Hydropericardium syndrome (HPS)

History

HPS was first reported in 1987 in 3- to 6-week-old broiler chickens in Karachi, Pakistan [57, 66]. HPS was described as an accumulation of a straw-colored “jelly”-like fluid in the pericardial sac, and discolored and enlarged liver with basophilic intranuclear inclusion bodies. Kidneys were congested and high mortality rates (up to 70%) were observed [5, 13, 48]. The disease was observed in rapidly growing broilers [11, 24, 57]. Occasionally, the disease was also reported in 20-week-old layers and breeders and wild pigeons [14, 86].

Inclusion body hepatitis (IBH), which had previously been reported in the United States in 1963, has some clinical similarities with this emerging syndrome, but the accumulation of fluid in the pericardial sac was not found in inclusion body hepatitis. Therefore, researchers differentiated this syndrome from inclusion body hepatitis and a new name, “infectious hydropericardium” was given [2, 80]. Later, HPS was observed in various parts of the world, and a few other names were coined for this syndrome, such as Angara disease [57] and Litchi heart disease [42].

Clinical signs/pathology

Diseased birds exhibit different signs, including lethargy, huddling with ruffled feathers, loss of appetite, and mucoid yellow droppings. Reduction in weight gain has been recorded in infected flocks, which results in a poor feed conversion ratio [13]. Asrani et al. [14] indicated that the birds remained active until death, and in the terminal stages of infection, the chicks were dull, depressed with ruffled feathers, and reluctant to move, and they tended to gather in corners.

Parameters of clinical pathology indicate severe anemia and significant reduction of all hematological values in birds with HPS [14, 89]. Serum protein profiles were also found altered, with a decrease in albumin and an increase in β-globulin [73]. Reduction in the osmotic pressure of colloidal plasma results in the leakage of fluid into the pericardial sac. Blood glucose and plasma protein levels are decreased, while serum uric acid, potassium, calcium and triglycerides levels are significantly increased, which appears to be due to accumulation of fluid in the pericardial sac and abdomen [19]. Serum enzyme activities of AST, ALT and CPK are low in normal birds and high in HPS-infected birds [56, 128]. All of these changes indicate involvement of the liver, kidney and heart in HPS.

Necropsy findings

Gross lesions found during necropsy include enlarged, pale, friable liver, occasionally with necrotic foci. Accumulation of fluid in the pericardium is the most prominent macroscopic lesion [13, 24, 96]. Lesions are observed in vital organs, including the liver, heart, kidneys and lungs. In addition, an enlarged spleen and atrophy of the thymus are observed in most dead birds with HPS. Up to 20 mL of clear, straw-colored fluid is observed in the pericardial sac, giving it a balloon-like shape [14, 24, 70]. The liver is swollen, congested, friable and yellow colored with areas of necrosis and patechial hemorrhages. Kidneys are pale, swollen and friable with urates in tubules [95]. Researchers have observed the same lesions during experimental induction of HPS [12, 31].

Etiology

Initially, a nutritional disorder was considered to be the cause of HPS. The causative factors were thought to be fishmeal, rancid fat, and vitamin and mineral imbalances [57, 96]. Anjum [12] attempted to reproduce the disease using various strategies, including using feed samples from farms where natural outbreaks of the disease had occurred and feed with high levels of mycotoxins (100 ppm), but subcutaneous inoculation with infected liver homogenate was able to transmit the disease successfully, which supported the possibility of an infectious nature of this disease. A viral etiology of the disease was indicated by the presence of basophilic intranuclear inclusion bodies in hepatic cells [13, 41, 42]. The detection of icosahedral-shaped adenovirus particles in a liver extract by electron microscopy verified the viral etiology [24]. Later, Mazaheri et al. [80] reported biotypes of fowl adenovirus 4 to be the cause of the disease. It was found that fowl adenovirus 12 alone or in association with fowl adenovirus 4 was responsible for inclusion body hepatitis/hydropericardium syndrome in poultry flocks in India [99]. In Pakistan, adenovirus serotype 4 was isolated from field cases of HPS [125]. Mazaheri et al. [80] characterized field isolates of fowl adenovirus from several outbreaks of HPS and found that a serum neutralization test showed a positive reaction against antibodies of serotype 4 and 10. It was revealed using immunological and molecular techniques that fowl adenoviruses 4 (FAdV-4) and 10 are closely related [33]. Zhang et al. [129] have reported that HPS is prevalent in many countries, including China. They found that FAdV-4 has been the main causative agent of HPS in China for many years. However, they observed that the virulence of this virus has been increasing since 2015.

Natural transmission

Hydropericardium syndrome is a highly pathogenic disease [66], that can spread rapidly from flock to flock and farm to farm [27]. It is a contagious disease that can be transmitted horizontally either mechanically or by the oral-fecal route [2, 22, 107]. Cowen et al. [28] have demonstrated a role of wild birds in the spread of the disease. Hafez [45] showed that vertical transmission can occur through eggs and that the virus can spread from parent to progeny.

Experimental transmission

The infectious nature of the disease was demonstrated by experimental transmission of the disease by subcutaneous inoculation with an infected liver homogenate [12, 14, 23]. Anjum [12] and Naeem et al. [87] attempted to reproduce the disease experimentally using an infected liver homogenate via the subcutaneous route, the oral route and by direct contact with infected birds. Successful experimental reproduction of disease was observed within 2-5 days after subcutaneous inoculation, with typical symptoms of the disease. However, the oral route was suggested to be the natural route of infection.

Epidemiology

The first epidemic of HPS was reported in 1987 on poultry farms in Angara Goth, which is an extensive broiler-producing area near Karachi, Pakistan [57, 65]. Later, the disease was reported in different countries, including Iraq [1], India [42, 85], Mexico, Ecuador, Peru, Chile [28], South and Central America [117], Canada [43], Hungary [59], Poland [91], Korea [26], Russia [20], China [129] and Japan [3, 78]. Apart from the broilers, rare outbreaks were also reported in other avian species, including pigeons and quail [60, 86]. Asthana et al. [15] reported that FAdVs are commonly present in chickens worldwide and that HPS is the most severe disease associated with FAdV serotype 4.

Zhang et al. [129] reported that during the past few years, the incidence of HPS has increased in many countries, including China. It was found that FAdV-4 was present in China before 2014 but no severe HPS outbreaks were reported. However, recent outbreaks of HPS have been reported with high mortality rates on broilers in small and medium-sized chicken farms in China. Moreover, duck farms have also experienced heavy economic losses due to HPS. It has been shown that strains circulating in China before 2014 and after 2015 had different ancestors and that new strains circulating in China were derived from earlier Indian strains, with some genetic differences.

Adenoviruses

Nicklin et al. [90] and Robinson et al. [104] have shown that adenoviruses (Ads) have non-enveloped virions with a diameter of 70-90 nm. Adenoviruses were discovered in the 1950s in outgrowths of human adenoids and tonsils. It was found that humans and other vertebrates are infected by adenoviruses. Different isolates differ in their ability to agglutinate rat and rhesus erythrocytes. Based on these properties, adenoviruses have been classified into five subgroups [30].

Aviadenoviruses

Aviadenoviruses are a diverse group of pathogens that cause a variety of infections in birds [34, 105]. The aviadenoviruses are further divided into three groups. Group I comprises 12 serotypes of avian adenoviruses from chickens, geese, turkeys and other species that have a common group of antigens [64, 82, 121]. The aviadenovirus that causes HPS is classified as group I, serotype 4 [29, 52, 88, 122]. The virus responsible for acute inclusion body hepatitis is classified as group I, serotype 8 [33, 44, 101].

The second group of avian adenoviruses is known as group II, and its members which cause infections such as hemorrhagic enteritis in turkeys, marble spleen disease in pheasants, and splenomegaly in chickens. The group II viruses also share a common group of antigens that distinguishes them from other groups [32].

The third group of avian adenoviruses is known as group III, and its members are responsible for egg drop syndrome in laying chickens [83].

Morphological characteristics

Aviadenoviruses are non-enveloped virus with icosahedral morphology and a diameter of approximately 90 nm. The viral capsid is composed of 720 hexon subunits arranged as 240 trimers and 12 vertex penton capsomers with one or two fibers protruding from the surface. Aviadenoviruses are easily identified by their typical icosahedral shape and triangular facets of capsomers. This symmetry is typical for all members of the family Adenoviridae [124].

Shah et al. [114], Nicklin et al. [90], and Laver et al. [71] observed aviadenoviruses by electron microscopy and found hexagonal-shaped viral particles with a diameter of 70 to 90 nm.

Molecular characteristics

The aviadenovirus genome is a linear, non-segmented, dsDNA that is 35–36 in kbp length. The DNA has guanine/cytosine content of 53–59%. Viral genome encodes about 40 proteins, and it has terminal redundant sequences with inverted terminal repeats (ITR). Both ends of the viral genome are covalently attached to terminal proteins (TP) [124].

Russel [108, 109] reported that adenovirus genome encodes about 13 structural proteins. He found that the capsid of the viral particle consists of three major proteins, i.e., hexon, penton base and knobbed fiber. The remaining proteins are minor structural proteins including cement proteins (VI, VIII, IX, IIIa) and core proteins (V, VII, Mu, terminal protein, IVa2, protease).

Immunological characteristics

Balamurugan et al. [17] and Kumar and Chandra [68] studied the protein profiles of FAdV-4 and reported eight to twelve protein fractions of isolates with molecular weights between 13.8 and 110 kDa. Among these protein fractions, seven immunogenic polypeptides were detected (with molecular weights from 15.8 to 110 kDa) using western blot analysis with FAdV-4 serum. Hexon, penton base and fiber were found among the highly immunoreactive fractions. Fingerut et al. [36] and Jucker et al. [58] found that structural capsid proteins of adenovirus, i.e. penton and fiber, interact with cell receptors during penetration of the virus into the cell. Among the nonstructural proteins, 100K has importance, because Hong et al. [53] described that this protein may have a role in intracellular transport and folding of viral proteins during viral replication.

Phylogeny and genetic organization

According to Davison et al. [30], adenoviruses are non-enveloped, icosahedral viruses that belong to the family Adenoviridae which is divided into five genera, i.e., Atadenovirus, Siadenovirus, Mastadenovirus, Aviadenovirus and Ichtadenovirus. They replicate inside the nucleus. They have double-stranded, 35- to 36-kbp linear DNA. They are classified as medium-sized DNA viruses. The genomes have inverted terminal repeats (ITR) ranging from 36 to 200 bp, and their 5’ ends have a terminal protein (TP).

Mase et al. [77] compared the nucleotide sequences of the short fiber genes of various fowl adenovirus 4 isolates from Japan, India and Pakistan. Phylogenetic analysis showed that the fowl adenovirus 4 isolates from chickens with HPS grouped into a different cluster from fowl adenovirus 4 strains isolated from non-HPS cases. It was therefore suggested that fowl adenovirus 4 strains can be distinguished on the basis of the nucleotide sequence of the short fiber gene.

Harrach et al. [46] suggested a new classification for adenoviruses in which turkey adenovirus was assigned to the genus Aviadenovirus and the species in the family were renamed by adding a letter designation, i.e., “Possum adenovirus A, Goose adenovirus A, Canine adenovirus A, Tree shrew adenovirus A and Frog adenovirus A”.

Proteins encoded by aviadenoviruses

During viral replication, aviadenoviruses penetrate into the nucleus of host cells. Transcription of early and late genes encoding viral proteins and assembly of new virions occurs in the nucleus. The capsid consists of three major structural proteins, the hexon, fiber, and penton base. The hexon forms the majority of the structural components of the capsid, which has 240 trimeric hexon capsomeres and 12 pentameric penton bases. The trimeric fiber protein protrudes from the penton base at each of the 12 vertices of capsid and has a knobbed rod-like structure. The major difference in the capsids of fowl adenovirus C is the presence of two thin fibers, as most other aviadenoviruses have one fiber [76]. The structure and function of some important viral proteins are described below.

Hexon

The icosahedral capsid of adenovirus is mainly composed of hexon protein. There are four types of hexon, designated as H1, H2, H3 and H4 [21]. H1-type hexons are sixty in number, and they are associated with pentons at the 12 apices, so they are also called peripentonal hexons. The remaining hexons are designed as ‘group of nine’ (GON) on the 20 faces of the icosahedra. The H2-type hexon are on the twofold axes, H3 are on the threefold axes, and the remaining ones are H4. The size of hexon is different in the different serotypes, the largest hexon comprises of 967 amino acids. Each hexon has up to nine hypervariable regions, and they are situated at the top of the molecule [111]. These regions represent the type-specific antigens of the hexon, and at least one of them has a major role in virus-neutralizing activity [79, 103, 110].

Penton base

The penton base monomer comprises approximately 471 amino acids. The covalent complex of two proteins (homopentameric penton base and homotrimeric fiber protein) forms the penton capsomere. The penton base is situated at strategic positions at the apices of the icosahedral capsid, and it plays a key role in stabilization of the capsid as it interacts with the neighbouring capsomeres, peripentonal hexons, and other associated proteins. Moreover, the penton base is sensitive to heat, trypsin, pH and changes in ionic strength [126]. It has been found that the penton base, in association with some other proteins plays a major role during penetration and entry of the virion into the cell [35]. At the post-entry stage, the role of the penton base is not clear, but it has been found that the penton base protein interacts with cellular components, and neutralizing antibodies against the penton base have been detected in the sera of patients [54].

Fiber

The fiber polypeptide comprises approximately 582 amino acids, and it binds noncovalently to the penton base from its N-terminal side [130]. The fiber has three regions, including the tail, shaft and knob. The fiber is the first component of the virus that interacts with host cells/tissues. The ability of the virus to bind to the host cell is a key feature of the infection process and affects the pathogenicity of the virus. Variable sequences on the fiber knob determine the haemagglutinating properties of adenoviruses, and these properties are used to classify the species from A to F [92]. The fiber knob also has an important role in fiber protein synthesis and encapsidation [49].

Studies on the morphology of the penton of CELO virus revealed that aviadenoviruses have a different pentameric base structure, which is associated with two fibers, one long and one short. It was found that the two fibers have different receptors, so it was suggested that both fibers are required, one for virus attachment and the other for internalization [51, 119]. Mase et al. [77] determined the nucleotide sequence of the short fiber of fowl adenovirus serotype 4, and its amino acid sequence was deduced. They found that the short fiber comprises 479 amino acids, and its nucleotide sequence can be used to differentiate various fowl adenovirus serotype 4 strains. It was also found that epitopes affecting virulence, cell-surface-binding receptors, and receptors for tissue tropism exist within the fiber. Thus, the main role of the fiber protein is tethering (attachment) of the viral capsid to the host cell surface by its interaction with cellular receptors [90, 109].

100K

100K is an important nonstructural protein (NSP) of the virus. Xie et al. [127] found that antibodies specific for the 100K and 33K nonstructural proteins were detected in chickens experimentally infected with FAdVs, but not in chickens vaccinated with inactivated FAdVs. They also found that antibodies against NSPs can be used to distinguish FAdV-infected from vaccinated chickens [116]. This protein has been shown to play an important role in transport of viral proteins during viral replication in host cells. The 100K protein of adenoviruses of subgroups B and C has been identified as a chaperone protein that assists in trimerization and nuclear localization of hexons. Experiments have been conducted in which the hexon protein was expressed alone in insect cells or co-expressed in the presence of the 100K protein. When expressed alone, the hexon protein was detected in the cytoplasm in inclusion bodies, but when the hexon and 100K proteins were co-expressed, surprisingly, the hexon was found in a soluble trimeric form. It has been suggested that the 100K protein acts as a scaffold protein for the hexon, because evidence suggests that it plays a role in folding, self-assembly and nuclear import of the hexon in insect cells [53].

Diagnosis

Different techniques have been used by various researchers for accurate and precise diagnosis of HPS, which include conventional microbiological and molecular techniques.

Conventional microbiological techniques

Clinical diagnosis of HPS is a difficult task because of the sudden onset and acute nature of the disease [37, 38, 72]. Usually, initial diagnosis has been done by observing gross lesions and by histopathology by detection of intra-nuclear inclusion bodies of hepatocytes [13]. The diagnostic significance of electron microscopy has been discussed by Cheema et al. [24]. The presence of icosahedral viral particles detected by negative staining electron microscopy in an infected liver homogenate supported the view that the disease was caused by an aviadenovirus. Isometric, roughly spherical particles with morphology resembling that of adenoviruses were also found using transmission electron microscopy [23, 39].

Microbiological and serological methods that are used for diagnosis of subclinical HPS include agar gel precipitation test [47], indirect haemagglutination assay [75, 102], dot immunobinding assay (Rabbani et al., [98], enzyme-linked immunosorbent assay [47, 112], immunoperoxidase test [106], and tissue culture and embryonated hen egg culture along with virus neutralization test [97].

The ability of the virus to be propagated in embryonated hen eggs confirmed the viral etiology of the disease [24, 61, 62]. Mahmood and Hassan [73] propagated the fowl adenovirus in embryonated duck eggs. They injected the virus through the yolk sac and chorioallantoic sac. Death of embryos along with stunted growth and hemorrhages were reported by both routes. They also reported intranuclear inclusion bodies in hepatocytes of inoculated embryos.

The virus has also been propagated and isolated from cell culture [8, 17, 18, 22]. Khawaja et al. [66] propagated and isolated the virus in cell culture of chicken embryo kidney cells. They reported cytopathic effects in cell culture, e.g., degeneration, detachment of cells from the surface, and basophilic intra-nuclear inclusion bodies. Afzal and Ahmad [4] made some efforts to propagate the HPS-associated virus in QT 35 and Vero cell lines, but the virus could not be successfully cultured in these cell lines and the virus was unable to produce any cytopathic effects. Roy et al. [106] adapted the HPS virus to Vero cells by subjecting it to four blind passages. Later, they observed characteristic cytopathogenic effects (CPE) 96 hours after inoculation of Vero cells.

Kumar et al. [69] and Mahmood et al. [72] cultivated the HPS-associated virus in chicken embryo hepatocyte suspension and found a biological titer of 103.6 LD50 units per mL, while the same virus showed a biological titer of 105.6 LD50 units per gram of infected liver homogenate. Thus, it was found that 100 times more virus can be harvested from infected liver homogenate.

Molecular diagnostic techniques

In the case of adenoviruses, the use of serological techniques is associated with problems in interpretation of results, because antibodies are commonly present in both healthy and infected birds [45, 120]. Molecular techniques target and detect the pathogen itself rather than the antibodies induced by it, and in this way, early detection of the disease is ensured. The invention of the polymerase chain reaction (PCR) technique has provided high sensitivity and specificity for the diagnosis of HPS virus by targeting the hexon gene [29, 40, 120, 123]. Ganesh et al. [40] extracted viral DNA from infected liver tissue or purified virus and used it to amply a variable part of the hexon gene by PCR using specific primers. The results revealed the successful amplification of a 700-bp fragment of the hexon gene. This fragment of the hexon gene was subsequently also used as a probe for detection of the virus by dot blot hybridization of viral DNA. Rau and Hess [100] and Hess [50] amplified a 1319-bp portion of the hexon gene using specific primers. They also studied the restriction fragment length polymorphism (RFLP) after digestion of the PCR product with the enzyme HpaII. Mase et al. [77] found that PCR-RFLP analysis of the short fiber gene using the enzyme AluI was useful to distinguish various fowl adenovirus 4 strains. Thakor et al. [120] amplified an 890-bp DNA fragment of the hexon gene using gene-specific primers for detection of HPS-IBH virus in field samples.

In developing countries, in spite of keeping in view the economic importance of the disease, none of the techniques described above have been applied in the field for diagnosis and control of disease [65].

Prevention and control

Various strategies have been used by researchers for prevention and control of HPS since the initial outbreaks in 1987. It has been suggested that maintaining good husbandry practices, such as disinfection, maintaining strict biosecurity, and proper ventilation, may significantly reduce the chances of infection [10]. The addition of iodophor solution to drinking water at a concentration of 0.07-0.1% has been found to be efficacious in reducing the course and mortality of HPS in broilers [1, 2]. In cases of outbreaks, there is no specific treatment available against this viral disease; however, antibiotics are used for prevention of secondary bacterial infections [84, 118].

Vaccines

Standard vaccines are available against many other poultry diseases, but there are few against HPS. Two types of vaccines are currently reported in the literature against HPS. These include parenteral inactivated (killed) whole-cell vaccine and oral live attenuated vaccine. A list of the various types of available licensed vaccines against HPS are given in Table 1.

Table 1 Licensed vaccines available against HPS/IBH in different countries
Full size table

Parenteral inactivated (killed) whole-cell vaccines

Initially, the disease was controlled by an inactivated liver homogenate vaccine that was formulated after the first outbreak, and many researchers reported that the vaccine had successfully prevented the disease [7, 9, 25]. It was found that vaccination at 15-18 days or two vaccinations at 10 and 21 days of age gave the best protection [6, 47].

Kataria et al. [63], and Chandra et al. [22] reported that HPS can be brought under control by inactivated liver homogenate vaccines (0.25 mL/bird) or by inactivated cell culture vaccines (103.5 LD50/bird) injected subcutaneously at 10-15 days of age. Moreover, they found that the vaccine is effective in cases of outbreaks and that it significantly reduces mortality.

Khan et al. [65] and Mahmood et al. [72] suggested that outbreaks of HPS mostly occur post-vaccination in Pakistan. For this reason, the local vaccine production has been minimized. The poultry industry is still at the same risk as it was in 1987. It was suggested that the virus should be propagated on specific-pathogen-free embryonated hen eggs and cell cultures to produce killed and live attenuated vaccines. They also suggested that the unhealthy liver homogenate vaccine should not be used.

Kim et al. [67] showed that cell-culture-propagated inactivated oil-emulsion FAdV-4 vaccine conferred broad cross-protection against various serotypes of FAdV field isolates to vaccinated birds as well as the progeny of vaccinated breeders. It was concluded that cell-culture-propagated inactivated oil-emulsion FAdV-4 vaccine could be effective in preventing not only FAdV-4 infection but also other serotypes of FAdV. Previously, Icochea et al. [55] evaluated the efficacy of inactivated vaccines in Peru. They also reported that oil-adjuvanted cell culture IBH vaccine provided better protection when compared to the autogenous vaccine.

Oral live attenuated vaccine

Mansoor et al. [74] developed a live attenuated vaccine against HPS. They adapted a field isolate of HPS virus to chicken embryonated eggs by subjecting it to four blind passages. The chicken-embryo-adapted virus was further passaged up to 12 times for its complete attenuation. They tested this attenuated virus by giving it through the oral and parenteral routes to broilers at 14 days of age. They compared this attenuated vaccine with the old liver homogenate vaccines. It was found that the antibody response measured by ELISA was significantly higher in the group of broilers that were immunized with 16th-passage attenuated HPS virus at 7, 14 and 21 days post-immunization. In a challenge protection test at 24 days post-immunization, it was found that broilers immunized with 16th-passage attenuated virus were conferred with 95% protection, while the broilers immunized with the liver homogenate vaccine showed significantly lower protection (55%). Thus, the attenuated vaccine was more immunogenic and effective against HPS virus in broilers.

Recombinant (subunit) vaccine

Currently, inactivated vaccines [12] and attenuated vaccines [74] are available against HPS. The lack of full attenuation or inactivation and the oncogenic potential of adenoviruses have prevented their use in routine vaccines. A subunit vaccine has the advantage that it eliminates the danger of disease outbreaks caused by incomplete inactivation or attenuation [36].

The effectiveness of subunit vaccines against infectious bursal disease (IBD) virus in chickens [93], egg drop syndrome (EDS) virus in chickens [36], hemorrhagic enteritis virus (HEV) in turkeys [94], and hepatitis B virus in humans [81] have been demonstrated previously. Balamurugan and Kataria [16] and Khan et al. [65] suggested that modern practices of recombinant DNA technology should be applied to meet the needs of the present era for diagnosis and prevention of HPS. Pitcovski et al. [94] also suggested that subunit vaccines might be useful against adenoviruses. Shah et al. [115] developed a subunit vaccine against HPS. For this purpose, the penton base protein of HPS virus was cloned, expressed in Escherichia coli and used as subunit vaccine in broilers. The immunogenicity of the recombinant penton base protein and a challenge protection test against pathogenic virus demonstrated the ability of the recombinant penton base protein to confer 90% protection. The results suggest that the recombinant penton base protein is a candidate for a subunit vaccine against HPS. Schachner et al. [113] compared the immunogenicity of two viral recombinant fiber proteins (fiber-1 and fiber-2). The recombinant fiber-2 was identified as a protective immunogen, so it was proposed as a candidate for a subunit vaccine. A challenge protection test revealed that the fiber-2-vaccinated group was protected against challenge. However, in spite of this protection, fecal excretion of viral DNA was detected by real-time PCR, demonstrating that the vaccine had prevented clinical symptoms, but not infection.

Conclusions

HPS has been affecting poultry throughout the world since 1987. In most cases, FAdV-4 has been reported with some genetic differences. However, some researchers have found FAdV-4 in association with FAdV-10 or FAdV-12 to be the cause of HPS. Recently, researchers have also found some genetic changes in the viral genome, and it is suspected that the virulence of FAdV-4 has been increased over the years. Moreover, the worldwide scarcity of vaccines has made the situation alarming. However, it has been reported that cell-culture-propagated inactivated oil-emulsion FAdV-4 vaccine gives better protection than other available vaccines. Therefore there is a need to develop effective vaccines against this disease using modern molecular techniques. Recently, the effectiveness of subunit vaccines has been demonstrated, and subunit vaccine candidates against HPS have been identified, including recombinant viral surface proteins, e.g., penton base and fiber-2 (short fiber). Biotechnological approaches may be used for further understanding of FAdV evolution, which will provide essential information for the development of vaccines against HPS.