Inactivated vaccines are safer than live vaccines because they cannot replicate at all in a vaccinated host, resulting in no risk of reversion to a virulent form capable of causing diseases. However, they generally provide a shorter length of protection than live vaccine and generally elicit weak immune responses, in particular cell-mediated immunity, as opposed to live viral vaccines. For this reason, inactivated vaccines are administered with potent adjuvant, and require boosters to elicit satisfactory and a long-term immunity. Vaccines of this type are generally created by inactivating propagated viruses by treatment with heat or chemicals such as formalin or binary ethyleneimine. This procedure can destroy the pathogen's ability to propagate in the vaccinated host, but keeps it intact so that the immune system can still recognize it. Although inactivated virus vaccines have been used for preventing various types of viral diseases over the decades, they need further development for controlling newly emerging diseases.

For examples, influenza virus vaccines are continually improved to contain all serotypes because many new serotypes emerge in new outbreaks. As with other approaches, many studies have been focused on searching for better adjuvants which enhance immune responses in accordance with inactivated vaccines as well as help to overcome the inhibitory effects of maternal antibody. For live AIV vaccines, the possibility of reassortment between live vaccine strain and field isolates and of back mutation from low-pathogenic to highly pathogenic viruses lead to serious concerns for vaccine safety. Thus, prior stimulation of the immune system using some immunomodulators followed by vaccination with inactivated vaccines may be needed to confer better protective immunity within a short period of time and may be promising in controlling LPAI H9N2.

About 81,000 people received national support (120,000 won per person; about 100 USD) for immunoglobulin administration, antigen and antibody tests for hepatitis B to prevent vertical infection from infected mothers. The participation rate was 60% in 2002, 89% in 2003, 96% in 2004, 98% in 2005, and 98% in 2006.

Although live viral vaccines are produced in several ways, the most common method for creating vaccine strains is made through passing viruses in cell cultures, embryos, or suitable materials. For instance, a selected virus strain is serially passed in chicken embryos, resulting in better replication in chick cells but with a lost ability to replicate in animals cells of the target host. Also, the live vaccine viruses can be generated by inducing random mutations on viral genome and followed by selecting a non-virulent mutant incapable of causing clinical diseases. An alternative to creating attenuated viral strains is that viruses are serially passed in a non-adapted host until they can effectively propagate, and the loss of their pathogenicity in the original host is confirmed. All of these methods involving passing virus in suitable matter can create a new version of the virus that can still be recognized by animal immune systems but cannot replicate well in a vaccinated host. This produces the necessary immune response in the host if the host is challenged with infection by the original pathogenic virus. Most importantly, protection efficacy derived from a live attenuated vaccine typically outlasts that provided by a killed or inactivated vaccine. Nevertheless, these vaccines still have a residual virulence or a risk of reversion to a virulent phenotype. A single point mutation on certain gene may tend to induce attenuation of virus but may lead to back mutation, resulting in the wild type virulent virus. Considering the relatively high mutation rate of RNA viruses, it is taken into consideration that live vaccine viruses would need to have multiple mutations on various genes of the viral genome when developing an attenuated vaccine strain. Despite these drawbacks of live vaccines, live vaccines play an important role in preventing and eradicating viral diseases in industry animals. Interestingly, a potent adjuvant is not necessary for the formulation of live vaccines because live vaccine viruses are capable of infecting target cells and provoking immune responses to injected viruses. Additionally, live vaccines can easily be administered by various routes, such as injection, drinkable water, or instillation into the nasal cavity or eyes.

For examples, modified live vaccines for avian influenza virus (AIV) have been used in many countries to control AIV infection since killed vaccines are moderately effective but multiple injections are needed to develop protective immunity. Although vaccination is not perfect, it would be the most promising control means for the low pathogenic avian influenza (LPAI) H9N2 to date. Porcine epidemic diarrhea virus (PEDV) is an agent causing severe entero-pathogenic diarrhea in pigs, which leads to significant economic losses in Asia. PEDV strain DR13, a field isolate, was attenuated by serially passing the virus on Vero cells and tested for its virulence in piglets and sows. Vero cell-adapted virus showed reduced pathogenicity and induced protective immunity in pigs, indicating that this attenuated virus may be a vaccine candidate.

The goal for managing influenza is decreasing morbidity and mortality rate to achieve reduced disease burden. The vaccination program selected the high risk groups for vaccination with priority. The influenza surveillance system operated and monitored daily and weekly surveillance for influenza and influenza like illness altogether with laboratory surveillance.

The route of administration and delivery method used in vaccination may affect vaccine-induced immune responses, antigen presentation, and type of MHC molecule involved in the resultant response. Live attenuated IB vaccines have gained wide application via injection orally and through aeronasal spray. Killed or inactivated DNA vaccines and peptide-based vaccines are commonly used via injection routes. Some improved methods have been used to deliver recombinant proteins, plasmid DNA, and peptide vaccine. For example, an IBV-DNA vaccine carrying S1- and/or N-protein of IBV has been delivered orally using attenuated Salmonella enterica serovar Typhimurium strain. Interestingly, both humoral and mucosal immune responses were shown to significantly increase following oral and intranasal immunization. Vaccinated chickens were protected against homologous challenge. Other approaches recorded success using a Lactococcus lactis bacterial system to deliver IBV vaccine, and this approach led to an efficient mucosal immune response [99, 100].

Virus-like particle (VLP) has been a new focus of interest in vaccine development. This technology utilizes the immunogenic properties of a live virus without potential to retain pathogenic effects. A VLP-based IBV vaccine has been developed using the IBV-M- and IBV-S-genes. Immunization of mice with the candidate vaccines demonstrated high levels of cell-mediated immunity, comparable with the results obtained using H120 live attenuated virus vaccine. Similarly, a chimeric VLP vaccine has been synthesized using M1 protein of avian influenza H5N1 virus and fusion protein “NA/S1” derived from IBV-S1 protein and the cytoplasmic and transmembrane domains of H5N1 avian influenza NA protein. The chimeric vaccine induced significant S1-specific antibodies in mice and chickens, neutralizing antibody in chickens, and increased IL-4 secretion in immunized mice. Putting together these findings, there is a huge potential for VLP-based vaccines as innovative candidate and their use may provide a delivery system for the newer IBV vaccine.

Only one isolate, defined as parrot/Indonesia/BX9/16, was sequenced for the partial S1 gene of IBV using XCE2+/XCE2− primers (Table-1). Nucleotide sequencing of 323 nucleotides from the partial S1 gene showed that there was no difference in the nucleotide sequence of the parrot/Indonesia/BX9/16 gene when compared with IBV 4/91 Israel variant 1 (AF093794.1) and the 4/91 vaccine strain (KF377577.1) (Figure-1). The nucleotide and amino acid pairwise distance also showed 100% homology with the IBV 4/91 Israel variant 1 (AF093794.1) and the 4/91 vaccine strain (KF377577.1). However, differences were observed between the sequenced gene, the H120 (FJ888351) positive control, and the non-chicken IBV-like peafowl/GD/KQ6/2003 virus (AY641576) (Table-4). A phylogenetic tree (Figure-2) of the aligned nucleotide sequence of the partial S1 gene was constructed using the maximum likelihood method with Mega 7 software with 1000 bootstrap value. The tree showed a close relatedness of viral isolate, parrot/Indonesia/BX9/16, to the IBV strain 4/91 variant 1 Israel (AF093794.1), the 4/91 vaccine strain (KF377577.1), CK/CH/YN/SL 1301-1 (KX107779.1), chicken/Attock/NARC-786/2013 (KU145467.1), and gammaCoV/Ck/Poland/G193/2015 (MK576138.1), whereas there were differences observed when compared with the H120 vaccine (FJ888351.1) positive control.

With the aim of improving prevention and control of viral outbreaks, the Chinese government has been investing continually in the advancement of science and technology since 2003, including the appropriation of more than 12 billion RMB for research and development related to combating SARS, influenza, and other major infectious diseases. Meanwhile, China has built 11 national technology platforms, 11 national research centers, 6 national key laboratories, and 2 national engineering laboratories. In 2010, the Chinese National Influenza Centre was designated as a WHO Collaborating Centre for Reference and Research on Influenza. All these laboratories and funding contributed to application of advanced technologies in preventing and controlling infectious diseases.

Above all, quick identification of pathogens is a prerequisite to controlling emerging epidemics. To achieve it, China has developed state-of-the-art pathogen isolation and identification technologies such as high-throughput sequencing method. In contrast to the SARS-Cov debacle, H7N9, H10N8, and H5N6 were identified within China [28–30]. BGI, a Chinese company, helped Germany sequence the pathogen Escherichia coli O104:H4 within a week using high-throughput sequencing technology in 2011. Meanwhile, Chinese researchers exploring the genesis and source of emerging viruses have found that bats are natural reservoirs of SARS-like coronaviruses and have demonstrated that domestic fowl play an important vector role for H5N1 and H7N9 [4, 32, 33].

The government encourages the development of diagnostic reagents, vaccines, and medicines as well as prophylactic equipment (e.g., infrared thermometers). China's national vaccine regulatory system was confirmed to meet WHO standards in 2011. China has developed SARS, H5N1, H1N1, and H7N9 vaccines (Table 1) and became the first country to use an H1N1 vaccine. China now produces oseltamivir (like Tamiflu®) and peramivir (like Rapivab®), obviating the need to import antivirals.

China's improvements in research funding and technical capabilities have led to a series of important findings. For example, Chinese researchers have revealed the crystal structures of key viral proteins (e.g., SARS-Cov protease, H1N1 neuraminidase N1, and H5N1 polymerase PAC-PB1N complex) [36–38], which is useful for drug design, and discovered an oseltamivir-resistance mechanism in H7N9. A traditional Chinese medicine (TCM) herbal formula was confirmed to reduce H1N1 influenza-associated fever safely and with efficacy similar to that of oseltamivir in a randomized clinical trial.

In recombinant or subunit vaccines, consideration is given to the presence or absence of posttranslational modification associated with the vaccine antigen. However, thorough knowledge of the chemistry and biology of the immunodominant antigen is needed to guide selection of a suitable expression system, since outcomes may differ from bacteria, yeast, mammalian, baculovirus, and plant expression systems. Different expression systems have been used to generate recombinant protein antigen. An attempt, using a vaccinia virus-based IBV vaccine, failed to produce antigen enough to induce significant antibody responses in mice. It was proposed that the use of vaccinia virus-based vaccines may be hindered by issues of safety regarding vaccinia virus itself, as well as its poor replication ability in avian cells. In another study, a baculovirus-based vector was used to express the S1-glycoprotein of Korean nephropathogenic KM91 strain. Immunization of chickens with the KM91 vaccine resulted in 50% kidney protection following a homologous challenge. Similarly, an S1-glycoprotein of IBV has been expressed in a transgenic potato under the control of a cauliflower mosaic virus (35S) promoter gene. This success could be useful in designing food-based oral IB vaccines for use in poultry.

An improved “BacMam” virus surface display technology, a modified strategy from baculovirus vectoring, was used recently to display the S1-glycoprotein of IBV-M41 serotype. Subsequent experimental trials with the vaccine resulted in significant humoral and cell-mediated immune responses. About 83% of the challenged birds were shown to be protected, which is comparable to 89% protection obtained in birds immunized with commercial inactivated vaccine.

All challenge experiments with H5N1 HPAIV were performed under the guidelines of the Animal Care and Use Committee. Our experimental protocol, describing that animals could die of the HPAIV infection and that animals would be euthanized if they manifested severe symptoms, such as inactivity, loss of appetite, loss of 20% or more body weight, etc., for 24 h, was reviewed and approved by the Faculty of Veterinary Science Animal Care and Use Committee. All experiments with live HPAIVs were performed in a biosafety level 3 containment laboratory at Mahidol University, Thailand, after approval by the faculty.

Sera were collected from all chickens before inoculation and from virus-infected and mock-inoculated chickens at 10 days post infection, and the sera were treated with receptor destroying enzyme to remove any nonspecific inhibitors. The HI test was performed using treated sera, chicken red blood cells, and four hemagglutination units of H5N1 HPAIV. Pre-inoculation sera from all chickens used in this study were determined to be serologically negative for H5-specific HI antibodies.

Promptly after the SARS epidemic, the Chinese government accelerated the establishment of an effectual and national unified management system for public health emergencies and enacted two laws: the Regulation on Public Health Emergency and the Measures for the Administration of Information Reporting on Monitoring Public Health Emergencies and Epidemic Situation of Infectious Diseases [18, 19]. In addition to defining the standards and grades of public health emergencies, these laws support the construction of command systems and clarify the responsibilities and the leadership role of the chief executive of central and local governments previously held by the Centers for Disease Control and Prevention (CDC). Accordingly, the executive capacity of the command systems has been much improved. Importantly, China also established an emergency information dissemination system to enable timely (within 2 hours), accurate, and comprehensive release of information. Moreover, both central and local governments are now expected to be prepared for a public health emergency response (e.g., techniques, personnel, materials, and management preparedness).

In 2004, China revised the Prevention and Treatment of Infectious Diseases Law, adding SARS and avian flu as notifiable diseases and revising the law to comply with the principal rules of infectious disease prevention and control (i.e., infection source control, interruption of route of transmission, and susceptible people protection). The law adheres to the “five early” principle of early detection, diagnosis, reporting, isolation, and treatment. Early isolation can restrain contagion. The law applied China's experience in emerging epidemics to prevent and control 37 infectious diseases.

The Chinese Ministry of Health (CMH) issued a technical guide for avian flu prevention and control in 2004. It requires that suspected and confirmed cases be handled quickly at designated hospitals with the equipment to prevent nosocomial infection. The epidemiological and etiological data of patients should be acquired to enable determination of human-to-human transmission capacity. The guide suggests that persons exposed to dead poultry infected by avian flu virus be isolated and observed for 7 days. In order to control zoonotic infectious diseases, China revised its Law on Animal Disease Prevention in 2007, adding an animal epidemic surveillance and reporting system for timely disclosure of animal epidemics and providing compensation to farmers for economic loss due to culling infected or potentially infected poultry.

Guided by the aforementioned laws, a series of social innovations enacted after the SARS epidemic have improved China's ability to combat emerging diseases. The CMH issued a swine flu prevention guide on April 29, 2009, 12 days before the first reported H1N1 case. On April 3, 2013, 4 days after the first H7N9 confirmed case, the CMH also issued a nosocomial H7N9-infection prevention guide. Administrative reforms resulted in better handling of H5N1, H1N1, and H7N9 relative to SARS. Importantly, in keeping with its move toward greater transparency, after confirmation of the first H1N1 case on May 11, 2009, China posted patient zero's travel information publicly on the same day. Likewise, after confirming the first H7N9 avian flu case on March 30, 2013, China published detailed information about the patient's medical consultation. Transparency helps to subdue rumors and maintain social stability.

The first clinical case occurred in the park in October 2010 (Fig 1, enclosure A1), where a mother (Tosha) was housed together with one son (Sheppard) and three daughters (Grey, Izzy, Split). The index patient was the 1.5-year-old son Sheppard. He presented with ulcerative skin lesions on the lower lip and nostril. The affected areas were biopsied and the animal received long-acting broad-spectrum antibiotic (8 mg/kg cefovecin once i.m.). The animal was isolated in the stable ten days after initial symptoms when histopathological results of the submitted samples indicated presumptive poxvirus infection.

Eleven days after the first lesions had been noticed in the male, a fissure was observed on the tongue of the mother (Tosha), and on the following day one of its sisters (Izzy) had a lesion on the upper lip. The next day, respiratory distress became obvious in another sister (Grey). At that time, the entire group was isolated and put inside the stable. Cefovecin was given by remote injection to all of them in order to prevent secondary bacterial infections.

The female Grey with respiratory symptoms died 12 days after onset of symptoms and was found partly eaten by the others. The other animals recovered uneventfully. Only one of the five animals (Split) did not show any symptoms.

The methods performed in this research have been approved by the Ethical Committee of the Faculty of Veterinary Medicine, Bogor Agricultural University, Indonesia, which were validated with the certificate number 058/KEH/SKE/IV/2017.

Vaccination continues to have a major impact on the health of humans and animals. Furthermore, vaccination of animals is proving to be effective in reducing transmission to humans. Understanding linkages between innate and adaptive immunity are improving formulations of new, as well as existing, vaccines, making them more effective.

Vaccination has saved more lives than many other therapeutic interventions combined. Prominent examples are smallpox and polio, where prior to immunization, millions of people died annually. Indeed, the World Health Organization estimates that vaccination has prevented paralysis in over eight million people since polio eradication programs began in 1988. These are just two examples of many diseases that have been effectively controlled by vaccination and thus have saved millions of lives. As a result, our children today are protected from diseases, such as measles, pertussis, tetanus and diphtheria to name a few. However, even with such overwhelming statistics, a strong anti-vaccine lobby exists to dissuade parents from vaccinating their children. This is both wrong and ill-informed placing individuals and communities at risk. These individuals benefit from being surrounded by vaccinated children by an effect commonly referred to as herd immunity. Unfortunately, this is being ignored by the strong lobby groups, who base their rhetoric on a few very selected falsehoods and ignore the benefits of immunization. The classical falsehood is that vaccines cause autism. This has been disproved many times but still gets brought up by the anti-vaccine lobby groups as well as the popular press. Thus, communicating the benefits of immunization to the broad public represents an important challenge for all of us.

Another big challenge to vaccination today is that most vaccines are delivered by needle injection. This often results in local mild reactions and these minor adverse events are being used as a reason to dissuade individuals not to vaccinate their children. Indeed, in our society, all forms of ‘preventive medicine' are looked upon less favorably than therapies. Many of our therapeutic drugs cause significantly greater adverse reactions than vaccines. However, they are accepted because they are treatments. Anti-cancer drugs are one of the best examples, which may have many side effects. The reason for this dichotomy is that our society is much less accepting of preventative medicine versus therapeutic approaches to disease management. If the focus continues to favor expensive therapeutics over economic preventative medicine, escalating costs of health care will bankrupt society.

Important areas for the use of vaccines are the emerging zoonotic infections that can cross the species barrier and that are transmitted from humans to animal, or vice versa. Examples include the recent pandemic influenza, severe acute respiratory syndrome and avian influenza, to name a few. In fact, over 70% of new emerging and re-emerging diseases are zoonotic in nature. Since drugs do not exist to control these diseases, vaccines are the best choices for disease control. Indeed, we should place more emphasis on immunizing the animal species concerned to reduce the chance of transmission to humans. Similarly, contamination of food and food products with disease causing organisms, such as Salmonella, Escherichia coli or Campylobacter, are responsible for billions of dollars in losses every year. The recent Listeria outbreak in Europe caused over 40 deaths and millions of euros in direct and indirect costs, highlighting the importance of food safety and the need for vaccines that can enhance the safety of our food and food products (food safety vaccines). An example of such vaccines is the development of an E. coli 0157:H7 vaccine for cattle to reduce shedding of the bacterium into the environment and contamination of meat and meat products, thereby reducing the chance of human infection.1 Thus, one can control infection rates in humans by immunizing animals. Similarly, immunization of humans can protect animals since diseases can move from humans to animals, as recently shown for influenza virus H1N1 transmission from humans to animals,2 providing support for the ‘One World One Health' concept.3

The majority of vaccines used today have been developed by conventional methods and fall into two categories, live vaccines and killed vaccines.

In the case of live vaccines, the pathogen is passaged in culture multiple times resulting in specific mutations that render the pathogen less virulent than field strains of the agent. These vaccines are then administered to individuals; the agent replicates and induces a full array of immune responses leading to protective immunity upon subsequent exposure to the pathogen.4,5 In the case of killed vaccines, the pathogen is chemically inactivated to prevent its replication but not so dramatically as to interfere with the antigenic components of the pathogen, which then induce a more restricted immune response, although often sufficient to prevent disease. This later group of vaccines are generally mixed with immune stimulants (adjuvants) to enhance the immunity to the killed pathogen. Killed vaccines can induce a mild reaction in some vaccinated individuals since they are often administered by needle injection.

To reduce side effects of vaccines, and improve efficacy, the focus over the last few years has been on developing novel vaccines, delivery systems and adjuvants. The merging of molecular biology and immunology has dramatically enhanced our ability to improve vaccine efficacy and safety. For example, by identifying and deleting virulence genes in a virus or a bacterium, one can reduce the ability of the agent to cause disease. Back mutations are almost impossible to occur, which make these vaccines extremely safe. More importantly, such vaccines can be delivered by the natural route and induce a wide array of immune responses generated by infection leading to solid immunity. For example, if delivered mucosally, they induce both mucosal and systemic immunity, which is critical, since most pathogens enter by the mucosal surfaces. This not only reduces the disease in the vaccinated individual but also dramatically reduces the quantity of pathogens secreted into the environment should this vaccinated individual get infected.6 This approach has been further improved by using molecular biology to produce vectored vaccines or killed vaccines. For most pathogens, only a few (1–5) specific antigenic components are required for induction of protective immunity. Thus, in the case of bacteria, the other 1000+ proteins are irrelevant to induction of protective immunity and indeed, some of these proteins may actually be detrimental to protective immunity. Using what is called reverse vaccinology,7 one can screen for these protective antigens and insert them into a vector—examples include the yellow fever virus or pox virus and adenovirus vectors for HIV vaccination8,9,10 or introduce them into a bacterium or yeast to produce large quantities of killed antigens in bioreactors. These so-called subunit antigens cannot replicate, they are safe, and since the response is specific for the selected antigens only, they allow us to distinguish between vaccinated and infected individuals and animals, so called marker or differentiate infected from vaccinated animals vaccines. In fact, global trade of animals and animal products is largely regulated by the policies around the absence of antibodies to specific infectious diseases.11

Unfortunately, most subunit vaccines are not very immunogenic and need to be formulated with immune stimulants (adjuvants). The majority of killed vaccines in humans were formulated with alum. The regulatory agencies favor alum as an adjuvant because of extensive experience with it. Unfortunately, it produces a skewed immune response that favors systemic antibody production and gives little mucosal or cellular immunity. In many cases, cellular and mucosal immunity is required to control an infection; thus, there is room for improvement of these vaccines. Indeed, much can be learned from the development of vaccines for animals and many different adjuvants and delivery strategies have been successfully used for decades. The development of safe and effective adjuvants for humans is a hot topic in vaccine research, and as a result we are starting to see licensure of novel adjuvants such as MF59, AS01-AS03, ISOMS, etc.

Due to a better understanding of the immune system, we are now able to tailor the quality as well as the magnitude of the immune response. If one then combines adjuvants with appropriate formulations that can be introduced at mucosal sites, it provides the best chance of inducing mucosal immunity with a safe killed vaccine without needle injection.12,13,14 This is critical in resource constraint environments where expensive needles are difficult to obtain. Furthermore, such an approach helps to reduce the number of immunizations required to provide protective immunity, something that is urgently needed in developing countries where access to vaccines is limited, as well as lowering the cost by reducing the amount of antigen needed (antigen spearing).15 The best example occurred following the outbreak of H1N1 influenza where the ability to produce large quantities of vaccine was limited due to time constraints between the appearance of the virus and need for vaccination of the population. Thus, if one can expand the number of individuals immunized by 10-fold, that would dramatically improve the control of the disease.

Based on the recent advances in understanding immune responses and identifying antigens involved in inducing protection from a number of infectious agents combined with the willingness of regulatory agencies to begin licensing combination, adjuvants gives us confidence that we will be able to develop new vaccines to new agents and also improve existing vaccines that can even be safer than our vaccines used today. In this way, all members of society will benefit.

Serum samples were tested for antibodies by immunofluorescent assay (IFA) by using CPXV-infected HEp-2 cells and a FITC-conjugated goat-anti-human IgG (H+L) as described previously or by indirect ELISA by using vaccinia virus-infected HEp-2 cell lysate as antigen. To this aim, UV-inactivated cell lysate was prepared by infecting HEp-2 cells (ATCC, CCL-23™) with vaccinia virus strain New York City Board of Health (ATCC, VR-1536™) by using RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) as described before.

For ELISA, PolySorp microwell plates (Nunc) were coated with 100 μL of lysate of infected or non-infected HEp-2 cells at 4 μg/mL in 0.1 M carbonate buffer (pH 9.6) over night at 4°C. Between each step, plates were washed with 4 × 300 μL of washing buffer (100 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Plates were blocked at room temperature for 1 hour with 200 μL per well of 3% bovine serum albumin (BSA, Carl Roth) in washing buffer. Subsequently, 100 μL per well of non-inactivated sera were incubated at a 1:100 dilution in washing buffer supplemented with 0.25% BSA for 1 hour before detection with either goat anti-cat IgG (H+L) HRP, goat anti-dog IgG (H+L) HRP, or goat anti-mouse IgG (H+L) (Dianova, used at a 1:5000 dilution). Finally, signals were developed for 15 minutes by using 100 μL of SeramunSlow TMB substrate (Diavita) per well before the reaction was stopped by addition of 100 μL of 0.25 M H2SO4. The resulting absorption was read at 450 nm referenced to 620 nm at an ELISA reader (Tecan). Each serum was tested in two replicate measurements on both infected and non-infected HEp-2 cell lysate. Binding signals against non-infected HEp-2 cell lysate were subtracted from signals against vaccinia virus-infected cells, and sera with a differential binding signal above 0.05 were considered positive.

The index patient was a retired man aged 60 with a history of hypertension for more than 10 years. He developed a fever, cough, and shortness of breath on 8 March 2013 and was admitted to a Chinese hospital (hospital A) on March 11 with a left upper lobe inflammation. Initial blood routine testing identified no abnormality, except for increased hypersensitive C reactive protein (28.5 mg/L). He was treated with azithromycin and piperacillin-sulbactam. Because of progressive respiratory distress, persistent hyperpyrexia, and hypoxemia, he was transferred to the hospital’s intensive care unit in the afternoon of 15 March with a diagnosis of viral pneumonitis and type I acute respiratory distress syndrome. He was again transferred to another tertiary hospital’s (hospital B) intensive care unit because of deterioration on 18 March and began to treatment with oseltamivir the next day. He did not develop diarrhoea during the course of the disease. He died of disseminated intravascular coagulation and multi-organ failure on 4 May.

The index patient’s daughter, an unemployed woman aged 32, was otherwise healthy without any underlying illnesses. She provided bedside care for her father until he was admitted to the second hospital’s intensive care unit. She developed fever with body temperature 39.6°C and cough on 21 March. On 24 March, she was admitted to the pneumology department of the same hospital (hospital B) with pneumonia in the left upper lobe. Initial testing showed leucocytopenia (2.0×109/L), lymphopenia (0.7×109/L), and slight hypoxia. She was treated with antibiotics (azithromycin and piperacillin-sulbactam). Oseltamivir (75 mg twice a day) was administered on 24 March. She was transferred to intensive care on 28 March because of persistent hyperpyrexia, respiratory failure, and acute respiratory distress syndrome. Although treated with mechanical ventilation, broad spectrum antibiotics, oseltamivir, immunological therapy, and fluid resuscitation, she died of multi-organ failure and cardiac arrest on 24 April. Table A in appendix 1 summarises the clinical characteristics of the two patients.

Since the highly pathogenic H5N1 avian influenza virus (AIV) was first transmitted from birds to humans in Hong Kong in 1997, other pathogenic AIVs, including H7N2, H7N3, H7N7, and H9N2 have been reported in China and other parts of the world.1,2,3,4 However, no human infections with the novel H7N9 virus have been reported until now from China. Here we report a fatal case caused by H7N9 AIV in the very early stage of this endemic.

A 52-year-old retired female resident in Shanghai was admitted to Fudan University affiliated Huashan Hospital due to 7-day history of pyrexia, accompanied by cough, chest stuffiness and dyspnea for the past two days. The patient had a sudden onset on March 27th, 2013 with rigors, and the highest temperature reached 40.6 °C but with no obvious symptoms of cough, pharyngalgia, stuffiness, dyspnea, nausea, vomiting, abdominal pain or diarrhea, and did not receive medication. The next day the patient visited emergency room and chest auscultation demonstrated rough breath sounds, absence of rales. Laboratory tests showed a leukocyte count of 5300/mm3, with 72% of neutrophils, and C reactive protein (CRP) of 26.8 mg/L. The patient was given antibiotics. On the third day, the patient took chest radiography and showed small patchy shadows in lower lobe of the right lung. The patient was given antibiotics intravenously for three consecutive days, still without cough, expectoration or shortness of breath, although her temperature was not resolved. On day 7 after onset of fever, due to quick progression of the symptoms, including cough, chest stuffiness and shortness of breath, the patient visited the emergency department of Fudan University affiliated Huashan Hospital again. Unfortunately, the arterial blood gas analysis showed severe hypoxemia, pH 7.54, PaCO2 4.33 kPa, PaO2 3.66 kPa, and saturation of oxygen 61.3% on room air. In the meantime, chest computed tomography (CT) demonstrated diffuse bilateral consolidation with right pleural effusion (Figure 1). Laboratory findings indicated a leukocyte count of 3290/mm3, with 92% of neutrophils and 5.5% of lymphocytes; platelets of 155 000/mm3; increased myocardial enzymes, prolonged prothrombin time and abnormal serum electrolytes. The patient was suspected severe flu with acute respiratory distress syndrome and thereafter was given endotracheal intubation and placed on a mechanical ventilator. Intravenous injection of methylprednisolone 40 mg was administered to inhibit inflammation and alleviate edema in the lung. On April 3rd (day 8), antimicrobial regimen as well as immune globulin therapy and the methylprednisolone were maintained. However, the patient's condition worsened and died of acute respiratory distress syndrome.

On April 4th, the throat swab was sent to the laboratory of Chinese Center for Disease Control and Prevention and the result revealed the presence of H7N9 avian influenza A virus. Meanwhile laboratory tests for pathogens, including respiratory syncytial virus, influenza B virus, human metapneumovirus, cytomegalovirus, herpes simplex virus 2, human immunodeficiency virus, and severe acute respiratory syndrome coronavirus (SARS-CoV), were all negative. This is one of the six laboratory confirmed fatal cases of H7N9 infection reported to World Health Organization.

To date (April 7th, 2013), a total of 21 cases have been laboratory confirmed with influenza A (H7N9) virus in China, including 6 deaths, 12 severe cases and 3 mild cases.5 An inter-government task force has been formally established, the animal health sector has intensified investigations into the possible sources and reservoirs of the virus. However no definite history of contact with livestock was found in this case. The patient also did not feed or eat poultry at households. Some other confirmed cases had close contact with poultry or with associated environment. It is interesting to note that the virus has also been found in a pigeon in a market in Shanghai. It is unclear how this case was infected by H7N9 AIV, similar to some other cases without known recent close contact with birds or poultry. However, influenza A H7 viruses are a group of influenza viruses that normally circulate among birds and the influenza A (H7N9) virus is one subgroup among the larger group of H7 viruses.6 Although the patient denied close contact with poultry, H7N9 virus was detected among poultry at local market. The most likely source of the virus in this case seems to be from the environment or food contaminated with this novel virus. The emergence of H7N9 AIV infections in humans suggests the avian influenza virus evolves to achieve adaptations including the ability to bind to mammalian cells and to break the species barrier. Fortunately, among close contacts of this case, the patient's husband was pyretic with a temperature of 38 °C, but negative for H7N9 AIV detection and recovered soon, indicating no evidence of human-to-human transmission up to this point. At this time there is no evidence of ongoing human-to-human transmission. The possibility of animal-to-human transmission is being investigated, as is the possibility of person-to-person transmission.

To date, the overall proportion of fatal cases among those reported 21 cases was 28.6% (6/21), lower than that in H5N1 AIV infection in humans (average 59%).7 According to the experience from H5N1 AIV treatment, cases with a fatal outcome were admitted to hospital later (median, 5 days) than those who survived (median, 1 day).7 All fatal cases in Shanghai including this patient were admitted to hospital very late until the symptom of shortness of breath developed. Meanwhile, due to unclear cause of the disease, fatal cases, including this patient, had not been given the anti-influenza drugs such as neuraminidase inhibitors (oseltamivir) as soon as possible and within 2∼4 days of disease onset, leading to loss of valuable salvage time for the severe cases. Since the laboratory testing conducted in China has shown that the influenza A (H7N9) viruses are sensitive to oseltamivir and zanamivir, and if these drugs are given early in the course of illness and the patients are hospitalized earlier, the survival rate of this new emerging infectious disease might be significantly improved. Future strategies to prevent fatal cases should include prompt laboratory diagnosis and early antiviral and steroid treatment, and good supportive care.

In the 1980s, hepatitis B surface antigen (HBsAg) positive rate was shown to be 6.6 to 8.6% in all populations12). However, since hepatitis B vaccine was first introduced in 1982, and then subsequently included in the immunization schedule table of the Korean Pediatric Society in 1991, and also in the national immunization program in 1995, HBsAg positive rate has significantly decreased9,13).

According to the 2008 National Health and Nutrition Survey in Korea, it has significantly decreased to 2.9% in populations aged 10 years or more (Fig. 4)14). Specifically, HBsAg positive rate in those aged 4 to 6 years was found to be 0.2%15) from a study with nationwide sampling in 2006. This data could be a basis for certification from the WHO that hepatitis B has been well-controlled in the ROK.

Nucleic acid extraction from clinical swab samples was performed with taco™ preloaded DNA/RNA Extraction Kit (GeneReach USA) on taco™ mini Nucleic Acid Automatic Extraction System (GeneReach USA) and according to the manufacturer's instructions. The Taco mini system can be driven by AC and DC current (net or battery power), and its 5 kilograms allows users to transport it to the field (Figure 1). The taco preloaded DNA/RNA Extraction Kit is a magnetic bead‐based total nucleic extraction reagent including Lysis buffer, Washing Buffer A, Washing Buffer B, and Elution Solution are all preloaded and sealed by foil. They can be easily transported under ambient temperature and used upon needed. Briefly, 100 μL of each swab sample was added to individual wells in the first row of preloaded 48‐well extraction plate. The preloaded 48‐well extraction plate was inserted into the instrument, and the “start” button was pressed. The extraction time was 25 minutes, and the eluted nucleic acid was available in the last row of the preloaded 48‐well extraction plate. The nucleic acid was used immediately or stored at −80°C until use.

Six-week-old BALB/c female mice were purchased from Southern Medicine University, Guangzhou, China, and were housed, fed in microisolator units according to the Veterinary guidelines of South China Agricultural University and all animal experiments were approved by the South China Agricultural University Institutional Animal Care and Use Committee.

Indirect enzyme-linked immunosorbent assay test (ELISA) was done to analyze the serum samples. Commercially available ELISA kits (BioChek®, Reeuwijk, Netherlands) of ART, ORT, ILT, and IBV were used to detect the antibodies. Serum samples were diluted at 1:50 dilution in dilution buffer, followed by 1:10 dilution, and final dilution of 1:500 was used as working samples for respective ELISA. 100 μl of negative and positive controls was added into antibody coated plate wells A1, B1 and C1, D1, respectively, remaining 92 wells were filled with samples. After that, plate incubated at room temperature for 30 min in case of IBV and 60 min in case of ART, ORT, and ILT. Meanwhile, conjugate and wash solutions were prepared according to manufacturer’s instructions. After incubation, contents of wells were aspirated and washed four times with wash buffer (350 μl). Then, the plate was inverted and tapped firmly on absorbent paper to remove the moisture. Then, 100 μl conjugate reagents were added on each well. Again, the plate was incubated for 30 min at room temperature in case of IBV and 60 min in case of ART, ORT, and ILT, respectively. After incubation, washed the plate with wash buffer following the procedure described previously. Then, the wells of microtiter plate were filled with substrate and incubated for 15 min at 22°C–27°C in case of IBV and 30 min for ART, ORT, and ILT. After incubation, the reaction was stopped by adding 100 μl stop solutions. Finally, the optical density value of each sample was measured at 405 nm within 15 min after adding stop solution, and the results were recorded by calculating sample to positive (S/P) ratio and antibody titer.

Purified virus particles were absorbed onto carbon-coated copper grids and incubated with a monoclonal antibody (mAb) against HA of H9N2 (prepared in our lab) for 1 h. The grids were incubated with goat anti-mouse IgG labeled with 5-nm gold particles (Sigma) for 30 min. After 3 additional PBS washes, the grids were stained with 2% phosphotungstic acid (Sigma, St. Louis, MO, USA) and examined under transmission electron microscopy (H-7500; Hitachi, Tokyo, Japan).

Controversy exists regarding the best method of protecting the public against the potential release of smallpox as a biological weapon (Bicknell, 2002; Fauci, 2002; Halloran et al., 2002; Kaplan et al., 2002; Mack, 2003). Infectious disease modeling plays an important role in this dialog, and the biology of the transmission pathway, the focus of this review, is critical to producing appropriate predictive models and understanding which controls will work best under varying conditions (Ferguson et al., 2003).

The rapidity with which smallpox would spread in a developed nation is not known and is a major source of uncertainty in models used for public health planning (Ferguson et al., 2003). The basic reproductive number (R0), which describes the tendency of a disease to spread, has been estimated for smallpox from historical data and outbreaks in developing countries (Gani and Leach, 2001; Eichner and Dietz, 2003). Because R0 is a function of the contact rate between individuals, it can be affected by changes in the environment (Anderson and May, 1991). A potentially important difference between contemporary environments and those used to estimate R0 is that today many buildings, including hospitals, mechanically recirculate air. If smallpox was almost entirely transmitted by mucosal contact with large droplets (aerodynamic diameters >10 μm), which can only occur following “face-to-face” exposure over distances of a few feet, then change in the built environment would not change the contact rate between individuals. If, however, smallpox was frequently transmitted from person-to-person by airborne droplet nuclei [fine particles with aerodynamic diameters of ≤2.5 μm capable of remaining suspended in air for hours and of depositing in the lower lung (Hinds, 1999)] then mechanically recirculated air systems would increase the contact rate, R0, the risk of epidemic spread, and the difficulty of hospital infection control. Unfortunately, leading authorities disagree regarding the relative importance of fine and large particle routes of transmission; some state that smallpox was transmitted primarily via airborne droplet nuclei, (Henderson et al., 1999) while others emphasize “face-to-face” contact and state that, airborne transmission was rare (Centers for Disease Control, 2002; Mack, 2003). This paper reviews the evidence for each of these modes of transmission.

32 BALFs were taken from patients in intensive care units with various diseases requiring bronchoalveolar lavage for diagnosis or treatment. After vortex for 2 min and centrifugation at 3000 rpm for 10 min, the supernatants of BALFs were tested by RRT-PCR with the CDC protocol. One BALF was determined to be pH1N1 positive. The remaining 31 negative BALF supernatants without viscous phlegm were used to prepare mock-infected BALFs by mixing 100 µl of the pH1N1SWL02 stock (10–4 dilution) with 900 µl of the BALF supernatant.

Polio occurred in 1,000 to 2,500 patients between 1955 and 1963, 100 to 300 cases had been reported thereafter until 1973. Only several to dozens of cases were reported between 1974 and 1983, and since 5 cases were reported in 1983, wild poliovirus infections have not been reported, thus far (Fig. 3)5).

According to a study of polio cases between 1962 and 1964, those aged 1 year were most common and those aged 3 years or more accounted for 70%. Inactivated vaccines for injection were used in 1962, oral live attenuated vaccines were added in 1965, and improved inactivated vaccines for injection have been used from 20045,9,11).