Univariate logistic regression models for each risk factor showed that older age, having a concurrent health condition, exposure to poultry, delayed confirmation and antivirus treatment, were associated with death caused by H7N9 and H5N1 virus (all p < 0.05). Four variables remained significant after we adjusted for all 5 variables in a multivariate logistic regression model except the median days from onset to antivirus treatment in H5N1 group and only one variable related with the risk for the deaths in H7N9 group (Table 4). However, a male patient seems to increase odds of death in H7N9 groups, this relationship was not significant. This suggested the gender was not an indicator for death.

High morbidity and mortality in influenza are seen especially among those at the extremes of age (elderly and very young), those with underlying health conditions and pregnant women.29 Underlying health conditions especially associated with an increased risk for complicated influenza are immune-compromised individuals, either due to the underlying disease, or to immunomodulatory treatment, like organ transplant recipients and those taking medication for autoimmune conditions.30 Furthermore, chronic pulmonary disease31, diabetes mellitus, cardiovascular disease and malignancies are also considered risk factors for developing severe influenza or complications.32

Currently, seasonal influenza vaccination in most guidelines is only advised for healthy travellers if they plan to attend large events or to travel by cruise ship. This is mainly because influenza is widely considered a relatively mild and self-limiting disease in most healthy individuals.2 However, over the past decade, reports of patients without co-morbidities who develop severe and even lethal influenza with apparently ‘normal’ seasonal influenza viruses, have steadily accumulated.7,32,36,37,59,60. Since seasonal influenza is the most frequent vaccine preventable infectious disease in travellers, influenza vaccination should be part of regular pre-travel advice for all travellers. This raises the more general question about what burden of expected disease during the envisaged travel would justify inclusion of vaccination advice in travel guidelines. The probability of acquiring influenza, severity of disease, expected effectiveness of the vaccine and cost are among the factors that should be taken into account. One could argue that the a priori chances of developing typhoid fever, hepatitis A or tetanus during a two and half week trip to an Asian destination are much lower than being infected with an influenza virus. However, at least according to most guidelines issued in industrialized nations, these three immunizations are usually recommended for most travellers going to many developing countries while influenza vaccination is often not even considered.61

The rate of poultry exposure was far higher for the index fatalities than for the secondary fatalities with regard to both the H591 virus [67.4% (29/43) vs. 45.2% (19/42), respectively, p = 0.039] and for the H7N9 virus [100% (9/9) vs. 50% (3/6), respectively, p = 0.018]; however, common exposure or human case contact was slightly lower for the index than for the secondary H5N1 fatalities [0.0% (0/43) vs. 28.6% (12/42), respectively, p < 0.001], with the same being observed with regard to H7N9 [11.1% (1/9) vs. 100% (6/6) in the index and secondary deaths for H7N9, p = 0.001] (Table 3). The results showed that there were no differences in the percentage of total deaths, the mean age, gender distribution or the median days between the index and secondary deaths with regard to the two viruses (Table 3).

Archived sera and data from 827 poultry workers, poultry retailers in agricultural-trade markets, swine workers, veterinarians, slaughterhouse workers, zoo workers and non-animal-exposed volunteers were studied (Table 1). The total numbers of the 827 animal workers and non-animal-exposed volunteers participating in the current study were 223, 125, 175, 104 and 200 from Guangzhou, Foshan, Qingyuan, Jiangmen and Zhongshan cities, respectively. The age distribution in the five regions was comparable. About 30% of the participants were female and the majority of animal workers were swine workers (42.5%) or poultry retailers in agricultural-trade markets (23.5%). Interestingly, approximately 11% of participants reported having influenza symptoms during the last three months before enrollment.

HI titers of ≥1:20 were detected in 21 (2.54%) of 827 study subjects (18 with a HI titer of 1:20 and 3 with a HI titer of 1:40). None of these 21 participants (all animal workers) reported having influenza symptoms during the three months before enrollment. Three of these 21 subjects also had MN antibody titers ≥1:40 against H10N8 AIV (Table 2) and were considered as having probable evidence of H10N8 infection: two with MN titers of 1:40 and one with a MN titer of 1:80. Occupations among the three (0.36%) of the 827 subjects with probable infections included two poultry workers and one veterinarian living in Guangzhou city, Guangdong Province (Table 1). Each had daily poultry exposure. No demographic, clinical or specific animal exposure risk factor associations were found in the three subjects with probable infections.

This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention.

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.

Coronaviruses are a large family of viruses found in animals and humans. In both populations, coronaviruses cause a range of symptoms varying from mild, such as the common cold, to those seen in more serious respiratory illnesses in humans such as SARS. The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a strain of coronavirus first identified in a specimen from a 60 year-old man in Saudi Arabia who developed severe respiratory disease, renal failure and died in June 2012 (43).

As of 14 June 2013, the total number of cases of MERS-CoV stands at 58 with 33 fatalities, resulting in a case-fatality proportion of 57%. These include 43 cases with 27 fatalities in KSA; two fatal cases from Jordan; two cases from Qatar; three cases with two fatalities from UK; two cases and one death from France; two cases from Tunisia; one fatal case from UAE and three cases from Italy (44). Clusters of cases have occurred in health care settings or among family contacts, but human-to- human transmission has not been sustained within the community (45).

This warrants close watching throughout 2013 because this previously-unreported coronavirus is causing severe illness in humans and the epidemiology of this pathogen remains largely undescribed.

This perspective describes five of the top global infectious disease threats of particular concern to the CDC as a ‘snapshot’ of what we monitored during 2012 and will guide subjective judgment when determining which threats will be most closely monitored during 2013. It does not necessarily describe those diseases that CDC finds most important or those that require the most resources. Fortunately, the majority of outbreaks remain localized, and the global spread of a truly novel pathogen is rare.

Influenza viruses from waterbirds can cross the species barrier and infect numerous other species (see Fig. 1). A recent example of bird-to-animal transmission is the mortality amongst harbour seals [Phoca vitulina] of the North-European coastal waters following infection with the LPAI H10N7 virus,,. Various outbreaks of LPAI H3, H4 and H7 viruses causing severe respiratory disease and mortality amongst harbour seals have also occurred in the past decades along the New England coast of the United States of America,,,. The exact transmission route between seals is unknown but it is likely to occur via the respiratory route, most probably whilst the seals are resting on land. It is currently unknown if adaptation of influenza viruses from waterbirds is needed to allow the virus to infect and transmit amongst seals. In addition to seals, LPAI viruses have been isolated from a long-finned pilot whale [Globicephala melas] and Balaenopterid whales (species unknown),, and serological evidence for infection was reported in various other marine mammal species (for review see). However, the available data is very limited and it is remains unclear if LPAI viruses can also cause outbreaks of disease in other marine mammals similar to harbour seals.

Cholera remains a top potential threat that we continuously monitor very closely.6 Cholera is an acute gastrointestinal infection caused by ingestion of food or water containing the bacterium Vibrio cholerae serogroup O1 or O139.35

V. cholerae continues to infect, and kill, large numbers of people very quickly; it spreads easily through crowded environments with fractured water and sanitation infrastructure, such as densely populated urban slums and settlements for internally displaced persons. In 2015, 42 countries reported to WHO a total of 172,454 cases with 1,304 deaths attributable to cholera, although an estimated 2.8 million cases and 91,000 deaths are thought to occur each year.36,37 Of the 172,454 reported cases, 71,176 cases (41.3%) and 937 deaths (74.6%) were reported from sub-Saharan Africa;38 none of these cases was imported.

Notably, cases were reported from Democratic Republic of Congo (DRC) (19,182 cases), Kenya (13,291), Tanzania (11,563), Mozambique (8,739), Somalia (7,536), and Nigeria (5,290). These 6 countries together reported 821 deaths, resulting in a case-fatality proportion of 1.25%; no cases were reported from northern Africa.38 Cholera continues to be a threat in sub-Saharan Africa, the region with the lowest coverage of improved water and sanitation and a multitude of health system challenges. Epidemic cholera creates an additional burden on health facilities and personnel already stressed by HIV/AIDS, malaria, tuberculosis, and other infectious and noninfectious causes of morbidity and mortality (eg, malnutrition).

Asia reported 64,590 cases with 30 deaths to WHO in 2015, with most cases reported from Afghanistan (58,064; 89.9%) and Iraq (4,965; 7.7%); of the 64,590 cases, 57 were reported to be imported cases, into Iran (36), Bahrain (8), Japan (7), Kuwait (5), and Oman (1).38

Of increasing concern over the past several years is the establishment of cholera in the Americas, beginning with Haiti, which first reported cases in association with an outbreak in October 2010 that ultimately resulted in 754,972 cases and 8,863 deaths through the end of 2015. A total of 36,045 cases were reported in 2015 alone, which represents an increase of 30% in case counts from 2014. Despite the continued efforts of the Minstère de la Santé Publique et de la Population, cholera has maintained a presence in the country since the 2010 outbreak. And like sub-Saharan Africa, with a less than ideal healthcare infrastructure, the well-proven risk of explosive epidemics in Haiti and of spread to other nations remains as long as cholera is present.39

To combat endemic and epidemic cholera, in 2013 WHO created the world's first oral cholera vaccine (OCV) stockpile, which continues to expand.40 Since operations began, a total of 21 oral cholera vaccine deployments of about 4 million doses to 11 countries have been used in various contexts: humanitarian crises in Cameroon, Haiti, Iraq, Nepal, South Sudan, and the United Republic of Tanzania; outbreaks in Guinea and Malawi; and in endemic areas such as Bangladesh and DRC.41 Also of note, to prevent international spread of cholera, particularly among travelers, is the recent licensure and availability of Vaxchora™, a single-dose, live attenuated, oral cholera vaccine in the United States, the first cholera vaccine to be licensed in the United States in many decades and the only one that is available in the United States.42

In 2016, GDDOC closely watched and reported on widespread and persistent epidemics of cholera in Haiti, Kenya, Tanzania, DRC, South Sudan, the Central African Republic, Burundi, Benin, Ethiopia (acute watery diarrhea), Djibouti, and Yemen and have responded to requests for assistance or otherwise provided technical assistance to Haiti, Kenya, Tanzania, and DRC. We will continue to monitor reports of cholera, particularly those reports that could signify exportation into a country with an already-weakened healthcare system, placing the country at risk for Vibrio cholerae to become established, as occurred in Haiti.

The first 3 human infections with low-pathogenic avian influenza A (H7N9) virus were reported in China in March 2013 from Shanghai Special Administrative Region (2 infections) and Anhui Province (1), and all were fatal.22,23 This was the first time that infection with a low-pathogenic avian influenza A virus had caused severe and fatal human disease. Since then, infections in humans with low-pathogenic avian influenza A (H7N9) virus infection have been reported from throughout China.24

As of April 2017, there were nearly 1,400 infections in humans with avian influenza A (H7N9), with at least 530 deaths reported to WHO,25 including those reported from China's Special Administrative Region (SAR) of Hong Kong (21 infections). Of particular concern are those that were acquired in China and then subsequently exported to other countries and later diagnosed, allowing potential autochthonous transmission in those areas, including Taiwan SAR (5), Canada (2), Macao SAR (2), and Malaysia (1).25 Most reported infections with avian influenza A (H7N9) have been hospitalized with pneumonia, and decedents had multi-organ failure.26

Risk factors for H7N9 virus infection are visiting or working at a live poultry market and raising backyard poultry in China.27,28 Prevention measures have included the temporary closure and decontamination of live poultry markets.29,30 While studies have shown that the exposure potential of avian influenza A (H7N9) virus persists in live poultry markets, closure and decontamination of these markets may rapidly reduce the occurrence of infections of avian influenza A (H7N9) virus in poultry, thus preventing transmission of the virus to humans.31

During the fifth epidemic of this outbreak, which began October 1, 2016, there was a significant increase in human infections with avian influenza A (H7N9) compared with the first 4 epidemics.32,33 The number of provinces with H7N9 infections has increased, and some human infections with highly pathogenic avian influenza A (H7N9) virus have been reported in 2017, suggesting ongoing evolution of H7N9 viruses in poultry.34 We are constantly monitoring disease reports that not only report human illness, but that also report detection of H7N9 virus in poultry, including backyard poultry and in live poultry markets.

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.

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.

The current danger to people from avian influenza has been recognized. The World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC) have adopted measures to prevent the emergence of avian influenza in Asia and control its wider transmission.54 The CDC's response has been focused on enhancing surveillance and laboratory testing for human avian influenza (Table 2).55

The Chinese government also exerts great importance on the prevention and control of avian influenza. The Chinese Center for Disease Control and Prevention has published interim guidelines to limit the possibility of human infections during outbreaks of avian influenza in domestic birds and poultry in Mainland China.56-59 In addition, the Chinese government has taken a series of concrete measures in this regard:

First, the Chinese Ministry of Public Health has formulated a series of clinical management measures for human influenza A (Table 3).60-67

Secondly, China established a national command headquarters in January 2004, headed by the Vice Premier, in order to oversee the response to avian influenza.68 These command headquarters have formulated medium- and long-term measures, shown in Table 4.69

Influenza A (H5N1) virus is susceptible to Oseltamivir (Tamiflu) and Zanamivir (Relenza)70 but is resistant to Amantadine and Rimantadine.71,72 Treatment should be started within 48 hours of onset of fever, without waiting for laboratory confirmation.73,74 Mild cases are treated. A higher dose of 150mg twice daily and treatment for 7 to 10 days is required for the treatment of severe infections. Salicylate administration should be avoided in children younger than 18 years to prevent the possibility of Reye's syndrome. The effectiveness of Zanamivir in reducing the severity and duration of illness and in preventing complications has been proven in children from 5 to 12 years old.75

Currently, the H5N1 avian flu virus is limited to outbreaks among poultry and persons in direct contact to infected poultry. Avian influenza (AI) is endemic in Asia where birds often live in close proximity to humans. This increases the chance of genetic re-assortment between avian and human influenza viruses which may produce a mutant strain that is easily transmitted between humans, resulting in a pandemic. Unlike SARS, a person with influenza infection is contagious before the onset of case-defining symptoms. Researchers have shown that carefully orchestrated of public health measures could potentially limit the spread of an AI pandemic if implemented soon after the first cases appear. Both national and international strategies are needed: National strategies include source surveillance and control, adequate anti-viral agents and vaccines, and healthcare system readiness; international strategies include early integrated response, curbing disease outbreak at source, utilization of global resources, continuing research and open communication.

There were several zoonotic influenza strains that infected poultry and human during the past decade in Taiwan (Figure 2). In April 2009, a new strain of H1N1 from Mexico was found to be a novel strain of influenza for which current vaccines against seasonal flu provided little protection. This new H1N1 strain resulted from a reassortment of bird, swine, and human flu viruses. In June 2009, the WHO declared the outbreak of a pandemic which was named as pandemic H1N1/09 virus (pdmH1N1) in July 2009. It is possible that the virus has been circulating in human population since some time in the past and had not been detected.

The first case of H1N1 detected in Taiwan was confirmed on May 20, 2009, and another 9 positive cases in a row were identified within a week. The pdmH1N1 virus was isolated in late May 2009, causing a community outbreak in early July and then spreading islandwide. Taiwan CDC provided free seasonal influenza vaccine started from October; people received their vaccination at over 3,500 contracted hospitals and clinics. Then the Taiwan government approved an inactivated vaccine with influenza A/California/7/2009 (H1N1) strain known as AdimFlu-S (Adimmune Corporation, Taichung, Taiwan) and initiated mass vaccinations in November. Free AdimFlu-S vaccines were provided giving the priority to schoolchildren, the elderly, and front-line healthcare personnel. Due to the delay in vaccine development and delivery in 2010, health authorities used both vaccination and school closure to control pdmH1N1 [22–24]. Overall, the vaccine coverage rates were 76.9% for children and 24.6% for civilians in late July 2010 [22, 24, 25].

Confirmed case of pdmH1N1 influenza virus infection in Taiwan is defined as an individual with laboratory-confirmed pdmH1N1 influenza virus infection by one or more of the following tests: (1) real-time reverse transcriptase-polymerase chain reaction (RT-PCR), viral culture, or fourfold rise in pdmH1N1 influenza virus-specific neutralizing antibodies; (2) viral culture and (3) RT-PCR which can reliably identify the presence of pdmH1N1 influenza virus in specimens, especially RT-PCR, which has the highest sensitivity and specificity. With RT-PCR, pdmH1N1 influenza virus will test positive for influenza A and negative for seasonal H1 or H3. Meanwhile, commercially available rapid influenza diagnostic tests (RIDTs) detect influenza viral nucleoprotein antigen and are capable of providing results within 30 minutes. The sensitivity of RIDTs for detecting pdmH1N1 influenza varied from 10% to 70% and is directly related to the amount of virus in the specimen but inversely related to the threshold cycle value of the test. Rapid tests for influenza may detect the antigen from either influenza A or B in respiratory specimens with a high specificity (>95%), but the negative result from a rapid test does not rule out influenza infection, and most of rapid tests cannot distinguish pdmH1N1 from H3N2 influenza A viruses.

The oseltamivir-resistant pandemic influenza A (H1N1) virus strain in Taiwan was first isolated from a 20-year-old male in October 2009. The H1N1 virus isolated before the patient received oseltamivir treatment was sensitive to oseltamivir. However, three days after initiation of treatment, the virus isolated from the same patient shows a mutation involving the substitution of a histidine for tyrosine at position 275 (H275Y), which is resistant to oseltamivir. Oseltamivir treatment was not associated with statistically significant reduction in the duration of viral shedding, thereby proving ineffective in preventing viral spread in community. The role of swine in the genesis of this pandemic was again apparent. The lesson learned is that the pandemics of influenza can arise anywhere in the world and that global surveillance is merited.

The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers.

DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates–[11]. In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigen-specific and sustained T cell responses–[3],,,. DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis),, pigs (against classical swine fever virus and mycoplasmosis), and horses (against West Nile virus and rabies). In addition, DNA vaccines have been tested against avian plasmodium infection in penguins and against influenza and infectious bursal disease in chickens,,, duck hepatitis B virus in ducks, and avian metapneumovirus and Chlamydia psittaci in turkeys, (reviewed in ref.). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain, there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate); no protection with multivalent DNA vaccines against heterologous strains has been reported.

Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species–[27]. While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment,. Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses,,. In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies.

In this study, we used an automated high capacity needle-free injection device, Agro-Jet® (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses.

avian influenza?

To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8κB, which mediates high level expression and immunogenicity in vivo

[34],,. Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described,. We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice,. Significant neutralizing antibodies generated to homologous HAs were detected consistently by LAI with few exceptions, while cross reactivity to a standard isolate, A/Vietnam/1203/2004, was variable. For example, IC90 titers exceeding 1∶800 were observed against A/chicken/Nigeria/641/2006 and A/Hong Kong/156/1997, while a lesser response was detected for the A/chicken/Korea/ES/2003 strain (Fig. 1). Heterologous neutralization to A/Vietnam/1203/2004 was variable and did not fully correlate with the degree of relatedness of the specific HA. The ability of these immunogens to generate robust cross-reactive antibodies is consistent with previous observations,,.

Successive serological surveys in the poultry worker cohort provided evidence of seroconversions and some prior exposure (Table 2). At baseline sampling (January 2013), 125 participants were enrolled in the study, with one person testing positive to A/H5N1 antibodies and another testing positive to A/H9N2 antibodies. Participant retention was high throughout the study, with 117, 105 and 106 people resampled at the second (March 2013), third (June 2013) and fourth (November 2013) sampling missions, respectively. Seroconversions to A/H5N1 were detected for two participants at the third sampling and two participants at the fourth sampling. Seroconversions to A/H9N2 were detected for one participant at the third sampling. Overall seroprevalence was 4.5% for A/H5N1 and 1.8% for A/H9N2. Rates of seroconversion were 3.7 infections per 1000 person-months for A/H5N1 and 0.9 infections per 1000 person-months for A/H9N2. There was no serological evidence of exposure or molecular detection of A/H7N9 before or during the study.

There were no calls to the toll-free number reporting an incident with clinical signs or symptoms compatible with influenza infection.

During August 2007 and December 2012, we tested swab samples from 4,308 domestic waterfowl from four live bird markets. Most (86%) were adult waterfowl and 96% appeared healthy (Table 1). Most (93%) had been raised in backyards, and the remainder were from free ranging small-scale commercial flocks (Table 1). The average backyard flock had 18 birds (95% CI: 18–20), and the average small scale and commercial free ranging flock had 325 birds (95% CI: 293–358). Two hundred fifty (6%) of the sampled birds raised in backyards and 63 (19%) of the sampled birds raised as free ranging waterfowl flocks had at least one death from illness in their flock in the week preceding sampling. The poultry mortality ratio between the backyard and free ranging waterfowl were not significantly different (average mortality in backyard poultry was 1.2% vs. 0.9% among small-scale free ranging poultry, p = 0.07) (Table 1).

Out of 4,308 waterfowl samples, 191 (4.4%) were positive for influenza A viruses in the live bird markets, including 74 (1.7%) that were influenza A/H5 positive. Influenza A virus prevalence varied from 3% [95% CI: 1.9–5.3%] to 5% [95% CI: 3.7–6.2%] depending on the market and ranged from 0.5% to 3% for influenza A/H5 viruses with the highest percentage observed at Chittagong (Fig. 1). Besides influenza A/H5 viruses, we also identified H1 (n = 6, 0.1%), H3 (n = 9, 0.2%), H4 (n = 8, 0.2%), H6 (n = 2, 0.001%), H9 (n = 9, 0.2%), and H11 (n = 10, 0.2%) among the influenza A positive waterfowl samples (Fig. 2, Appendix - Table 1). Hemagglutinin type for the remaining influenza A positive samples (38%) were not determined due to inability to propagate virus, amplify genomic material by PCR, and/or co-detection of Newcastle Disease virus RNA. Compared to the samples collected from backyard raised waterfowl, the small-scale operation raised waterfowl had more influenza A virus detections in their samples (3.6% vs. 5.4%, P < 0.01). However, the backyard raised waterfowl samples yielded more diverse hemagglutinin and neuraminidase subtypes (i.e. H1N1, H3N2, H3N6, H3N8, H4N1, H4N2, H4N6, H5N1, H5N2, H5Nx, H6N1, H9Nx, H11N2 and H11N3) compared to the viruses identified in the small scale raised waterfowl (i.e. H1N1, H1N3, H4N6 and H11N6). We did not identify evidence of co-infection by multiple influenza A subtypes through individual waterfowl sampling.

From 2007 to 2010, the majority of the influenza A virus detections in the live bird market occurred during the colder months (4.8% during October–March vs. 2.0% during April–September, p < 0.01). We also identified H5N1 in the live bird markets more often during the colder months (78% of the total H5 test positive samples were collected during October–March) of the year, though the difference was not statistically significant (0.2% during April–September vs. 0.6% in October–March, p = 0.2) (Fig. 2, Appendix-Figure 4, Appendix - Table 1). The surveillance samples collected from waterfowl in live bird markets were positive for three clades: 2.2.2, 2.3.2.1a, and 2.3.4.2 (Fig. 2). While clade 2.2.2 H5N1 viruses were exclusively detected during 2007–2010; clade 2.3.2.1a H5N1 viruses became the predominant clade detected by this surveillance following the introduction of the virus in early 2011 (Fig. 2, Appendix-Table 1).

The domesticated waterfowl sampled during October-March were more likely to test positive for influenza A viruses compared to the waterfowl sampled during April-September (odds ratio (OR) 1.5; 95% CI 1.1–2.0) (Table 2). Domesticated ducks were more likely to be infected with influenza A virus compared to geese (OR: 5.2, 95% CI: 2.1–12.7), and when infected, they were more likely to appear healthy at the time of sampling (OR: 3.4, 95% CI: 1.1–10.6) (Table 2). Birds raised in larger flocks with >20 birds and the birds raised in backyard flocks (vs. raised in small-scale operations) were less likely to be infected with influenza A/H5 viruses (Table 2).

We collected 590 pooled environmental samples from May 2009 through December 2012. One hundred and seventy three (29.3%) were positive for influenza A viruses and 74 (12.5%) were positive for influenza A/H5 virus by rRT-PCR (Fig. 3, Appendix Table 2). Besides H5 subtypes, H3 (n = 1, 0.2%), H7 (n = 2, 0.3%), H9 (n = 59, 10.0%), and H11 (n = 1, 0.2%) RNA were identified (Fig. 3). Among the pooled, influenza A positive environmental samples, 11 (1.9%) had RNA from multiple subtypes (Fig. 3, Appendix–Table 3).

A low pathogenic avian influenza (LPAI) A/H7N9 virus was identified in one environmental sample. BLAST analysis19 confirmed the virus to be of Eurasian lineage descent closely related to other LPAI H7 viruses circulating in the region but phylogenetically distant to the Chinese lineage H7N9 viruses20.

Pooled environmental swabs collected from both urban and rural markets tested positive for influenza A viruses by rRT-PCR. In the four rural live-bird markets, 17.6% (95% CI: 11.8–24.7%) of the 148 environmental specimens were rRT-PCR -positive for influenza A viruses, and 4% (95% CI: 3.4–14.8%) were positive for H5 virus. In the 16 urban markets in Dhaka, 33.3% (95% CI: 28.9–37.9%) of the 442 environmental specimens were rRT-PCR positive for influenza A viruses and 69 (15.6%) were positive H5N1 virus RNA. The environmental samples collected from the urban live bird markets were twice as likely to contain influenza A viruses (33.3% in urban vs. 17.6% in rural, p < 0.001) and five times more likely to contain influenza A/H5 viruses (15.6% in urban vs. 3.4% in rural, p < 0.001) than samples collected from rural live bird markets. Until the end of 2010, the majority (n = 20, 80%) of the H5 positive environmental samples in urban Dhaka markets were identified between October and March. Following the identification of clade 2.3.2.1a in March 2011, this seasonal trend was no longer apparent in 2011 and 2012 (15% in October–March vs. 10% in April–September, p = 0.09) (Fig. 3).

The sponsor of the study had no role in the study design, data collection, data analysis, data interpretation, writing of the report, or the decision to publish. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. The views expressed are those of the authors and do not necessarily represent the policy of the China CDC or the institutions with which the authors are affiliated.

In this study, a global dataset spanning 18 years was systematically collated to investigate changes in the epidemiological characteristics of human H5N1 cases, and also focused on Egypt, given its unique situation of increasing incidence since November 2014.20,37,46 Our analyses suggest that the geographic extent of human H5N1 cases has expanded from East Asia to Southeast Asia, then to West Asia and North Africa during 2003-2009, which may be related to the global dispersal of the virus via bird migration.62-64 The bird migration network was shown to better reflect the observed viral gene sequence data than other networks and contributes to seasonal H5N1 epidemics in local regions.3,5,7 In addition, previous evidence demonstrated Siberia as a major hub for the dispersal of the virus via bird populations, and Southeast Asia and Africa as areas of local virus persistence and the major sources of genetically and antigenically novel strains.5,7,65,66 Therefore, the increasing range of virus dispersal and outbreaks among birds may also increase the risk of human exposure.3,67 However, some of the apparent geographical dispersal in cases may also be attributed to enhanced clinical and laboratory surveillance capacity in the past 15-20 years.

Human H5N1 infections were found to exhibit seasonality, related to the cooler season from December to March and across diverse climate zones in the Northern Hemisphere (Appendix Figure 4B and 5), which may correlate with the migration patterns of wild birds and the activity of virus in winter or cooler seasons.3,7,43 A recent study found that the timing of H5N1 outbreaks and viral migrations were closely associated with bird migration networks in Asia.5 In addition, the lower temperatures in Asia and North Africa across diverse climates were associated with increasing human H5N1 virus infection in winter, which is consistent with increased poultry outbreaks and H5N1 virus transmission during cold and dry conditions, and also overlapped with human seasonal influenza epidemics.3,43,68,69

Although most human populations are thought to have little or no immunity to influenza A(H5N1) viruses, most cases examined in this study were children and younger adults, and these age groups were also more likely to recover, whereas the fatality risk was higher in adults, which might be related to the immunological reaction of virus in different age groups.41 Consistent with previous reports,28,45 the cases with ≥3 days from onset of illness to hospitalization were more likely to be fatal than those admitted within 3 days of onset with a odds ratios (OR) of 3.6 and 95% confidence intervals (CI) of 2.5 - 5.1, which might be due to the early administration of antiviral treatment, or selection bias where the cases admitted later after onset were more likely to be severe.17 Compared with Indonesia, Vietnam, Cambodia, mainland China and Thailand , the lower CFR in Egypt (Chi-squared tests, p<0.001) might be related to a less virulent virus clade, less severe clinical disease, and earlier identification, hospitalization and early treatment with oseltamivir for H5N1 cases.20,44,70 However, the CFR might be underestimated because various government entities or reports may not have identified or updated which cases have died at the time we collated data. Additionally, almost all human cases of H5N1 infection had a recent history of close contact with infected live or dead birds, other human cases, or H5N1-contaminated environments, which reaffirmed reports that human H5N1 virus infection is typically preceded by exposure to sick or dead poultry in backyards, LBMs or farms.71-76

An increased number of animal-to-human infections has been reported by Egypt during November 2014 – April 2015 with the number of cases more than the total of the last 8 years from 2006-2014.20 The increase in the number of human cases in Egypt since November 2014 can be attributed to a mixture of factors, including increased circulation of H5N1 viruses in poultry, lower public health awareness of risks in middle and upper Egypt and seasonal factors, such as closer proximity to poultry because of cold weather and possible longer survival of the viruses in the environment.77 However, the increased numbers of human cases in Egypt are of major concern because of the continued potential pandemic threat from H5N1. A few cases of human-to-human transmission and family clusters have been reported in Egypt and other countries.40,46,78-82 Nevertheless, H5N1 viruses do not currently appear to transmit easily from person to person, and the risk of community level spread of these viruses remains low.20,27,39

H5N1 viruses have evolved from the 1996 progenitor strain and now comprise at least 10 clades, through a complexity of genetic changes, which have infected domestic poultry and wild birds in many countries.21,62,63,83,84 In this study, 4 clades (0, 1, 2, and 7) and 3 subclades (2.1, 2.2, and 2.3) of H5N1 virus strains have infected humans, all of which have been reported in human cases before 2006.41,85 Compared to clade 0, the cases with clade 1, subclade 2.1 and 2.3 were more likely to result in death with a crude OR of 2.8 (95% CI: 0.93, 9.6), 11.0 (95%CI: 3.5, 37.8) and 3.2 (95%CI: 1.0, 11.4) respectively (Appendix Table 3). However, the risk of death between cases with clade 0 and subclade 2.1 was not significantly different (OR: 1.0; 95% CI: 0.3, 3.3). Based on available information, the clades of viruses isolated from humans were the same as the clades circulating in local poultry.21,28 During the period from late 2003 to mid-2005, most H5N1 virus infections in humans were caused by clade 1 strains in Southeast Asia (i.e., Vietnam, Thailand, and Cambodia).85

Although the highly pathogenic H5N1 virus strains can be expected to continue evolving over time, preliminary laboratory investigation has not detected major genetic changes in the viruses isolated from the patients or animals in 2014-2015 compared to previously circulating isolates in the same regions,41,86 and the genetic diversity of the H5N1 virus decreased significantly between 1996 and 2011 in China, presumably under strong selective pressure associated with vaccination in poultry.56 However, other influenza A(H5) subtypes, such as influenza A(H5N2), A(H5N3), A(H5N6) and A(H5N8), have recently been detected in birds in Europe, North America, and Asia, and so far no human cases of infection have been reported, with the exception of three human infections with influenza A(H5N6) virus detected in China in 2014-15.39,77 However, the co-circulation of influenza A viruses in human and animal reservoirs can provide opportunities for these viruses to re-assort and acquire the genetic characteristics that facilitate sustained human-to-human transmission, a necessary trait of pandemic viruses.3,87

Vaccines and antivirals are the most effective approaches to prevent influenza virus infection and treat illness respectively.41,88,89 Vaccination of poultry has been implemented in many of the affected countries to control H5N1 in poultry, especially in those locations where H5N1 viruses have become enzootic in poultry and wild birds.90-92 Currently, 27 A(H5N1) candidate vaccine viruses for humans are available and a new candidate vaccine is in preparation to protect against the currently circulating H5 clade 2.2.1.2 of viruses, covering all the recent H5N1 virus isolates from Egypt.41,93 The first adjuvant vaccine for the prevention of H5N1 influenza has been approved by the United States Food and Drug Administration in November 2013, and this vaccine is being stockpiled for pandemic preparedness by the United States government.94 In addition, the antiviral drug oseltamivir can reduce the severity of illness and mortality when started soon after symptom onset and appears to benefit all age groups. Prompt diagnosis and early therapeutic intervention should therefore be considered for all H5N1 cases,89,95,96 though antiviral resistance continues to receive attention and there is a need for continued monitoring.97 The availability of antivirals and vaccines in the event of a H5N1 pandemic should be considered in advance.98

There are some limitations to this study. First, the data used were collated from different sources. The data quality may be influenced by key steps in public health surveillance or reports including the case definitions, reporting methods, availability of health care and laboratory diagnostics, under reporting, and the completeness and accuracy of data reported or announced by different countries or organizations. Compared to the areas where many cases were seen in this study, some countries with few cases or without cases reported might be attributed to the low availability and capability of public health services, serological testing, and surveillance. Second, detailed data on case characteristics and clinical management were unavailable to assess the association between clinical manifestation, treatment and outcome, and this study did not include the cases with subclinical H5N1 virus infection, which have been occasionally reported.72,99-101 Third, the findings might be influenced by missing data on exposure, outcome and hospitalization, and the misclassification of cases with presumed clade or subclade. In addition, this study only included data sources in English or Chinese, which might neglect data on cases reported in other languages, including announcements or reports from Egypt.

In conclusion, the high-risk areas, population groups and seasonality of human HPAI H5N1 infections have been systematically reviewed here, providing evidence for the planning of prevention and control. The geographic distribution of countries with human H5N1 infections has expanded, especially between 2003 and 2008, with variations in outcome, demography, seasonality and the clade or subclade of viruses across the region. The incidence of human infections increased dramatically in Egypt from November 2014 to April 2015, but remained at a low level in other regions, and the CFR in Egypt has not significantly changed. However, since avian influenza A(H5N1) viruses present a continuous threat to human populations, echoing the recommendations of WHO and other organizations on influenza at the human-animal interface,41,89,102-104 there is a need for sustained efforts and close collaboration between the animal health and public heath sectors at community, national, and international levels to monitor the dynamics in human, poultry and wild birds, and to conduct early clinical management, while downstream research is encouraged to develop vaccines and antivirals, explore the driving factors behind the epidemic, and evaluate the potential for future pandemics.