11).

Vaccination of pregnant women can help prevent influenza in newborns and young infants (12). In 2015, IIV3 coverage in this population exceeded 90% (3). A study carried out in Argentina using the influenza A/H1N1 MF59-adjuvanted vaccine (13) demonstrated that vaccinated pregnant women had a lower risk of 1) giving birth to low-weight babies (odds ratio (OR): 0.74) and 2) premature deliveries (OR: 0.79). Furthermore, vaccination was not associated with adverse perinatal or maternal events.

In Brazil, influenza immunization is routinely administered to children 6 months to 5 years old, adults 60 years old and older, pregnant or postpartum women, health care workers, the indigenous population, individuals in prison, and high-risk groups (e.g., those with chronic respiratory or cardiovascular diseases, diabetes, immunocompromising conditions, etc.) (14). Overall, in 2011–2014, vaccination coverage exceeded 80% in all groups (15), and in 2015 approximately 50 million people (25% of the population) were immunized (14). Most vaccines used during the 2016 influenza season were manufactured locally by the Instituto Butantan (São Paulo).

The median age of the 373 hospitalized patients was 10 y (range 2 mo- 86 y, mean 19.4 ± 20.1 y), and the male-to-female ratio was 1.1:1 (196/177). The total admission rate due to influenza infection was 8.4% (373/4,463). The age distribution of the patients admitted to hospital are shown on Figure 1 (black bar), and these demonstrated similar patterns to those of the total patient cohort except older age group. The admission rate of the 0-10 y was 8.8% (191/2,160), the 11-20 y was 4.7% (62/1,329), the 21-30 y was 9.2% (41/447), the 31-40 y was 8.9% (23/258), the 41-50 y was 12.1% (15/124), the 51-60 y was 16.7% (14/84), and the ≥ 61 y was 44.3% (27/61).

All inpatients received the recommended doses of oseltamivir and the majority of inpatients received a broad-spectrum antibiotic. In total, 338 patients (90.6%) received oseltamivir within 48 h of fever onset. Six adult patients (4 in the 41-65 y group and 2 in the ≥ 65 y group) with underlying diseases were infected during their hospital stay and these patients were excluded from the subjects. Additionally, four of the infected patients were pregnant women, and their clinical course was uneventful. We analysed the chest radiographs of the inpatients and found that 116 patients had pneumonia (80 children and 36 adults). Pneumonia was detected in 31.3% (116/373) of the admitted patients, and 2.6% (116/4,463) of the total infected patients, respectively (Table 1). No children were treated in the intensive care unit. However, six adult patients were treated in the intensive care unit, two had ARDS and 4 were at risk of deterioration because of underlying diseases. None of the infections in this study was fatal.

We followed the study participants who gave blood at time points A and C and reported (in the spring of 2010) their 2009–2010 pandemic vaccination status. At time point A, the levels of A/H1N1/California/7/2009-specific Abs and IFN-γ production in response to the flu antigens were comparable between participants who received (after time point A) the pdm flu vaccine and individuals who chose not to receive the vaccine (seropositive at SRH > 4 mm2, but below protective levels (SRH < 25 mm2)).

At time point C, the levels of A/H1N1/California/7/2009-specific Abs were above protective levels (SRH ≥ 25 mm2) in pdm flu vaccinated participants, the Ab levels were significantly higher (p < 0.001) than in non-pmd flu vaccinated study participants. We also observed a statistically significant increase in IFN-γ production in response to all flu antigens (except for B/Malaysia/2506/2004), including the flu matrix antigen M1, in blood from study participants who received the pdm flu vaccine in the winter of 2009–2010 compared to the non-vaccinated study participants (p ≤ 0.04) (Table 2; note that M1 is not a designated component of the flu vaccine).

The present study involved the collaboration of one government health institution in Mexico that performed the sample collection. Before each examination, each adult who had voluntarily come to the examination point and agreed to participate was informed about the microbiological process of his/her sample, and oral consent was obtained. Parents or guardians provided oral consent on behalf of all under age child participants. The ethical committee of the health secretariat of Mexico approved the use of oral consent, given that the studies were conducted as part of the national H1N1-surveillance program and thus part of a routine public health-monitoring program conducted by the Mexican government.

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.

We classified the admitted patients into 4 age groups, as mentioned previously. Interestingly, there were significant differences in the male-to-female ratios in the admitted patients and the pneumonia patients between children (≤ 15 y) and adults (16-86 y). In the admitted patients, the male-to-female ratio was 1.6:1(132:84) in children, whereas the ratio was 1:1.4 (64:92) in adults. Additionally, in the patients with pneumonia the male-to-female ratio was 3:1(60:20) for the children and 1:2(12:24) in the adults. There was a correlation between increased age and an increase in the admission rate and the frequency of underlying diseases (linear by linear association test), and the total duration of fever and the hospitalization (ANOVA test). In laboratory findings, the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values displayed similar correlation (ANOVA test) (Table 2). The ≥ 65 y group showed significant differences compared to other age groups in nearly all of the examined parameters (χ2 test and Student's t-test, data not shown). The frequency of underlying diseases was significantly different among age groups. Many patients had more than one underlying diseases and these patients were more prevalent in older groups. The underlying diseases of the admitted patients are shown in Table 3.

We previously found that children with pneumonia had higher leukocytes counts with lower lymphocyte differentials than the children without pneumonia. The adult patients with pneumonia had higher leukocytes counts, CRP and ESR values with lower lymphocyte differentials than the adults without pneumonia (Table 4).

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.

The mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days.

RT-PCR is considered to be one of the most sensitive and specific tests for the diagnosis of influenza. In addition, multiplex PCR methods can detect a panel of respiratory viruses or co-infections simultaneously. In this study, pharyngeal swab specimens were collected from ILI case-patients, who strictly complied with Chinese CDC standards.

The general population in Beijing, China has been exposed to the novel pandemic H1N1 influenza virus since mid May 2009. According to the Chinese CDC, as of May 2011, 136869 confirmed cases and 875 deaths from pandemic influenza H1N1 2009, have been reported nationwide in mainland China,.

In this study, approximately 29.4% of the samples were positive for at least one virus, which is consistent with the results of other studies, in which between 0.9–27%, and 44% of reported samples were positive. Our research suggests that FLU-A was the predominant viral pathogen among ILI patients in Beijing from August 2010 to May 2011, which was similar to that of 2009, 2008 and 2006, but different to 2007,,,. Almost all positive samples demonstrated FLU-A strains, while few FLU-B strains were detected. This result differs from that reported from the United States, which demonstrated that 26% of the positive specimens were influenza B viruses. This discrepancy may in part reflect the epidemic of influenza A virus in North China in 2010–2011. It is possible that FLU-B may not have caused ILI symptoms severe enough for the sufferer to seek medical attention. It is also possible that FLU-B was not circulating in this geographical area during this time. According to the CNIC, Influenza B virus-positive rate was about 1.6% in North China from June 2010 to May 2011. The percentage of tests that were positive for influenza which included FLU-A and FLU-B was 23.7% which was lower than the same period in 2009,.

FLU strains, based on our data, accounted for approximately 80% of the RT-PCR positive cases, and in August, September and October 2010, and Mar 2011, all infections were caused by FLU-A alone. The incidence of FLU-positive specimens was high. This may reflect the possibility that our sample collection was biased towards patients exhibiting ILI, which is a clinical or symptomatic definition of influenza to identify potential influenza cases, in other word, influenza-like illness case-definition make influenza viruses as the virus most commonly detected,,,. A study of human-to-swine transmission of pandemic influenza A virus concluded that the human ILI case definition has a high specificity and a low sensitivity for FLU-A. Influenza viruses usually account for a much greater proportion of positive specimens of influenza-like illness in adults than other respiratory viruses during the peak seasons.

A total of 7 samples (8.5% of the total number of RT-PCR positive cases) revealed the presence of co-infections. In five FLU-A-positive samples, viral co-infections were observed, including one co-infection with HRCV in July, one with HRV in November, one with HRSV-A, and one with HRSV-A and HRV in January, and one with HRSV-A and HRV in February. A population challenged by multiple infectious agents may result in an epidemic and restructure of various viruses.

Rates of ILI are an indicator of trends for influenza pandemics. Beijing is located in the temperate zone of the Northern Hemisphere, where influenza typically peaks seasonally once a year, and Beijing experience one peak of influenza activity and the peak occurred during December–January next year before 2009,,, but ahead to November in 2009,. That the peaks in ILI and the increase in acute respiratory infections (ARI) are due to influenza is supported by the seasonal pattern of high-probability ILI, the low level of respiratory syncytial virus infections, and laboratory results in the influenza season,,. During the 2010–11 influenza season, a seasonal pattern in ILI activity was observed and influenza activity peaked in late January 2011 in Beijing. Compared with the previous pandemic year (2009), lower outpatient numbers were observed during 2010–11(Figure 2). Overall, the rates of influenza-like illnesses in outpatients were lower during the 2010–2011 season, than during the 2009–10 pandemic influenza season (Figure 3).

As a result of the requirement for fever in our definition of ILI, our calculated incidence may underestimate the true incidence of ILI in the cohort. Our study shows that the positive rate of influenza virus was consistent with changes in the ILI rate during the same period.

More than 70% of ILI-case patients were infected with p-H1N1 between June 2009 and January 2010. Compared to the previous year, the age of FLU-A patients ranged from 18 to 61 years with a mean age of 36.7 years, which is older than the age of 2009 ILI-case patients (mean age was 23.4 years).

In 197 (70.6%) specimens, no viral etiology was identified or the virus was not detected. This may reflect the fact that only viruses known to cause respiratory symptoms were tested, and therefore the remainder may have been caused by other respiratory viruses or by other micro-organisms, which could also have been additional pathogens in the positive specimens. It is also possible that some viruses could not be detected due to low levels of shedding.

In this study, we attempted to identify symptoms associated with influenza A infections. However, it is widely known that a clinical diagnosis of influenza is not straightforward, and it is difficult to find symptoms or combination of symptoms specific for influenza,. We found that cough was significantly associated with influenza A (χ2, p<0.001). This is consistent with the results of Boivin, of Ohmit and of Monto, who reported that cough and a fever>38°C were associated with a positive PCR test in the influenza population, when influenza was prevalent within the community,,. Our specimens were collected during the influenza season in Beijing and the cough appears to be specific for influenza in this cohort. This is also consistent with the view of Call et al, who believe that both fever and cough are more specific for influenza among elderly individuals when influenza virus is circulating in an area. We did not find any statistically significant difference in the occurrence of fever (temperature >39°C) between the FLU-A-positive group and the FLU-A-negative group (χ2, p>0.05). A study that involved all age groups demonstrated that muscle and joint pain and headache were associated with influenza. However, we did not find any statistically significant differences in the occurrence of muscle and joint pain, and headache between the FLU-A-positive group and the FLU-A-negative group (χ2, p>0.05).

This study has limitations. No testing for other etiologies of acute respiratory illness was performed. As is generally known, respiratory viruses, bacteria and other micro-organisms can cause respiratory illness with influenza-like symptoms. Without doubt, other micro-organisms could have been additional pathogens in the positive specimens.

In conclusion, pH1N1 did not affect typical influenza seasonal peaks, although FLU-A remained the predominant virus in Beijing in 2010. Symptomatically, cough was associated with FLU-A infection. The positive rate of influenza virus was consistent with changes in the ILI rate during the same period and there was a significant reduction in the incidence of ILI in 2010 compared to 2009. The findings of this study may facilitate the clinical discrimination of influenza A virus infection, as well as providing data and distribution information for virologic surveillance of influenza.

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 viruses A and B are the main pathogens responsible for the onset of epidemics because of their evolving nature. They are RNA viruses that have a high mutation rate and ability to make “drift” changes; however, only influenza A viruses are responsible for pandemics. Worldwide, influenza A viruses are the cause of severe infections in 3–5 million people annually, and these viral infections kills 0.25–0.5 million people annually.44 As such, influenza outbreaks produce high morbidity and mortality rates with great economic and social impact.44

Early findings in relation to the most recent influenza pandemic occurred in April 2009 in Mexico and soon spread to other countries. The pandemic was caused by an H1N1 variant, which came from two genetic recombination events. The first occurred in 1998, when an avian virus, an American pig virus, and virus fragments of humans had exchanged genetic materials. The following recombination with a European swine virus strain resulted in the pandemic swine origin influenza virus.2,30 In Mexico in recent years, this has caused at least four outbreaks with high mortality rates compared with that presenting in other countries.6 During the winter of 2017–2018, influenza activity increased in Mexico, and 2,855 cases of influenza and 73 deaths were confirmed by March 02, 2018, of which 46 cases were A(H3N2), 11 cases A(H1N1) pdm09, 10 cases B, and the remaining six cases were not subtyped.39

Results emanating from different studies have shown that influenza outbreaks are characterized by high severity of symptoms with increased mortality,6,8,13,25,32 Several factors have been associated with H1N1 disease severity, such as factors due to the virus (ie, viral pathogenic mutations, resistance to antivirals), factors inherent to host susceptibility (eg, age, sex, race), including physiological immunosuppression or acquired diseases (ie, diabetes, hypertension, obesity, asthma), factors associated with available medical services and public health facilities, and factors arising from the presence of bacterial coinfections.1,3,6,8,9,16,18,25,32,34,35,41

Seasonal and pandemic influenza often have complications arising from bacterial coinfections. Cillóniz et al12 documented that in H1N1 patients with community-acquired pneumonia, the most frequently isolated bacterial pathogens were Streptococcus pneumoniae (26, 62%) and Pseudomo-nas aeruginosa (6, 14%). Staphylococcus aureus was rarely found, and Haemophilus influenzae was not found.12 During the 1918 pandemic, most deaths had bacterial coinfections. Globally, more than 34% of influenza virus infections needed intensive care among hospitalized patients, from which 0.5% of all cases of influenza corresponded to healthy young individuals and at least 2.5% of total cases the elderly group and those with coinfections harbored the bacteria.27 Symptoms of influenza cases with bacterial coinfections are similar to those with severe influenza, but the former may have a higher risk of death. Identification of coinfections should be considered in patients with influenza-like illness (ILI) presenting symptoms suggestive of pneumonia, such as dyspnea, tachypnea, and hypoxia, or with evidence of septicemia.27 Many copathogens are known to be colonizers of the respiratory mucosa, ie, S. pneumoniae, H. influenzae, and Neisseria meningitidis, including the upper and lower respiratory tracts. Distinction between copathogenic colonization and coinfection is critical, because a proven coinfection is clinically correlated with signs of pneumonia and bacterial contagion producing increased acuity or severity of disease.27

Empirical antiviral treatment should be considered and managed in such critically ill patients. The most commonly isolated bacterial pathogens are those that colonize the nasopharynx, and this complex of virus–bacteria contributes significantly to the pathogenesis of the disease, mainly in periods of endemic influenza.26,34 There have been studies reporting copathogens between influenza and other viruses, but few cases have observed that this produced severe complications because of coinfection.15,33,42 Two studies have hypothesized a “viral interference”, suggesting that a rhinovirus infection may interfere with the A(H1N1pdm09) influenza, but this is still not fully understood.24,33

The precise identification of infectious pathogens responsible for acute respiratory infections, primarily influenza, is a critical factor for proper treatment of the disease and control during outbreaks and for the appropriate use of antibiotics and antivirals. For these reasons, continuation of investigations into the pathogens commonly associated with influenza cases is urgently needed. Here, the presence of bacterial and viral copathogens are identified using clinical samples for the molecular diagnosis by resequencing microarray in study patients with confirmed influenza A(H1N1)pdm09 in Oaxaca, Mexico. We also document an association between influenza A(H1N1pdm 09) and symptoms of disease severity in dead patients with multiple-microbial infection.

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.

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.

Among the 279 patients tested by RT-PCR, 69 (24.7%) had influenza A virus and 13 (4.7%) had other respiratory viruses detected. Complete information concerning clinical presentation and routine examination was available for 190 patients (68.1% of the total), 61 of the FLU-A-positive cases and 129 of the FLU-A-negative cases. Data on the number of samples, clinical and univariate characteristics, P value and odds ratio (OR) (as well as the 95% confidence intervals (CIs)) in FLU-A-positive and in FLU-A-negative cases can be seen in Table 2. In comparison to FLU-A-negative ILI-case patients, cough was more likely to be reported in patients whose RT-PCR tested positive for influenza A virus (Table 2). Patients infected with FLU-A experienced more cough than patients infected with other viruses or those viruses free (χ2, p<0.001). Other clinical characteristics (such as sore throat, headache) appeared to be more frequent in those infected with the FLU-A virus than those non-infected, but the difference was not statistically significant. FLU-A-infected patients presented with a mean temperature of 38.6°C [95% CI (38.4, 38.7)] which on average, commenced 1.3 days before admission [IQR 1–2]. Approximately 30% of FLU-A-infected patients reported hyperpyrexia (temperature >39°C). The median age of patients who tested positive for influenza A was 32 years [IQR 27–47], and 44.3% were male. There was no significant difference in male and female rates. There was no difference in the neutrophil percentage between those whose RT-PCR tested positive for influenza A and those who tested negative. The clinical and univariate characteristics of influenza-A virus ILI-case patients are showed in Table 2.

For RT-PCR-positive patients, we undertook telephone follow-up to collect further data (Table 1). In 20 FLU-A-positive cases, 4 (20%) received antiviral treatment within three days of the onset of symptoms. A further 21.2% (11 of 52) of these RT-PCR-positive patients had been vaccinated. A further 11.3% (8 of 71) reported chronic comorbid conditions (diabetes mellitus in four and hypertension in four). The median duration of fever was four days [IQR 3–5]. The median time for disappearance or abated of systemic symptoms in the presence of influenza virus infection, or other viral infections was 4 days [IQR 3–7] and 3 days [IQR 2–6] respectively.

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.

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.

Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.

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.

Around 20% of the respondents believed that H1N1 is highly fatal (20.6%) or could cause severe irreversible bodily damages (18.9%; Table 2). Respectively, 7.1% and 47.6% believed that Hong Kong has a higher or a lower chance of having a large scale H1N1 outbreak in the future year, as compared to other countries. Close to 10% of the respondents perceived a high or very high chance for himself/herself (8.6%), his/her family members (8.7%) or the general public (12.5%) to contract H1N1 in the next year (Table 2).

Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified.

During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season.

Patients’ sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5–15 years, 63% 15–65 years and only 3% were 65 and older.

The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) (Table 1).

Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis.

Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2).

Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed.

A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1). Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation.

Regarding negative swabs (Fig 2), we observed no seasonality during the study period with a similar proportion whatever the season.

Respectively 54.9%, 44.0% and 63.4% of the respondents currently avoided going to crowded places, avoided going out or avoided visiting hospitals. Around 15% (15.8%) of the respondents were currently much worried that either they or their family members would contract H1N1; 6.0% showed signs of emotional distress (i.e. panicking very much or felt much depressed or were very much emotionally disturbed due to H1N1).

Main Influenza A viruses (IAV) cause acute respiratory diseases in humans, birds, and other mammals, representing one of the major threats to public health. Wild birds are the reservoir of influenza A viruses. An avian strain can adapt to the human host and attain human-to-human transmission capability through acquired mutations. An unexpected human adaptation of an influenza subtype or strain rather than currently circulating influenza viruses may cause pandemic flu. The pandemics of 1918 H1N1 (Spanish flu), 1957 H2N2 (Asian flu), 1968 H3N2 (Hong Kong flu) and 2009 H1N1 (swine flu) symbolize the devastating public health and socioeconomic impacts of pandemic flu and keep us alert to any such outbreak. Additionally, seasonal flu is responsible for about 50,000 deaths per year. The H5N1 type IAV, which infected 18 patients in Hong Kong and caused 6 death in 1997, is a potentially serious threat to human health in the near future because of its high mortality (about 60%) and potential human-to-human transmission.

Influenza viruses mutate frequently because of their segmented RNA genome, making it almost impossible to produce a timely and sufficiently effective vaccine to prevent the potential oseltamivir-resistant H5N1 influenza A viruses epidemic outbreaks. Therefore, it is the only way to use anti-influenza agents for treatment and prevention at the beginning of pandemic outbreak of a virulent influenza strain, which gives time for the development and widespread dissemination of an effective vaccine. There are two classes of anti-influenza drugs up to now available in the clinic, which targeting the M2 ion channel and neuraminidase (NA) expressed on the virus envelope, respectively. Adamantanes block the ion channel formed by the M2 protein, which is critical in the release of viral ribonucleoprotein complexes (vRNPs) into the cytoplasm. Although ion channel inhibitors can be effective against influenza virus infection, they have been reported to cause central nervous system (CNS) side effects. Also, currently circulating IAV strains are mostly resistant to adamantanes. Thus adamantanes are not recommended for a general and uncontrolled use.

Two neuraminidase inhibitors, oseltamivir and zanamivir, were both approved in 1999 for treatment and prevention for acute uncomplicated flu caused by influenza A and B. Neuraminidase inhibitors interfere with the enzymatic activity of the NA protein, which is critical for the efficient release of newly synthesized viruses from infected cells. However, resistant virus strains are constantly emerging, especially to oseltamivir. Different from the oral administration oseltamivir, zanamivir can only be inhaled due to its low bioavailability, which makes the limited use of this drug. In 2009, a new NA inhibitor, peramivir, was authorized for the emergent treatment of certain hospitalized patients with known or suspected 2009 H1N1 influenza.

It seems quite pressing to seek for new anti-influenza medications. Up to now, the life cycle of influenza virus has been well understood, allowing for the validation of several therapeutic targets. Among them, hemagglutinin (HA) is one of the most appealing ones. Till now 16 subtypes of HA have been identified and can be further subdivided into 5 clades and 2 groups (Figure 1). This malleable nature of HA imposes a great difficulty to conduct rational drug design. Furthermore, the variety of HA may be even strengthened by antigenic drift and antigenic shift. Here, we described the functional and structural studies leading to the discovery of HA as a new anti-influenza target, and also how structural information is facilitating the rational design of new IAV entry inhibitors targeting HA.