Rhinoviruses are small, single-stranded RNA viruses in the picornavirus family that are responsible for more than half of all upper respiratory tract infections. In addition to exacerbating asthma and chronic obstructive pulmonary disease, rhinoviruses have also been associated with acute respiratory hospitalizations among children (30). In a large prospective study of US pneumonias, rhinoviruses have been identified as the second most prevalent etiology of pneumonia in children after respiratory syncytial virus and the first most common etiology among adults (31). There are more than 150 unique types of rhinoviruses. Among the three genotypes (A, B, and C) types A and C are most often associated with increased morbidity and bacterial secondary infection. In animals, rhinovirus type C has been associated with morbidity in chimpanzees (32). With an array of unique serotypes no vaccines or approved antiviral therapies have been commercially produced; however, experiments have suggested that vaccines and antiviral therapy may be possible (33, 34).

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.

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

Waterbirds and shorebirds of the orders Anseriformes (mainly ducks, geese and swans) and Charadriiformes (mainly gulls, terns and waders) are considered the natural host reservoirs of LPAI viruses (see Fig. 1). In wild birds LPAI viruses predominantly infect epithelial cells of the intestinal tract, and are subsequently excreted in the faeces. However, infection of wild birds with LPAI viruses is typically sub-clinical and occurs in the absence of obvious lesions,,. Every year, LPAI viruses cause outbreaks amongst waterbirds. These outbreaks are most commonly associated with the increased presence of juvenile, immunologically naïve birds in the population and occur during migration when contact rates between, and within, populations are high. The relatively high virus prevalence in waterbirds may be due, in part, to virus transmission through the faecal–oral route via surface waters.

Enterovirus D68 has caused sporadic respiratory disease outbreaks across Asia, Europe, and USA since 1960s; however, in 2014, a nationwide outbreak of D68 was associated with severe respiratory illness in USA, resulting in 14 deaths out of a known 1,150 cases (35). The CDC found 36% of all EVs tested during this outbreak were D68 and that patients with a history of asthma were found to be at a disproportionately increased risk of infection (36). One study of the 2014 outbreak found 59% of patients seen with EV-D68 in hospitals across Missouri, Illinois, and Colorado were admitted to intensive care units and 28% received ventilator support (35). In a study evaluating EVs in non-human primates, EV-D68 was detected as a recombinant zoonotic strain (37).

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.

Important viruses in the Paramyxoviridae family include the human parainfluenza viruses (PIV), human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV). These pathogens are transmitted via the respiratory route and all are causing agents of acute respiratory tract infections in humans, particularly young children, elderly and the immunocompromised. Infections with these viruses are among the leading reasons for pediatric hospitalizations (for review see). At present, there are no licensed vaccines for effective prevention of these infections, which has spurred the evaluation of first recombinant MVA vaccines against PIV and RSV.

Recombinant MVA co-producing the fusion (F) and hemagglutinin-neuramidase (HN) proteins of PIV3 have been generated for preclinical testing in animal models. In the cotton rat model, recombinant MVA elicited high levels of PIV-specific antibodies upon immunization by intramuscular or intranasal application. Upon challenge, MVA-HN was more efficient in inducing protection as determined by a substantial reduction of PIV loads in the nasal turbinates and lungs. This result favorably compared to responses achieved with an attenuated live PIV candidate vaccine. Furthermore, when used in rhesus macaques the recombinant MVA vaccines also induced protection against PIV challenge, although intranasal vaccinations could not completely prevent infections of the upper respiratory tract.

First generation candidate MVA vaccines against RSV expressed recombinant gene sequences encoding for either the RSV fusion protein (F) or the glycoprotein (G) or both envelope antigens together. In mice, all recombinant MVA induced RSV-specific antibodies and levels of MVA vaccine induced circulating antibodies were even higher than those found after experimental RSV infection. A follow-up study with these MVA vectors in a mouse model also demonstrated the induction of strong RSV-specific T cell responses, resulting in clearance of RSV from the lungs of the vaccinated animals, although it also associated with weight-loss in vaccinated animals. An enhancement of RSV-mediated lung eosinophilia was not seen upon challenge infection of MVA vector vaccinated animals. A parallel immunization study in infant cynomolgus macaques also suggested that vaccination with recombinant MVA did not predispose for an RSV associated immunopathology. However, the combined intramuscular/intranasal immunization of these infant (<1 year old) macaques with the recombinant MVA failed to provide protection against RSV replication in the lower respiratory tract.

To evaluate the safety and efficacy of new approaches in RSV vaccine development the infection of cattle with bovine RSV (bRSV) provides an excellent alternative model using a highly related pathogen in its natural host. Recombinant MVA delivering bRSV F and G glycoprotein antigens (MVA/bRSV) were tested to protect calves against bRSV challenge. Intramuscular vaccination of calves with MVA/bRSV induced bRSV specific IgG antibody and CD8+ T cell responses, but no detectable IgE antibodies. Upon challenge with bRSV the MVA/bRSV vaccinated calves compared to control animals demonstrated less severe lower respiratory tract symptoms, reduced pulmonary virus loads and no signs of bRSV-associated immunopathology (eosinophilic infiltrations). However, complete protection against bRSV infection or replication was not achieved.

Overall, these previous studies suggested the safety and at least partial efficacy of first generation recombinant MVA vaccines against PIV or RSV. Other applications of recombinant MVA including mucosal delivery or the use of prime-boost strategies may contribute to further improve the effectiveness of preventive immunization against these respiratory diseases.

Respiratory viruses, such as seasonal and pandemic influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses, cause substantial burden of disease globally. These pathogens cause respiratory tract infections, mainly in young children, the elderly and immunocompromised individuals. In contrast to seasonal influenza, currently no licensed RSV and hPIV vaccines are available.

For influenza, it is recommended to annually vaccinate people at risk to protect them against infection with seasonal influenza viruses. However, as a result of selective pressure exerted by virus‑specific antibodies induced by previous infections and/or vaccination, seasonal influenza viruses accumulate mutations in the antigenic sites of the two main surface proteins: hemagglutinin (HA) and neuraminidase (NA). Consequently, antigenic drift variants emerge that evade host immunity.

Occasionally, avian or swine influenza viruses are introduced into the human population. Since neutralizing antibodies to these novel viruses are virtually absent, the human population at large is susceptible to infection. Last year alone, several avian influenza viruses caused human infections. From February 2013 to February 2014, 335 human cases of infection with H7N9, of which some viruses display signs of adaptation to humans, have been reported. One hundred and twelve of these cases had a fatal outcome. Although sustained human-to-human transmission of these viruses has not been reported, it is possible that they acquire this ability with just a few mutations as was shown experimentally for H5N1 viruses in ferrets. In addition, human cases of infections with avian viruses of the H10N8 and H9N2 subtype have been reported, some with a fatal outcome. If one of these viruses becomes transmissible from human-to-human, it can cause a widespread outbreak that could evolve into an influenza pandemic with considerable morbidity and mortality.

In terms of pandemic preparedness, procedures should be in place to respond rapidly and produce tailor made vaccines on short notice. Furthermore, there is a need for the development of universal influenza vaccines that induce broad protective immunity against human influenza viruses and potentially pandemic viruses of various subtypes.

Influenza viruses cause annual epidemics and periodic pandemics. We have experienced three pandemics in the 20th century (i.e., Spanish flu in 1918–1919, Asian influenza in 1957, and Hong Kong influenza in 1968) and one in the 21st century (i.e., pandemic influenza A (H1N1) 2009 in 2009). These influenza pandemics have a huge impact on public health and the global economy1. Additionally, recent sporadic human infections with H5N1 and H7N9 avian influenza viruses have raised the pandemic threat of these viruses2–5. The continued circulation of H5N1 viruses in birds provides opportunities for them to infect humans. Indeed, human cases of H5N1 infections have been reported in several countries, with a total of 860 confirmed cases and 454 fatalities as of 30 Oct 2017, which is a case fatality rate of approximately 53% (http://www.who.int/influenza/human_animal_interface/2017_10_30_tableH5N1.pdf?ua=1). Therefore, concern over the pandemic potential of H5N1 viruses is clearly justified.

Influenza virus can be transmitted from person to person through several transmission modes, including direct contact, indirect contact with a contaminated object (fomite), droplet transmission (droplets of >5 µm in diameter), and droplet nuclei transmission, also referred as airborne or aerosol transmission (droplet nuclei of <5 µm in diameter, which can remain suspended in the air for prolonged periods). Experimental infection of animal models with influenza viruses is an essential component of the characterization of influenza viruses. Mice, ferrets, and nonhuman primates (NHPs) are commonly used to examine the pathogenicity, replicative ability, and transmissibility of influenza viruses, although each model has its advantages and limitations. NHPs have been frequently used as a model to characterize the virulence of various influenza viruses, such as highly pathogenic H5N1, and the 1918 and 2009 pandemic viruses, because of their close genetic relationship to humans6–8. Intranasal and/or intratracheal inoculation with a liquid inoculum of influenza viruses has been the primary method used to infect NHPs; however, a liquid inoculum does not reflect the natural infection.

Aerosol inhalation methods for inoculation of mice and ferrets have been established with several influenza viruses, including H5N1 viruses9–13. In the ferret model, these studies demonstrated that the inoculation of animals with highly pathogenic avian influenza H5N1 virus via the aerosol route led to higher nasal wash virus titers, earlier onset of clinical signs, and/or a broader spectrum of disease compared with infection via intranasal inoculation despite no difference in lethality9–11. In a murine model, Belser et al.12 demonstrated that inoculation of mice with H5N1, H7N9, or influenza A(H1N1)pdm2009 viruses via the aerosol or intranasal route resulted in comparable levels of morbidity and mortality among the infected mice. In contrast, few reports have evaluated inoculation routes in the NHP model. A recent study demonstrated that challenge of NHPs with pandemic 2009 viruses (A/California/04/2009; H1N1pdm) through the aerosol route resulted in efficient distribution of virus in both the upper and lower respiratory tracts, whereas intranasal infection led to limited distribution of virus in the upper respiratory tract14. Wonderlich et al.15 recently established aerosol infection with highly pathogenic H5N1 influenza virus in the NHP model; however, they have not compared the outcomes in the NHPs infected via the aerosol route with those in NHPs infected via the conventional method of liquid virus inoculation. Therefore, in this study, we compared the virus distribution, pathogenicity, and disease outcome in NHPs infected via the aerosol route with these parameters in NHPs infected through multiple routes (e.g., the intranasal and intratracheal routes).

Four groups of 6 ferrets each received 30, 10, 3 or 1 mg/kg CR6261 via an intravenous injection and were challenged the next day with the highly pathogenic avian A/Indonesia/5/2005 (H5N1) virus. A control group of 6 ferrets received 30 mg/kg of the irrelevant isotype-matched antibody CR3014.

All ferrets that received 30 or 10 mg/kg CR6261 survived, compared to only 33.3% of the control animals (Figure 1A). A further reduction of the dose to 3 mg/kg was clearly correlated with a lower survival rate of 66.7%. Though not statistically significant different from survival observed in control animals, there is a 50% mortality reduction. The lowest dose of 1 mg/kg CR6261 was not associated with survival benefit compared to control animals. Survival times differed significantly between groups receiving 30 or 10 mg/kg CR6261 and the control group (p = 0.020).

Moribund animals showed general depression, anorexia and lethargy, and exhibited clinical signs of respiratory disease, including dyspnoea. Animals treated at efficacious dose levels (30 and 10 mg/kg) did not loose body weight, whereas the mean weight loss in the control group was 10.5% by the time the ferrets died or were euthanized (Figure 1B). An exception was one control ferret that succumbed to infection within 48 hours after challenge and lost hardly any weight before. Ferrets that received 3 or 1 mg/kg CR6261 showed similar declines in body weight as the control animals.

One day after challenge the maximum body temperature was observed; for each ferret the maximum body temperature is depicted in figure 1C. The groups treated with 30 or 10 mg/kg had mean temperatures of 39.4°C and 40.7°C, respectively, which were significantly lower than the mean of 41.7°C observed in the control group (p<0.001 and p = 0.015, respectively). The mean temperature observed in animals treated with the lower doses of CR6261 (41.3°C and 41.5°C for 3 and 1 mg/kg, respectively) did not differ significantly from the control group.

The mean temperature in animals observed 3 days before challenge was similar across the 5 groups, ranging from 37.6°C to 38.4°C. Individual body temperature varied considerably within one healthy ferret over 24 hours (standard deviation 0.7°C).

Ferrets treated with 30 or 10 mg/kg CR6261 did not shed infectious virus in the upper respiratory tract at any time, whereas animals in the control group did (Figure 1D). Treatment with 3 and 1 mg/kg did not prevent shedding, but reduced the proportion of ferrets with infectious virus in nasal and/or throat swabs.

After necropsy, all ferrets were assessed for viral load in the lungs (figure 1E), and the animals that received 30 mg/kg CR6261 or control antibody were also assessed for viral load in the brain, liver, spleen, blood and kidney. The levels of virus replication in the lungs of ferrets treated with 30 and 10 mg/kg CR6261 were 3.9 and 2.9 log10 TCID50/g lower than that in the control group (both p<0.001). No infectious virus was detected in any of the other organs of the ferrets that received 30 mg/kg CR6261, whereas infectious virus was found in the brain of 2, the liver of 3, and the spleen of 5 of the 6 control animals (data not shown). There was no significant difference in lung viral load in the groups receiving 3 or 1 mg/kg CR6261 compared to the control group (p = 0.74 and p = 0.91, respectively).

Histopathological results were in accordance with the findings described above; animals that received 30 and 10 mg/kg CR6261 showed much less pulmonary lesions such as primary atypical pneumonia, subacute bronch(iol)itis, emphysema and congestion, or showed such lesions at a lower grade of severity, compared to animals from the other groups. Bronchiolitis obliterans was not observed in any animal that received 30 mg/kg CR6261. Compared to this higher-dose group, animals that received 10 mg/kg CR6261 showed more regenerative response (diffuse grey/red area and bronchioloalveolar hyperplasia) in the lungs, and more inflammatory changes in the trachea. Pulmonary oedema was not observed in animals that received 30 or 10 mg/kg CR6261, but was observed frequently in all other groups. These findings were in agreement with mean lung weights, which were lowest in the animals treated with 30 or 10 mg/kg CR6261 (6.3 g and 7.6 g, respectively) and significantly lower in this group than in control animals (15.0 g; both comparisons p<0.001). No significant difference in lung weight was found between the groups receiving 3 or 1 mg/kg CR6261 (14.1 g and 15.4 g, respectively) and the control group (15.0 g).

These findings show that, in a dose dependent way, prophylactically administered CR6261 confers protection against lethal H5N1 challenge, prevents morbidity and viral dissemination and reduces pulmonary pathology.

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.

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.

avian influenza?

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.

To assess the therapeutic efficacy of the monoclonal antibody CR6261, two groups of 10 ferrets were challenged as above and given 30 mg/kg of CR6261 either 4 or 24 hours later. A comparator group of 10 ferrets received 30 mg/kg of the control antibody 4 hours after challenge.

Survival rates in the groups receiving CR6261 at 4 and 24 hours after challenge were 100%, whereas only 20% of the animals in the control group survived (p<0.001) (Figure 2A). Mean decline in body weight at the end of the experiment was 6.2% in the group of ferrets that received CR6261 4 hours after challenge (Figure 2B), which was significantly less (p = 0.025) than the 10.1% observed in control animals. Animals treated 24 hours post challenge showed a mean body weight loss of 8.4%, which was not significantly different from the control animals (p = 0.427). The group of ferrets treated with CR6261 4 hours post challenge had a mean maximum temperature of 40.0°C, compared to 41.8°C in the control group (p<0.001). In line with the rapid rise in temperature after challenge observed in the prophylaxis experiment ferrets treated with CR6261 24 hours after challenge showed a mean maximal temperature of 41.5°C before CR6261 was administered (p = 0.15 versus 41.8°C of the control group, figure 2C).

Ferrets treated with CR6261 at 4 hours post challenge did not shed infectious virus in the upper respiratory tract throughout the study (figure 2D). In the group treated with CR6261 at 24 hours post challenge, one ferret had a low concentration of infectious virus (2.8 log10 TCID50) in the throat on day one, but no virus was detected on subsequent days. In contrast, all animals in the control group shed virus during one or more days. Accordingly, the mean viral loads in the lungs of ferrets treated with CR6261 at 4 and 24 hours post challenge were considerably lower than that in the control group (differences were 3.9 and 4.5 log10 TCID50/g, respectively, both comparisons p<0.001; Figure 2E).

The lungs of animals that received CR6261 at 4 hours post challenge showed less pulmonary lesions (alveolar oedema, bronchiolitis obliterans, congestion, emphysema, bronchioloalveolar hyperplasia and primary atypical pneumonia), or showed such lesions at a lower grade of severity, compared to the lungs of animals from the other two groups. Animals of the control group were most affected by primary atypical pneumonia. These findings were in accordance with the observation that the mean lung weights of ferrets treated with CR6261 at 4 hours post challenge were lower compared to the control group (5.7 g versus 14.9 g, p<0.001; Figure 2F). Animals that received CR6261 at 24 hours post challenge showed most regenerative response (bronchioloalveolar hyperplasia) in the lungs, suggesting damage to the lung parenchyma with subsequent regenerative response. The mean lung weight in this group was significantly higher than that of the group receiving CR6261 at 4 hours post challenge (8.4 g versus 5.7 g), but lower than that of the control group (p<0.001).

From the study outset, one animal had been added to each of the two treatment groups to be sacrificed for gross-pathology and histology on lungs as soon as 50% of the control animals died. The purpose was to test for possible bias due to differences in the timing of death. Infectious virus titres in the lungs of the treated ferrets sacrificed at day 3 were identical to those in treated animals sacrificed at the end of the study (open circles in figure 2E). Similarly, there were no differences in lung weight and pathology between animals sacrificed at day 3 or after day 5 (figure 2F). This indicates that the results were not biased by differences in the timing of euthanasia or spontaneous death.

The WHO Vaccine Composition Meeting for the 2014-2015 season held in February 2014 in Geneva approved that A/California/7/2009 (H1N1)pdm09-like virus, A/Texas/50/2012 (H3N2)-like virus, and B/Massachusetts/2/2012-like virus should be included in the trivalent vaccine formulation in the Northern Hemisphere.52 Consequently, the aforementioned novel avian strains are out of scope of the current vaccine formulation indicating the severity of the situation if the H7N9 influenza has global dissemination.

Laboratory testing in preliminary cases showed that neuraminidase inhibitors (oseltamivir, zanamivir) were effective against H7N9 infections, but the adamantanes were not. In addition, early treatment with neuraminidase inhibitors have been reported to restrict the severity of illness.34,53

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.

When an outbreak comes to an end, it is a matter of great concern. According to the WHO, an EHF outbreak in a country is reported to be over when 42 days have passed, and new cases have not been observed. The maximum incubation period for EHF was 21 days. The 42-day period was set by the WHO (twice the maximum incubation period) to provide a strong margin of security.27 Currently, the USA, Spain, Mali, Senagal, and Nigeria are categorized as affected countries.21 Currently, EHF epidemic appears to be ending after the slowing of transmission.22

Influenza viruses are known to constantly evolve and cross species barriers. The genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. The purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. In addition, we discussed the growing complexity of influenza virus–host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses.

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,,.

We compared in a first step recent H5N8 virus isolates collected during the epizootics in Germany in 2014 and 2016 (DE14-H5N8A and DE16-H5N8B), whose HA genes represented groups 2.3.4.4A and B, respectively, in chicken. After intravenous infection with DE14-H5N8A and DE16-H5N8B, all 10 inoculated chickens died within 2 days, resulting in an intravenous pathogenicity index (IVPI) of 2.81 and 2.93, respectively12. Thus, by legal terms, both viruses were highly pathogenic in chickens without marked differences in virulence and induced clinical signs. Also by contact with the i.v. inoculated chickens, the mortality of sentinel chicken reached 100% for both viruses.

Oculo-nasal infection of adult Pekin ducks and geese with HPAIV DE14-H5N8A did not induce any clinical signs (Fig. 1A). In contrast, application of DE16-H5N8B caused death of 2 out of 10 inoculated adult ducks within 4 and 5 days post infection (dpi), resulting in a clinical score of 0.6 (Fig. 1A). Interestingly, also the two sentinel ducks of the H5N8B trial co-housed with the infected animals died on day 4 and day 8, respectively, i.e., 3 and 7 days after having contact to inoculated animals. These differences in the pathogenicity between DE14-H5N8A and DE16-H5N8B became more pronounced following i.m. inoculation in 10 1-week-old Pekin ducks. Whereas only one of the DE14-H5N8A inoculated ducklings died at 3 dpi, all 10 ducklings inoculated with DE16-H5N8B succumbed to infection by 2 dpi, including 2 sentinel animals (Fig. 1A, p = 0.0003; compare Pekin, i.m. upper panel to Pekin, i.m. lower panel). The clinical course in Muscovy ducklings after i.m. inoculation of DE14-H5N8A was much more severe compared to Pekin ducklings (Fig. 1A, p = 0.0007; compare Pekin, i.m. upper panel to Muscovy, i.m. upper panel). Interestingly, progressive disorders of the central nervous system like tremor and opisthotonus prevailed in Muscovy ducks with a delayed onset of mortality after infection with H5N8B.

At the indicated times, swab samples were taken from the oro-pharynx and separately from the cloaca of individual birds and tested for viral excretion. Despite a healthy appearance of adult ducks and geese after infection with DE14-H5N8A, both species shed virus already 2 dpi: pharyngeal excretion was more pronounced compared to cloacal shedding (Fig. 1B; compare upper panel to lower panel). Viral load in the pharyngeal swab samples stayed high on 4 dpi, with a slight increase in viral genome load from 8.5 × 105 VE/ml to 3.7 × 106 VE/ml for ducks, and from 1.6 × 106 VE/ml to 3.1 × 106 VE/ml for geese (Supplementary Fig. S1). At that time also 7 from 8 cloacal swab samples were AIV-positive. On 7 dpi viral titers declined, but still 4 out of 6 duck samples were AIV-positive, and shedding persisted until 14 dpi for individual birds, with minute amounts of viral RNA detectable in pharyngeal (geese 14 dpi n = 3, see Supplementary Fig. S1; duck n = 1, data not shown) and cloacal swabs (geese 14 dpi n = 1, see Supplementary Fig. S1) of geese and/or ducks, respectively. Virus was transmitted efficiently to sentinel ducks and geese, with a similar course of infection as seen in the inoculated birds. In addition, 3 out of 4 sentinel chickens became infected and died within 7 days of contact (4, 5 and 7 days post contact (dpc)).

Remarkable, however, is the observation that on 14 dpi both sentinel geese still yielded viral RNA in pharyngeal swab samples, indicating virus replication and possibly shedding for up to 2 weeks.

Following infection with DE16-H5N8B virus, viral shedding started at 1 dpi, readily detectable in pharyngeal swabs from all 10 oronasally (o.n.) inoculated adult ducks, but only in one cloacal swab sample. By 2 and 3 dpi, cloacal swab samples from 6 and 9 ducks, respectively, yielded virus RNA, reaching the peak of shedding on 4 dpi with 8.7 × 105 VE/ml and 4.6 × 105 VE/ml for pharyngeal and cloacal swabs, respectively (Fig. 1B). Viral loads in swab samples declined, by 7 dpi, all animals alive shed virus by pharyngeal and cloacal routes. Thereafter, none of the samples tested positive. In line with the shedding data was the finding that DE16-H5N8B virus was transmitted efficiently to sentinel ducks (Fig. 1B, triangles) and to 10 sentinel chickens, that died within 7 days after contact (Fig. 1A). In line with the high level of virus replication, antibody responses developed fast in both waterfowl species and were already detectable on 7 dpi (Supplementary Fig. S2).

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.

The use of animal models is essential for studying the biological properties of influenza viruses such as pathogenicity, replicative ability, and disease outcome. In most cases, intranasal administration with a liquid containing the virus is used to infect animals with influenza virus; however, a liquid inoculum does not reflect the natural influenza virus infection. Recently the aerosol inhalation method has been tested in several mammalian models, including mice, ferrets, and NHPs due to its resemblance to human exposure to influenza virus. Here, we inoculated NHPs with a highly pathogenic H5N1 influenza virus via the aerosol route and showed that aerosol infection did not affect clinical outcome, but did cause more widespread infection throughout the lower respiratory tract compared with the conventional approach. The uniform infection in all of the lung lobes and the bronchial epithelium afforded by the aerosol infection system offers an advantage in some experimental settings. For example, when researchers take lung tissues for multiple purposes (e.g., virus titration, histopathology, and various OMICs analyses), they need to take samples from different parts of the lungs. Therefore, infection via the aerosol route would minimize experimental variability.

Recently, Wonderlich et al.15 demonstrated that aerosol infection of NHPs with a highly pathogenic H5N1 avian influenza virus caused fulminant pneumonia that rapidly progress to acute respiratory distress syndrome, and some of the infected animals died or were humanely sacrificed due to respiratory failure. The same phenomenon has also been observed in the ferret model9–11; however, findings by Wonderlich et al. are not consistent with our findings here. The differences in disease outcomes between their study and ours may be caused by the different conditions used for the aerosol infection; that is, they used a head-only exposure chamber (into which the NHP’s head was inserted), which allowed the animals to inhale the aerosolized H5N1 virus with an exposure duration ranging from 15 to 34 min. In contrast, we used a standard inhalation mask, which is not a tight-fitting mask and allows the animals to breathe the aerosol mist through the nose and mouth spontaneously; the exposure duration was approximately 5 min, potentially leading to virus infection with a lower dose compared to Wonderlich’s study. In addition, other parameters, such as origin, gender, and age of the macaques, as well as the conditions for virus stock generation (i.e., egg-grown virus15 vs. MDCK-grown virus (our study), which could affect the resulting infectivity of the propagated virus), may have influenced the experimental results. Since it is difficult to standardize the parameters for NHP studies, we should pay attention to the experimental conditions when comparing results across different laboratories.

In summary, here we established a system for aerosol infection of NHPs with highly pathogenic H5N1 influenza virus. We found that experimental infection with aerosolized H5N1 virus led to a wider distribution of virus in the lower respiratory tract compared with liquid virus inoculation via the conventional method. However, no difference in clinical outcome was observed between the two inoculation methods.

Bivalent NDV-vectored vaccines, which have been developed to prevent diseases of economic importance to the poultry industry, have advantages over traditional vaccines (Table 3). Examples include infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotrachitis virus (ILTV), and avian metapneumovirus (aMPV).

IBDV, a birnavirus that infects chickens, is an important pathogen that causes severe immunosuppression and high mortality in young chickens. Live attenuated vaccines of moderate virulence (especially widely used intermediate plus vaccines) are used widely to prevent infectious bursal disease (IBD); however, they can cause severe side effects (symptoms consistent with IBD) in young chickens. Huang et al. developed a NDV-vectored IBDV vaccine (rLaSota/VP2) expressing the VP2 gene of IBDV, which is responsible for protective immunity against IBDV. The VP2 gene is inserted into the 3'-end non-coding region of the NDV genome. The live IBV vaccine is very safe in young chickens and protects SPF chickens against virulent NDV and virulent IBDV.

IBV, a coronavirus that infects birds, causes respiratory disease and renal disorders (the nephropathogenic strain) in poultry and poor egg production in laying hens worldwide. Currently available live attenuated IBV vaccines risk giving rise to new variants through recombination with field IBVs. This often reduces the efficacy of IBV vaccines. Importantly, live IBV vaccines interfere with the live attenuated NDV vaccine. To overcome the limitations of currently available live vaccines, Toro et al. developed a NDV-vectored IBV vaccine (rLS/IBV.S2) expressing the S2 subunit of the IBV S glycoprotein. Oculo-nasal immunization of chickens (1.0×107 EID50/dose) provided complete protection from clinical disease (mortality) after challenge with a lethal dose of virulent NDV (CA02). The protective efficacy of the rLS/IBV.S2 vaccine was also assessed using a heterotypic protection approach based on priming with a live attenuated IBV Mass-type vaccine followed by boosting with rLS/IBV.S2. The vaccine protected chickens against clinical disease after lethal challenge with a virulent Ark-type IBV strain, leading to a significant reduction in virus shedding when compared with that in unvaccinated/challenged chickens.

ILTV, a herpesvirus that infects birds, causes respiratory disease in chickens. Currently available live attenuated ILTV vaccines are effective, but there are concerns about safety in chickens because of the risks of virulence acquirement and latent infections during bird to bird transmission. Bivalent NDV-vectored vaccines against ILTV have been developed to overcome side effects associated with the live ILTV vaccine. Kanabagatte Basavarajappa et al. developed a NDV-vectored ILTV vaccine (rNDV gD) expressing glycoprotein D (gD) of ILTV. The protective efficacy of the rNDV gD vaccine against challenge with virulent ILTV and virulent NDV was then evaluated in SPF chickens. Immunizing chickens with rNDV gD (106 TCID50/dose) via the oro-nasal route induced a strong antibody response and provided a high level of protection against subsequent challenge with virulent ILTV and NDV, indicating that rNDV gD has potential as a bivalent vaccine.

From 2013 to 2015, 800 nasal swabs were collected from canines presenting primarily with respiratory symptoms at veterinary clinics in 11 of the 14 prefectures in Guangxi Zhuang Autonomous Region (Guangxi) (Fig. 1B). Of the 800 samples, 116 (14.5%) tested positive for influenza virus by PCR (Table 1). The influenza virus-positive animals were primarily pets (115/116) (see Table S1 in the supplemental material). Most were pure breeds, including Alaskan malamute (n = 16), bichon frise (n = 3), border collie (n = 3), chihuahua (n = 3), German shepherd (n = 7), golden retriever (n = 9), Siberian husky (n = 6), and poodle (n = 20). The majority of influenza virus-positive animals (~67%) were young dogs less than 1 year of age (median age of 5.5 months; range, 34 days to 9 years of age). The age profile of the influenza virus-positive animals did not differ significantly from influenza virus-negative animals (median age of 4 months; range, 2 days to 15 years). Twelve influenza virus-positive animals were rural Chinese dogs, including a 3-year-old female that died during treatment. It is unclear whether the death was caused by influenza. Approximately 70% (81/116) of the canines that tested positive presented with respiratory symptoms. Other animals presented with diarrhea, fever, vomiting, or injuries. Seven of the influenza virus-positive dogs were otherwise healthy. The first influenza virus-positive samples were identified in Liuzhou prefecture in April 2013 (Fig. 1C and Table S1). The first successfully isolated virus was collected from a 2-month-old female Samoyed presenting with respiratory symptoms in the Wuzhou district in October 2013 (Table 2).

Influenza virus-positive samples were identified in 9 of the 11 sampled prefectures and in each of the 3 years of sampling (2013 to 2015). Sampling was not conducted evenly across prefectures or across time (Fig. 1C and Table S2), so the percentage of positive samples in a location (Table 1) is likely to be biased and not appropriate for quantitative spatial-temporal comparisons. Sampling was conducted in multiple veterinary clinics in two prefectures (Nanning and Liuzhou), which explains the larger number of samples available from these locations (Fig. 1C). Sixteen viruses (2.0%) from seven prefectures spanning multiple regions of Guangxi were successfully isolated, and the whole genomes of the viruses were sequenced (Fig. 1B and Table 2). CIVs were isolated from five prefectures in the fall of 2013: Wuzhou (WZ), Hechi (HC), Fangchenggang (FCG), Qinzhou (QZ), and Liuzhou (LZ) (Fig. 2B). In 2014, CIVs were isolated in two additional prefectures: Chongzuo (CZ) and Nanning (NN). In 2015, CIVs were again isolated in Liuzhou and Nanning.