In the host, initial infection occurs at epithelia of Harderian gland, trachea, lungs, and air sacs. The virus then moves to the kidney and urogenital tract, to establish systemic infection [33, 35]. In this regard, the severity and clinical features of IB depend on the organ or system involved. Infection of the respiratory system may result in clinical signs such as gasping, sneezing, tracheal rales, listlessness, and nasal discharges. Affected birds appeared listless and dull with ruffled feathers (Figure 1). Other signs may include weight loss and huddling of birds together under a common heat source.

Other clinical outcomes associated with IB infection include frothy conjunctivitis, profuse lacrimation, oedema, and cellulitis of periorbital tissues. Infected birds may also appear lethargic, with evidence of dyspnoea and reluctance to move. Nephropathogenic IBV strains are most described in broiler-type chickens. Clinical signs include depression, wet droppings, and excessive water intake. Infection of reproductive tract is associated with lesions of the oviduct, leading to decreased egg production and quality. Eggs may appear misshapen, rough-shelled, or soft with watery egg yolk (Figure 2). Unless effective measures are instituted, decline in egg production does not return to normal laying, thus contributing to high economic loss [1, 37].

Infectious bronchitis virus (IBV) is, by definition, the coronavirus of the domestic fowl. Although it does indeed cause respiratory disease, it also replicates at many nonrespiratory epithelial surfaces, where it may cause pathology, for example, kidney and gonads [1, 2]. Strains of the virus vary in the extent to which they cause pathology in nonrespiratory organs. Replication at enteric surfaces is considered to not normally result in clinical disease, although it does result in faecal excretion of the virus. Infectious bronchitis (IB) is one of the most important diseases of chickens and continues to cause substantial economic losses to the industry. Infectious bronchitis is caused by IB virus (IBV), which is one of the primary agents of respiratory disease in chickens worldwide. All chickens are susceptible to IBV infection, and the respiratory signs include gasping, coughing, rales, and nasal discharge. Sick chicks usually huddle together and appear depressed. The severity of the symptoms in chickens is related to their age and immune status. Other signs of IB, such as wet droppings, are due to increased water consumption. The type of virus strain infecting a flock determines the pathogenesis of the disease, in other words, the degree and duration of lesions in different organs. The upper respiratory tract is the primary site of infection, but the virus can also replicate in the reproductive, renal, and digestive systems. The conventional diagnosis of the IBV is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. Since two or three blind passages are often required for successful primary isolation of IBV, this procedure could be tedious and time consuming. Alternatively, IBV may be isolated by inoculation in chicken tracheal organ cultures. Furthermore, IBV may be detected directly in tissues of infected birds by means of immunohistochemistry [6, 7] or in situ hybridization. The reverse transcription-polymerase chain reaction (RT-PCR) has proved useful in the detection of several RNA viruses [9, 10]. Outbreaks of the disease can occur even in vaccinated flocks because there is little or no cross-protection between serotypes [2, 11]. The necessity of IB prevention in chicken regarding the nature of the virus with a high mutation rate in the S1 gene dictates the necessity to develop effective vaccines. The first step is to study the virus strains distributed in the geographical region and determine their antigenicity and pathogenicity in order to choose a suitable virus strain for vaccination. This virus was isolated from a flock suspected of IB suffering from severe respiratory distress and experiencing high mortality. The objective of the present study was to clarify some aspects of pathogenesis of the disease caused by IRFIBV32 (793/B serotype) in experimentally infected broilers. RT-PCR test was performed to detect the presence of the virus in body tissues and samples. The clinical signs, gross lesions, and antibody response of the affected chicks were also monitored.

Clinical monitoring of infected chicks reveals at first, apparent respiratory symptoms beginning at day 2 post-inoculation (dpi). Respiratory clinical signs were predominant in all of the inoculated groups and were intense and more severe until 7 dpi, with no clear differences in the pathogenicity of the three strains. The most prominent clinical signs were characterized by gasping, depression, sneezing, difficulty in breathing, cough, pulmonary and tracheal rales, with high scores were reported for all the tested strains with clinical score of 108, 126 and 140 for IBV/RA, IBV/MN and IBV/TU strain respectively, (Tables 1, 2 and 3).

Nasal discharge and watery eyes were also observed but were transient in some of infected by IBV/RA and IBV/MN chicks. These clinical symptoms were persisted in all groups until 12dpi. The birds infected by IBV/MN and IBV/TU appeared lethargic, reluctant to move (Fig. 1), whereas, chicks infected with by IBV/RA were not as apathetic. At autopsy, all infected chicks that were killed at 5 dpi to prevent their suffering were examined for the macroscopic lesions in the trachea, lung and kidney. These gross lesions consisted in hemorrhagic tracheitis, mucosal congestion and catarrhal exudates that mainly progressed. For the three strains used, the signs were observed in the most infected chicks with dominance in the lungs that were hemorrhagic and sometimes cyanotic at one lobe. Therefore, samples were taken at 14 dpi just for a macroscopic examination of organs to confirm whether the absence of clinical signs is correlative with the gross lesions. Whereas, gross lesions of kidney, were not observed in all inoculated chicks. During the experiment, the non-infected control group stayed normally without clinical signs or gross lesions. The statistical analysis was not performed as the three tested strains are phylogentically related to each other and the difference in clinical and tissues scores is not very significant.

Infectious bronchitis (IB) causes significant economic losses to the poultry industry worldwide [1, 2]. The disease was first identified in North Dakota, USA, when Schalk and Hawn reported a new respiratory disease in young chickens. Since then, IBV has been recognized widely, especially in countries with large commercial poultry populations. Apart from respiratory infections, IB affects the kidney and reproductive tract, causing renal dysfunction and decreased egg production, respectively. Although the disease first was believed to occur primarily in young chickens, however, chickens of all age are also susceptible.

Infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens characterized by respiratory signs, and in young chickens by severe respiratory distress and a decrease in egg production in layers.1 The chicken was considered the only natural host of infectious bronchitis virus (IBV) but recently pheasants has been introduced as the other natural host for IBV.2 The disease is transmitted by the respiratory route, direct contact and indirectly through mechanical spread.3 The virus belongs to Coronaviridae, Order Nidovirales. The IBV and other avian coronaviruses of turkeys and pheasants are classified as group 3 coronaviruses.4 Its genome consists of about 27 kb and codes for four structural proteins: the spike (S) glycoprotein, the membrane (M) glycoprotein, the nucleocapsid (N) phosphoprotein, and the envelope (E) protein.5,6 The spike glycoprotein (S) is anchored in the viral envelope and is post-translationally cleaved into two proteins S1 and S2.7 The S protein is very diverse in terms of both nucleotide sequence and deduced primary protein structure, especially in the upstream part of S1.8 Three hypervariable regions (HVRs) have been identified in the S1 subunit.9-11 The S1 subunit induces neutralizing, serotype-specific, and haemagglutination-inhibiting antibodies.12-17 Amino acid changes in the spike (S) glycoprotein lead to the generation of genetic variants.18,19 The high frequency of new IBV variants is a distinguished characteristic of this virus among other coronaviruses.20 Many IBV serotypes have been described probably due to the frequent point mutations that occur in RNA viruses and also recombination events. Therefore, the characterization of virus isolates which exists in the field is very important.21 More than 50 serotypes of IBV have been identified and new variants continued to emerge despite the use of live attenuated and killed IBV vaccines.22-24

The usage of live attenuated vaccines is the most important preventive measure of the disease, but anti-genically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks.18,19 The IBV Massachusetts (Mass) type was first detected in Iran by Aghakhan et al.25 In 1998, a virus similar to the European 793/B type was isolated in Iran (Iran/793B/19/08).26 In recent years, new variants of IBV have been reported from different part of the country.27-29 The aim of this study was to provide information on the molecular characteristic and the phylogenetic relationship of prevalent IBV genotypes circulating in chicken flocks in Bushehr province, Iran.

Infectious bronchitis (IB) is a highly contagious viral disease of the upper respiratory and urogenital tract of chickens, which is caused by infectious bronchitis virus (IBV). The disease is prevalent in all countries with an intensive poultry industry, affecting the performance of both broilers and layers, thereby causing the considerable economic loss in poultry industry worldwide.1 The virus is the coronavirus of the domestic fowl that is mainly observed in chicken. It possesses a positive sense single–stranded RNA genome that ranges from 27 to 31 Kb.2 The number of IBV serotypes that exist throughout the world is unknown. More than 50 different serotypes have been listed and new IBV variants continue to emerge.3 It is now well documented that a considerable number of different serotypes with antigenic and pathogenic differences exist in poultry industry of different parts of the world.4

The D274 type was the most common type of IBV in several western European countries in the early and mid-1980s.5 IBV strains of the 4/91 type, which are also known as 793B, were first reported and characterized in Britain, 1991,6 and have been the dominant genotype in Europe.7 The Serological survey has revealed a high incidence of IBV infection of the 793/B type in layer and broiler chickens worldwide.8 Infectious bronchitis still causes serious problems in the Iraqi poultry industry due to the inability of the vaccines to protect the different genotypes. Due to the limited network of poultry diagnostic laboratories in Iraq, differential diagnosis is only made according to clinical signs and gross lesions.

The characterization of IBV has raised additional problems in terms of both epidemiology and control. Although IBV in the poultry farms in Iraq (with H120 and 4/91 strains) is presently controlled by both inactivated and live attenuated vaccines, the outbreaks of IB have still been observed on broiler farms.9,10 In Iraq, the first report of identification and genotyping of IBV isolates has been from Kurdistan-Iraq, which indicated the circulation of 793/B ( with the prevalence rate of 25 %) along with a new IBV variant (Sul/01/09) in vaccinated (Ma5 , or 4/91) broiler farms.10 So far, there has been no report on the prevalence rate of IBV genotypes in the south of Iraq.

The aim of the present study was to detect of three IBV genotypes including (Massachusetts; Mass), 793/B and D274 in the south of Iraq.

Infectious bronchitis (IB), also called avian infectious bronchitis, is a common, highly contagious, acute, and economically important viral disease of chickens caused by coronavirus infectious bronchitis virus (IBV). The virus is acquired following inhalation or direct contact with contaminated poultry, litter, equipment or other fomites. Vertical transmission of the virus within the embryo has never been reported, but virus may be present on the shell surface of hatching eggs via shedding from the oviduct or alimentary tract. Dozens of serotypes and genotypes of IBV have been detected, and many more will surely be reported in future. The highly transmissible nature of IB and the occurrence and emergence of multiple serotype of the virus have complicated control by vaccination (Saif et al. 2008). To monitor the existing different IBV serotypes in a geographical region, several tests including virus isolation, virus neutralization, hemagglutination inhibition, ELISA and RT-PCR have been employed (Haqshenas et al. 2005; Saif et al. 2008). The ELISA assay is a convenient method for monitoring of both the immune status and virus infection in chicken flocks. Several commercial ELISA kits for IBV specific antibodies detection are already available, which used inactivated virions as coating antigen (Zhang et al. 2005). PCR on reverse transcribed RNA is a potent technique for the detection of IBV. In comparison with classical detection methods, PCR-based techniques are both sensitive and fast (Zwaagstra et al. 1992). Samples for IBV isolation must be obtained as soon as clinical disease signs are evident. Tracheal swabs are preferred and are placed directly into cold media with antibiotics to suppress bacterial and fungal growth and preserve the viability of the virus (Swayne et al. 1998). In Iran, IB is one of the most important viral respiratory diseases of broiler chickens. However, only the Massachusetts vaccine strain is officially authorized. Despite the use of the IBV vaccine it is common to find IBV problems in vaccinated chickens, causing a tremendous economic impact (Nouri et al. 2003). Several serotypes of infectious bronchitis virus have been reported from different parts of Iran (Seyfi-Abad Shapouri et al. 2002; Nouri et al. 2003; Shoushtari et al. 2008). There is no report about the serotypes and molecular detection of IBV in Zabol in the southeast of Iran. The aim of this study was molecular detection of IBV and the IBV serotypes in Zabol.

Infectious bronchitis (IB) is primarily a respiratory disease of chickens but with potential to cause more widespread infection in the urinary and reproductive tracts in chicken leading to significant production losses in commercial broiler and layer flocks worldwide. The causative infectious bronchitis virus (IBV) belongs to the family Coronaviridae. The disease is usually characterized by high morbidity and low mortality in mature birds, whereas in naive young birds (2–3 weeks of age), mortality up to 100% can be observed. Being an RNA virus with the ability to mutate and recombine, IBV persist as numerous serotypes and strains. The control of IB relies on vaccination. Vaccines are available for commonly occurring serotypes and strains but they are not necessarily antigenically similar to the wild-type viral strains circulating in poultry barns. Although, these vaccine strains may provide some degree of protection for some related strains known as protectotypes, the commonly available vaccines may not elicit protective immune responses in a flock if the field strains are antigenically very different from the vaccine strains. Owing to this reason, vaccination against IBV is not currently considered to be a very effective control method and other biosecurity measures are necessary to prevent the introduction of IBV into poultry production facilities.

IBV is known to replicate in the respiratory tract leading to changes in the muco-cilliary clearance mechanism, as such, expose the IBV infected birds to secondary bacterial infections. Additionally, IBV has tropisms for a variety of tissues. However, the mode of dissemination from the common route of entry, i.e. the respiratory route, to the rest of the body systems could potentially be due to the initial viremia. Once disseminated, IBV infects epithelial cells of the reproductive and urinary systems, particularly the oviduct and kidney depending on the infecting strain. Recently, it has been shown that a nephro-pathogenic strain of IBV (B1648) could replicate in peripheral blood monocytes leading to viremia. The infection of circulating monocytes could potentially disseminate IBV to the urinary tract, liver and spleen.

Macrophages play roles in innate immune responses, as well as in mounting adaptive immune responses by functioning as antigen presenting cells, as such they are critical in protecting animals from microbial infections. Although it is known that macrophage numbers are elevated in the respiratory tract in response to IBV infection, the role played by macrophages in IBV infection, particularly if they serve as a target cell for viral replication is not known. Macrophages have been implicated to play in an important role in the pathogenesis of some animal and human viruses including Marek’s disease virus in birds, feline corona virus in cats, and human immunodeficiency virus (HIV). It was also shown that coronaviruses such as severe acute respiratory syndrome (SARS)-coronavirus (CoV) can replicate within human macrophages thereby interfering with macrophage functions leading to severe pathology. However, a single report based on in vitro studies indicated that IBV, particularly nonpathogenic Beaudette and Massachusetts type 82822 strains do not replicate in avian macrophages.

Therefore, in this study we investigated the interaction of IBV with macrophages in lungs and trachea in vivo and macrophage cell cultures in vitro using two IBV strains, Connecticut A5968 (Conn A5968) and Massachusetts-type 41 (M41) which are known to induce clinical disease and pathological lesions in chickens. As implicated in some other viruses, we hypothesized that these two strains of IBV replicate within avian macrophages leading to productive replication and interfering with selected macrophage functions in the process.

Avian infectious bronchitis virus (IBV) belongs to the genus Gammacoronavirus of the Coronaviridae family and is the etiologic agent of infectious bronchitis (IB), which is a major, highly complex infectious disease of poultry caused by multiple serotypes of IBV (1). IBV possesses a single-stranded positive-sense RNA genome (approximately 27.6 kb) encoding 4 structure proteins (phosphorylated nucleocapsid (N) protein, small envelope protein (E), integral membrane glycoprotein (M), and spike glycoprotein (S)) in the order of 5′-Pol-S-3a-3b-E-M-5a-5b-N-UTR-3′ (2). The S glycoprotein is cleaved into S1 and S2 subunits posttranslationally. S1 protein involves in infectivity, contains serotype-specific sequences, hemagglutinin activity, and virus neutralizing epitopes. The mutations, deletions, insertions, and recombination events that have been observed in multiple structural genes, especially in the S1 gene, of IBV isolates recovered from natural infections have been considered to contribute to the genetic diversity and evolution of IBV, and consequently, to the development of a number of IBV serotypes (3, 4). IB affects chickens of all ages, and IBV replicates primarily in the respiratory tract and in some epithelial cells of the kidney, gut and oviduct, resulting in reduced performance, reduced egg quality and quantity, increased susceptibility to infections with other pathogens, and condemnations at processing. IBV is a major poultry pathogen that is endemic worldwide and leads to serious economic losses (5, 6). IB has been reported in peafowl, teal, partridge, turkey, pheasant, racing pigeon and guinea fowl (7). Therefore, serological and molecular characterization of the field isolates of the IBV is highly important. IB was firstly described in North Dakota, USA, in 1930 (8). The first isolation of IBV in Iran was reported by Aghakhan et al. in 1994. The isolate showed the antigenic relationship to the mass serotype (9). IB is still a serious problem in Iran. Some newly emerging IBV isolates have recently been found. Backyard chicken is considered an important source of spread and persistence of different diseases (IB, Newcastle disease and avian influenza) among the chickens in poultry farms, playing a major role in the epidemiology of avian infectious diseases. Most household flocks are small and of mixed age and feed mainly by scavenging. Chickens from different households may mix, potentially exposing them to different diseases.

Moreover, no preventive and controlling strategy has been undertaken against IB in backyard chickens in Iran (10–12).

Avian infectious bronchitis virus (IBV) is a highly contagious pathogen of chickens that replicates primarily in the respiratory tract and also in some epithelial cells of the gut, kidney and oviduct. IBV is a virus member of genus Coronavirus, family Coronaviridae, order Nidovirales. The virus possesses a positive stranded RNA genome that encodes phosphorylated nucleocapsid protein (N), membrane glycoprotein (M), spike glycoprotein (S) and small membrane protein (E). The spike glycoprotein is post-translationally cleaved into two subunits, S1 and S2. The S1 protein forms the N-terminal portion of the peplomer and contains antigenic epitopes mainly within three HVRs. Neutralizing and serotype specific epitopes are associated within the defined HVRs.

Variation in S1 sequences, has been recently used for distinguishing between different IBV serotypes. Diversity in S1 probably results from mutation, recombination and strong positive selection in vivo. Antigenically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. The generation of genetic variants is thought to be resulted from few amino acid changes in the spike (S) glycoprotein of IBV.

In Egypt, isolates related to Massachusetts, D3128, D274, D-08880, 4/91 and the novel genotype; Egypt/Beni-Suef/01 were isolated from different poultry farms. The commonly used IBV attenuated vaccine is H120 while the Mass 41 (M41) strain is commonly used in inactivated vaccines.

In the present study, Egypt/F/03 was isolated from 25-day-old broiler chickens in Fayoum Governorate, identified by Dot-ELISA, RT-PCR and sequenced to determine its serotype. Pathogenicity test to 1-day-old chickens and protection afforded by the commonly used H120 live attenuated vaccine were also performed.

Avian infectious bronchitis virus (IBV) is a positive-sense single-stranded RNA virus belonging to the genus Gammacoronavirus, family Coronaviridae. IBV can infect the respiratory and urogenital systems in growing chickens, causing high mortality, slowing down growth, and reducing egg production in the flock. Therefore, IBV may result in a considerable economic loss in the global poultry industry. In Taiwan, IBV first appeared in 1958, and the disease has occurred frequently, although an IBV vaccination program has long been implemented. IBV strains in Taiwan include two genotypes, namely Taiwan Group I (TW-I) and Taiwan Group II (TW-II), which are prominent because of their capacity to cause nephritis and respiratory symptoms in chickens. Because of its frequent gene mutations and recombination events, IBV variants in different serotypes have been isolated from the field constantly. Due to serotype differences in Taiwan, extensive use of commercially available Massachusetts (Mass) serotype vaccines did not effectively prevent IBV outbreaks in chicken flocks.

Molecular studies reported that IBV contains four major structural proteins: The spike (S) glycoprotein, the phosphorylated nucleocapsid protein (N), the envelope protein (E) and the membrane glycoprotein (M). The S glycoprotein is believed to be related to the host range, and it can be post-translationally cleaved into S1 and S2 subunits. The S1 protein is highly variable and is the primary target for neutralizing antibodies. Moreover, the N protein is a relatively conserved structural protein that reveals high identity among various strains, and its highly immunogenic nature can induce antibodies as well as the T-cell mediated immunity in chickens. Therefore, the S and N proteins, in parallel development, are widely used as antigen targets for IBV detection.

Monoclonal antibodies (mAbs), with the characteristic of high specificity, simple purification, and stable sources, are broadly utilized in the field and research as a reliably powerful tool for grouping and differentiating virus serotypes. The antigenic characterization of IBV strains by using mAbs directed against different epitopes on the S, N and M proteins could provide valuable information regarding antigenic relationships and variations. Moreover, mAbs specific to Taiwan IBV strains have been used in a serum blocking ELISA, providing a simple and rapid method for detecting the wild-type IBV infection. In addition, nucleic acid tests such as RT-PCR and other modifications of ELISA have been commonly used to identify strains of IBV in the field. However, these methods are costly and time consuming, requiring skilled technicians to perform respective procedures in an equipped laboratory.

The immunochromatographic strip (ICS) test, or lateral flow assay, is an antibody-based technique, referring to the migration of antigen-antibody complexes through a filter matrix such as nitrocellulose strips. During the test, gold nanoparticles-conjugated antibodies bind to the antigen of interest, and the complexes are then immobilized in the support matrix by unlabeled antibodies bound to the matrix. ICSs are widely designed for use at point-of-care as they provide cheap, simple and rapid tests desirable in many industries. The objective of this study is to develop an ICS test for on-site antigen detection of IBV based on virus-specific mAbs. The use of the detection strip was validated by samples collected from experimental infections. This rapid IBV test is anticipated to benefit disease surveillance and control.

Respiratory diseases are among the most devastating diseases in poultry industry because of their major economic losses. In most cases, there are more than one pathogen involving in the pathogenesis of the respiratory diseases.1 Among several avian viruses with predilection for the respiratory tract, infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) are the most important viruses of poultry worldwide. Similar respiratory signs of infectious bronchitis (IB) and Newcastle disease (ND) making differential diagnosis of these two diseases difficult.2

In broilers, IBV affects weight gain and feed efficiency, and, when complicated with bacterial infections like E. coli or S. aureus, it causes high mortality and increased condemnations.3-5 IBV, the causative agent of IB is a coronavirus readily undergoes mutation in chickens resulting in the emergence of new variant serotypes and genotypes.6 As new strains of IBV emerge, rapid detection of IBV is useful for implementation of control measures, research purposes, and understanding the epidemiology and evolution of IBVs.7

Newcastle disease classified as a list A disease by the Office Internationale des Epizooties (OIE), is caused by avian paramyxovirus 1 (APMV-1) or NDV.8 The virus is enveloped with a negative-sense, single stranded RNA genome of approximately 15 kb encoding six proteins (nucleoprotein, phosphorprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein, and large protein, respectively).9

Several laboratory methods such as virus isolation in embryonated eggs and organ cultures and serological tests are available for detecting and differentiating avian viral respiratory infections. However, these methods are time consuming and laborious.10-12 Molecular techniques such as reverse transcription-polymerase chain reaction (RT-PCR), sequencing and real time PCR, have been used for rapid and sensitive detection of IBV and NDV separately.13-17 However, those techniques detect only one specific pathogen at a time. The duplex PCR has the ability to amplify and differentiate multiple specific nucleic acids.18 The aim of the present study was to detect and differentiate two common avian viral pathogens using duplex RT-PCR for clinical diagnosis.

Avian influenza (AI) is a respiratory disease in poultry of zoonotic importance caused by influenza A viruses of the family Orthomyxoviridae. Type A influenza viruses are classified into different subtypes according to the two major surface glycoprotein: Hemagglutinin (H) and neuraminidase (N). Avian influenza virus (AIV) is divided into two groups based on their pathogenicity including highly pathogenic avian influenza (HPAI) in which mortality may be as high as 100%, and low pathogenicity avian influenza (LPAI) which causes milder respiratory disease. Laboratory examinations in specific pathogen-free (SPF) chicken demonstrate that H9N2 AIV is low pathogenic. It is almost one decade that the Middle East and Asian countries are facing frequent outbreaks of H9N2 infection. Mixed infection or coinfection of H9N2 AIV with other respiratory pathogens is one of the possible explanations for increasing the economic losses of H9N2 infection in commercial broiler chickens[6,7]. It was reported that infectious bronchitis virus (IBV) infection increased the pathogenicity and extended the period of H9N2 AIV shedding in broiler chickens.

In Egypt, serological evidence of H9 spread throughout Egypt was recorded on 2009-2010 before isolation of the virus in 2011. Therefore, there was serological evidence for the presence of H9N2 infection in chicken population during 2001. In Alexandria, isolated H9N2 virus from chicken flocks suffering from respiratory symptom with high morbidity rates (up to 70%) and mortality rate reaching 15% was analyzed. Blast analysis of the nucleotide sequences from the eight viral genes showed that the recently isolated Egyptian H9N2 strain was closely related to the other Middle East H9N2 strains. The virus shared the common ancestor - the A/Qa/HK/G1/97 isolate - which contributed to the internal genes of the H5N1 virus circulating in Asia.

The infections with H9N2 viruses in Egypt are higher in chicken than other species, mostly in apparently healthy broilers, and recorded in layers and breeders.

In the present study, two experiments were carried out for studying the pathogenicity of H9N2 avian influenza virus (AIV) in broiler chickens after vaccination with different live respiratory viral vaccines.

Poultry production in Algeria faces many zootechnical and health constraints, such as viral infections like avian infectious bronchitis (IB). The avian IB virus (IBV), a member of the Coronaviridae family (order Nidovirales and genus Coronavirus), frequently infects broilers and egg-laying hens and leads to severe economic losses to the poultry industry. Since its discovery in the 1930s, the IBV has been identified as the major cause of respiratory infections and poor zootechnical performances. Interestingly, it can also multiply in the renal tissue and cause nephritis, a phenomenon first described in the United States. More recently, IBV-associated nephritis has been accepted as the most pressing problem in broiler flocks in many countries.

The most effective method of protecting poultry from IBV infections is through live or killed vaccines. However, nephritis associated with infectious bronchitis has been observed in several vaccinated flocks, suggesting that the current vaccination strategies against IBV may not provide adequate protection. In fact, outbreaks of IB are frequently caused due to the strains serologically different from those used for vaccination. Since its discovery in 1931, a large number of serotypes or variants of IBV have emerged, and little or no cross-protection occurs between these serotypes. Therefore, it is crucial to track epidemic-causing serotypes in each geographic region or country and produce new vaccines to control IB.

In Algeria, poultry flocks have been vaccinated against IB with the Massachusetts (Mass) strain combined with the IB 4/91 United Kingdom variant strain or 793/B since the past few years. However, kidney damage with suspicion of IB has also been reported in recent years in spite of vaccination but has not been confirmed so far. This has led to the speculation of the possible emergence of variant strains against which conventional vaccines are not completely effective.

The aim of this study was to investigate the presence of IBV among Algerian broiler flocks and its possible involvement in broiler kidney damage. Clinically diseased broiler flocks were sampled and analyzed by the hemagglutination inhibition (HI) test and reverse transcriptase-polymerase chain reaction (RT-PCR) followed by phylogenic analysis.

Coronaviruses are single stranded positive sense RNA viruses belonging to the order Nidovirales, and are known to infect a variety of hosts. Several human coronaviruses have been identified, causing mainly mild respiratory infections, with the exception of severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, coronavirus infections have an economic impact on livestock industries worldwide. The avian coronavirus, infectious bronchitis virus (IBV), causes infectious bronchitis (IB), a mild respiratory infection, but as a consequence is responsible for serious effects on the global poultry industries due to poor weight gain in broiler chickens as well as reduced egg production and egg quality in layers. In addition, some strains of IBV are nephropathogenic whilst others result in severe pathology in the reproductive organs. Bovine coronavirus (BCoV) causes respiratory infection and diarrhoea in cattle, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV) cause diarrhoea in pigs and porcine haemagglutinating encephalomyelitis virus (PHEV) causes vomiting and wasting disease in pigs.

Infectious bronchitis (IB) is a serious and highly contagious disease of chickens all over the world. Avian infectious bronchitis virus (IBV) was first reported in the USA for replicating in the respiratory tract and some other epithelial cells of gut, kidney, and oviduct. Subsequently, some strains of IBV caused pathology in non-respiratory organs (such as kidney and gonads) were documented. The clinical disease and production problems frequently cause catastrophic economic losses to the poultry industry, accompanied by decreased production performance in breeder flocks, diminished egg production and poor egg quality in laying flocks. In China, IB has a more profound social impact for chicken industrial contributes to the rural economy. More importantly, there is accumulating evidence that nephropathogenic type IB has been more and more prevalent in China recently, but the strains isolated in earlier years mainly caused respiratory signs, which suggested that selecting and immunization with the appropriate vaccine strain is of great importance to control IB infection.

Some researchers reported that satisfactory cross protection could be provided by appropriate vaccine programs against genetically or antigenically unrelated IBVs. However, this symphysial vaccine manner was restricted by the diversity of the IBV strains. Since IBV strains were first isolated and identified in China in 1982, various live-attenuated and inactivated vaccines derived from respiratory-typed strains have been widely and extensively used in chicken farms to reduce the adverse effect of the IBV. However, the disease continues to emerge and cause serious production problems, even occurred in routinely vaccinated layer and breeder flocks in China, and the situation gets worse as time progressed. The most possible explanation for this phenomenon may be that the vaccine effectiveness is diminished by poor cross-protection against the circulating strains.

The spike tip glycoprotein (S1) of virus particle has direct relation to induce virus neutralizing antibody, and determines the cross-protection. Our previous research had confirmed that the predominant IBVs were nephropathogenic IBVs were mainly A2-like strains in China during 2008-2009. This study was to further investigate the prevalence of nephropathogenic IB under immune pressure with routine vaccine strains in China. Additionally, the effectiveness of vaccination program using the common field strains practically against IB was also verified.

Commercial poultry in Pakistan was established in the early 1960s, representing one of the largest agriculture-based segments of Pakistan’s economy with a significant contribution to national gross domestic product (1.3%). There are over 25,000 poultry farms spread across the country’s rural areas and the Pakistan poultry industry produces around 1220 million kg of chicken meat and 10,000 million eggs a year. In the past, regional studies on poultry disease surveillance and clinical surveys have been conducted to better understand the disease distribution pattern in different regions of Pakistan [3–5]. Recently, outbreaks of viral diseases with high morbidity and mortality were being reported consistently [6–8] and are possibly due to the intensification of commercial poultry production and lack of biosecurity measures. Pakistan’s poultry industry has indeed been growing continuously, facilitating the spread of multiple common viral respiratory diseases (CVRDs) such as Newcastle disease (ND), infectious bronchitis (IB), swollen head syndrome (SHS), infectious laryngotracheitis (ILT) and low pathogenic avian influenza (LPAI) infections, which are caused by Avian Avulavirus 1 (AAVV-1), infectious bronchitis virus (IBV), avian metapneumovirus (aMPV), infectious laryngotracheitis virus (ILTV) and avian influenza virus (AIV), respectively. These are highly contagious diseases of poultry, with worldwide distribution and they have serious economic impacts on the poultry industry. The causative agents of these diseases affect chickens of all ages except ILTV, which normally does not affect chickens before 3 weeks of age. These pathogens may interact with bacterial agents resulting in high morbidity and mortality in the infected chickens. The continuous emergence of new virulent genotypes from global epidemics and the frequent changes observed in the genomic sequence of these viruses lead to ineffective diagnostics and control measures. Outbreaks of CVRDs such as IB and SHS are not reported to the country’s ministry in charge of livestock and poultry production. Consequently, the distribution patterns of such chicken diseases are unclear in Pakistan. Moreover, CVRDs, such as LPAI (H9N2), are of great significance to public health due to their zoonotic potential [10–12]. Therefore, it is important to investigate the distribution pattern of CVRDs in different regions and different chicken production types to develop scientific and risk-based prevention measures of these poultry diseases.

The aim of this study was to detect and characterize chicken respiratory viruses found in commercial Pakistan poultry, which is the first step of control measures implementation. Five major chicken respiratory viruses were investigated: AIV, AAVV-1, IBV, aMPV and ILTV.

Infectious bronchitis (IB), an acute, highly contagious viral upper respiratory disease of chickens, is one of the most economically significant diseases hampering the intensive poultry industry worldwide. IB affects chickens of all ages, causing respiratory, reproductive, and renal manifestations. Although control of IBV infection is primarily achieved through live attenuated vaccines, the infection is difficult to contain because immunization with different serotypes of the virus do not necessarily cross-protect against other serotypes. Within an infected poultry flock, quick and accurate detection of the presence of the virus is imperative to properly vaccinate uninfected flocks. In addition, rapid differentiation of IBV infection from other upper respiratory tract diseases (e.g., avian influenza, Newcastle disease, infectious laryngotracheitis, avian mycoplasmosis) is important so that appropriate measures can be taken in a timely manner.

IB is a disease that negatively impacts the poultry industry of developing countries. For instance, in Morocco, IB continues to be an uncontrolled problem [4–6] due to the lack of in-country diagnostic capabilities that can be performed quickly and interpreted easily by local staff in potentially underequipped or otherwise challenging environments.

The causative agent of IB, infectious bronchitis virus (IBV), is a member of the species Avian coronavirus, genus Gammacoronavirus, family Coronaviridae. IBV is an enveloped, positive-sense, single-stranded RNA virus (genome length = 27.6 kb), expressing three major structural proteins: the nucleocapsid protein (N) surrounding the viral RNA, the membrane glycoprotein (M), and the spike glycoprotein (S) located on the surface of the viral envelope. The S protein contains two post-translational subunits, S1 and S2.

Current diagnostic assays for IBV include virus isolation in embryonated eggs, tracheal organ culture, cell culture immunoassays, and molecular assays that detect viral RNA. Virus isolation has been considered to be the reference standard. However, such isolations are expensive and time consuming because several passages may be required to detect the virus. Immunoassays use IBV-specific monoclonal antibodies to detect the virus in direct or indirect fluorescent antibody and enzyme-linked immunosorbent assay (ELISA) formats. Although faster and simpler than virus isolation, immunoassays tend to lack specificity and sensitivity. None of these immunoassays detect all strains or types of IBV [11–13]. Molecular assays, such as reverse transcriptase-polymerase chain reaction (RT-PCR), for the detection of IBV are commonly used because highly specific and sensitive results can be obtained in a timely manner. Molecular assays detect viral RNA directly from a clinical sample or from virus isolated in a laboratory host system. When RT-PCR is used to amplify the spike glycoprotein (S) of IBV, the assay can be coupled with restriction fragment length polymorphism analysis or nucleic acid sequencing to identify serotypes of the virus [2, 10, 12–16]. More recently, many fluorescent probe-based real-time RT-PCR assays have been developed to detect IBV strains [2, 3, 9, 10, 15, 17]. Real-time TaqMan RT-PCR assays have been developed that amplify a fragment of the 5′ untranslated region of the IBV genome to detect turkey coronaviruses and IBV or that target the N gene for IBV detection.

Unfortunately, performing most of these assays requires highly trained staff, a sophisticated infrastructure, or considerable monetary funds, and are therefore not necessarily viable options for developing countries such as Morocco. SYBR green I-based RT-PCR assays have proven to be among the most effective tools in the rapid and differential detection of a variety of viral diseases such as avian influenza, Newcastle disease, and IB. These inexpensive and easily performed assays are important to rapidly identify the causative agent of any upper respiratory disease or changes in egg shell quality and egg production in chickens [17, 18].

However, an assay employing real-time RT-PCR with SYBR green I dye to target the N gene of IBV is lacking. Here we report the development of a real-time RT-PCR assay with SYBR green I dye for rapid detection of IBV viral RNA directly from Moroccan clinical samples. We also compared the assay with conventional RT-PCR and agarose gel electrophoresis to detect IBV PCR-amplified products.

Infectious laryngotracheitis (ILT) and infectious bronchitis (IB) are common respiratory diseases in poultry and are currently present at epidemic levels around the world, including in China, the United States, and India. Because ILT and IB have high incidence and infectivity in chickens at different ages (in days), they have caused huge economic losses to the poultry industry. Both diseases have similar clinical symptoms and pathological changes, leading to significant difficulties in clinical differential diagnosis, especially in cases of mixed infection [1, 2]. In addition, the use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases, such as infectious laryngotracheitis and infectious bronchitis. Therefore, it is important to establish a method to simultaneously detect ILTV and IBV antibodies for the differential diagnosis and immune response evaluation after vaccination.

Infectious laryngotracheitis virus (ILTV), an alphaherpes virus, possesses at least 10 envelope glycoprotein, two main proteins of which are the glycoprotein B (gB) and gD, respectively, which are highly conserved herpesvirus structural glycoproteins. The gD of herpes virus has an important role by binding to the host receptors [1, 5]. The gD protein has been demonstrated to be a candidate antigen for recombinant vaccines [6, 7]. The infectious bronchitis virus (IBV) genome encodes four major structural proteins: the spike glycoprotein (S), the membrane glycoprotein (M), the nucleocapsid (N) protein, and the envelope or small membrane protein (E). The N protein is thought to be an appropriate diagnostic reagent for antibody detection. In this study, gD and N proteins were selected as antigen molecules for the diagnosis of ILT and IB.

At present, the methods used for the diagnosis and effect evaluation of vaccine immunity of avian respiratory diseases primarily include virology detection, serological detection, and molecular biological detection. Traditional methods, such as virus isolation, animal inoculation experiments, ELISA, hemagglutination (HA), and hemagglutination inhibition (HI) assays are characterized by complex procedures and long periods of time required for diagnosis. Despite its unique, sensitive, simple features and rapidity, polymerase chain reaction (PCR) is unable to meet the requirements for high-flux quarantine and is not favorable for the urgent screening of bulk samples. Therefore, it is imperative to establish a new technology for the effect evaluation of vaccine immunity and the rapid differentiation or correct identification of important avian respiratory diseases.

A new high-flux detection technology, flexible xMAP (x = Unknown, MAP = Multi-Analyte Profiling) assay, uses variously colored polystyrene beads that is carboxylated to allow covalent coupling of protein. Conjugated beads can be used to capture specific antibodies in serum, and a fluorescent secondary antibody is incubated to bind to the captured serum antibodies. A red laser is used to determine the color of the bead and a green laser to detect fluorescence intensity of bound secondary antibodies through the Luminex 200 detection system. This method is applicable for the simultaneous and rapid detection of multiplex pathogen,s antibodies, with simple operation and high accuracy that are superior to conventional methods. In this study, a method employing Luminex xMAP technology to simultaneously detect ILTV and IBV antibodies in serum was established, optimized and used for the differential diagnosis of IBV and ILTV. This assay can also be used to simultaneous monitoring of IBV and ILTV antibody levels for the evaluation of immunity after vaccination.

Avian infectious bronchitis (IB) is caused by a virus in the Coronaviridae family, genera Gammacoronavirus. It is a highly contagious disease with a short incubation period. The Avian coronavirus was previously classified, and is most commonly referred to, as avian infectious bronchitis virus (IBV). The IBV is responsible for respiratory disease, which manifests in clinical symptoms such as sneezing and tracheal-bronchial rales that can lead to the development of more severe symptoms [2, 3]. Infected birds exhibit reduced performance, consequently leading to a reduction in weight gain and deterioration in egg quality and quantity. Secondary bacterial infections will also contribute to economic losses. Carcass condemnation due to the development of airsacculitis [4, 5] negatively impacts commercial sales of bird meat and eggs. Brazil was once the world’s largest exporter of poultry and currently the world’s third largest producer of bird meat. The consequences of IBV are a significant threat to Brazil’s poultry industry.

The IBV genome consists of a non-segmented positive-sense single-stranded RNA that is approximately 27.6 kb in length. It encodes non-structural (accessory proteins) and four structural proteins: the nucleocapsid protein (N), the spike protein (S), the envelope protein (E), and the matrix protein (M). The nucleocapsid protein, or N protein, consists of 409 amino acids. It has a molecular mass of approximately 50 kDa and directly binds with the viral genome to form the virion nucleocapsid [6, 7]. Its structure is highly conserved, with different strains of IBV sharing a high degree of identity (94–99%). The N protein is also known for its immunogenicity, inducing specific antibody and cytotoxic T-cells mediated responses [9, 10]. There is significant interest in the use of the IBV N protein as an important target for diagnosis since it possesses the antigenic characteristics required for the development of serological assays that can be applied to detect or quantify antibodies against the IBV.

The laboratory diagnosis of IB is dependent on direct and indirect techniques. The direct techniques are employed for viral isolation and genomic or phenotypic identification of the virus, while the indirect methods are used to detect specific antibodies. In addition to being applied for serodiagnosis, serological techniques can also be employed to evaluate the immune responses stimulated by vaccines. Commercial ELISA kits are typically used to indirectly diagnose IBV. These kits, however, are expensive when large number of samples require screening and they are not acessible for applications with the scale of the Brazilian poultry industry [13–15].

ELISA techniques currently available are designed to detect polyclonal antibodies that target the whole virion. The use of nucleoprotein as the antigen for diagnosis and evaluation of vaccine immune responses is an interesting target to explore since this protein plays a important role in IBV virus replication and the induction of a specific immune response in infected birds [16, 17]. The use of recombinant antigens in the design of a specific diagnostic technique facilitates the development of highly sensitive and specific assays that display a high antigen concentration and, thereby, reduce or eliminate background reactions. The use of recombinant antigens also represents a viable method of reducing immunoassay development costs. Easy production of antigens in expression systems leads to simple and efficient antigen development which can reduce the production costs associated with diagnosis. The aim of the current study was to evaluate the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein of IBV.

The microscopic tissues analysis was performed in the euthanized chicks at 5 dpi. This period correlates with the maximum intensity of clinical signs (which begins between 2 and 7dpi). These signs fade from the 7dpi with a total restoration at 12 dpi, that why we only conducted the macroscopic examination at this time. Histopathological lesions developed at 5dpi in the three groups of experimentally infected chickens, were compatible of those previously described for infectious bronchitis virus. And their scoring was mainly correlating with the ranking and scoring described in previous experiments.

In the trachea, most detected lesions in all infected groups were characterized by mucosal thickening, hyperplasia of the surface epithelium, loss of cilia, and mononuclear inflammatory cell infiltrate (lymphocytes, plasmocytes, and macrophage) of lamina propria (Figs. 2a and b). Mean total score of severity was 3,7; 4,7 and 6,7 in birds infected by IBV/MN; IBV/RA and IBV/14/TU strains respectively. This indicates that IBV/TU strain seems to have a higher degree of pathogenicity than the other tested strains (Table 4).

In the lungs, lesions in all infected groups were mainly confined to the primary and secondary bronchi and included epithelial hyperplasia, loss of cilia, and heterophils and mononuclear inflammatory cell infiltrate (lymphocytes, plasmocytes and macrophages) of the lamina propria and peribronchial space (Fig. 3a and b). Other changes were located in the atria and parabronchi and consisted of hypertrophy and/or hyperplasia of pneumocytes. The score of these changes were from mild to moderate degree of severity and there was no big differences in the mean total score of severity among tissues infected with the 3 tested IBV strains (7,3; 8,3 and 6), thus indicating no differences in the pathogenicity of the three strains for the lung (Table 5). However, no kidney lesions were observed in chicks infected with the three tested strains. Tracheas and lungs of control chicks samples throughout the experiment, showed normal histological features.

Myanmar lies in the western region of mainland Southeast Asia. Agriculture is the backbone of the Myanmar economy, and poultry farming is one of the country’s major industries. In association with the recent economic development of Myanmar, the total number of raised chickens has increased over the last decade. In order to provide a stable supply of poultry products, the development of farm biosecurity measures is required, and it is important that farmers and veterinarians are aware of these measures. Infectious respiratory diseases have severe impacts on the poultry industry. Avian influenza and Newcastle disease are major threats to the poultry industry, and these diseases have been reported in Myanmar [2–4]. Other respiratory pathogens, such as mycoplasmas and infectious bronchitis virus (IBV), have not been investigated in Myanmar, although clinical signs suggesting contagious respiratory diseases have been detected, according to local veterinarians’ observations. These diseases cause considerable economic losses worldwide, and vaccines for their prevention have been developed. It is important to determine the genotypes and/or serotypes of each pathogen circulating in Myanmar to inform vaccination programs.

Avian mycoplasmosis is caused by several pathogenic mycoplasmas. Among them, Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the most impactful to the poultry industry. MG infections usually cause chronic respiratory disorders and are characterized by sneezing, coughing, and snicks as well as nasal and ocular discharges [5, 6]. MS infections most frequently occur as subclinical upper respiratory tract infections and may cause air sac disease. MS results in infectious synovitis, an acute to chronic infectious disease of chickens. The co-infection by MG or MS with respiratory virus infections, such as IBV and Newcastle disease, can exacerbate the disease conditions. Both MG and MS infections cause considerable economic losses in the poultry industry by reducing weight gains and meat quality in broilers, causing severe drops in egg production in layers, and increasing embryo mortality in breeders.

Infectious bronchitis (IB) is a severe acute disease of poultry caused by IBV, which primarily infects the respiratory tracts, with respiratory disease being the most frequent sign. In addition, IBV can infect the kidneys and reproductive tracts and consequently cause kidney damage and decrease in egg production. Generally, IB is controlled by serotype-specific vaccines. The identification of field isolates is necessary for appropriate vaccinations because these vaccines exhibit little cross-reactivity among different serotypes [10, 11].

In this study, we performed molecular detection of MG, MS, and IBV in chickens from poultry farms at the outskirts of three large cities in Myanmar: Mandalay and Pyin Oo Lwin in February 2018 and Yangon in May 2018. In addition, by analyzing genetic characteristics, we detected at least three genotypes of IBV existing in Myanmar. To our knowledge, this is the first report using molecular analysis to detect MG, MS, and IBV in Myanmar.

The poultry industry is an important subsector of agriculture, has generated huge employment opportunity, increases the supply of good quality protein, ensured food security, involved in country’s economic growth and reduced poverty level in both urban and rural areas of Bangladesh. There are several constraints that hinder the development process in poultry sector; among them, disease is the major one. Various pathogens, such as bacteria, virus, fungus, parasite, etc., are responsible for causing diseases in poultry and they attack their different body systems. Respiratory tract, an important part of poultry body system, frequently affected by pathogens causing respiratory diseases. Several pathogens such as bacteria, viruses, fungi, and environmental factors initiate the respiratory diseases of chicken. Viral and bacterial pathogens are responsible for causing most of the respiratory diseases, namely, avian rhinotracheitis (ART), infectious laryngotracheitis (ILT), infectious bronchitis virus (IBV), Ornithobacterium rhinotracheale (ORT), etc., that lead to huge economic losses in poultry industry. Bacterial pathogens colonize the respiratory system after primarily introducing of viral or environmental stress for pathogens.

ART virus is also known as avian pneumovirus (APV), important respiratory viral disease affecting both chickens and turkeys. ART was first identified in Bangladesh at 2016 by Ali et al. in broiler breeder, layer, and Sonali chicken (cross-breed between Rhode Island Red cocks and Fayoumi hens). Sneezing, depression, coughing tracheal rales, swollen infraorbital sinus, ocular and nasal discharges, and foamy conjunctivitis are the major signs associated with the disease. This virus also causes swollen-head syndrome in broiler breeders and broiler and dropped egg production in layers. ORT, another important bacterial pathogen, belonging to the super family of RNA containing bacteria causes respiratory infections and affects air sac. It has been reported throughout the world except Bangladesh and mainly affect in turkey and chickens but other species can often be infected with this pathogen. This can act as primary or secondary agents depending on immune status, environmental factors, and pathogenicity of related strain and also the presence of other pathogens. ILT virus, an important virus, causes respiratory infection in birds belonging to the family Herpesviridae. Thus, virus mainly affects the chickens and characterizes by sneezing, nasal discharge, swollen infraorbital, and nasal sinuses and sometime affects eye leads to conjunctivitis. The disease more frequently occurs in the areas of intensive poultry production and outbreak causes’ high economic loss as it increases mortality, reduces egg production, and declines growth rates. Another important viral pathogens responsible for respiratory disease, namely, Avian IBV belonging to the genus Gammacoronavirus of the Coronaviridae family. It can affect chickens of all ages, and primarily it replicates in respiratory tract, and later, it can move to epithelial cells gut, oviduct, and kidney, results decreased egg production and growth performance and sometimes attract other pathogens. So far, it has been reported in chickens, turkey, pigeon, pheasant, Guinea fowl, and peafowl.

Despite the country with a large number of poultry farms, only a few reports are available in Bangladesh regarding respiratory infections. A few works have been done on IBV and ILT, but the amount is quite scanty. In view of this, the present research work was conducted to perform a comparative serological study to check the presence of several viral and bacterial pathogens antibodies in chickens with special emphasis on ART, ILT, IBV, and ORT, as well as to determine the distribution of its specific antibody in respect of the types of birds (broiler and sonali), age groups, and locations of farms of different districts of Bangladesh.

Viruses belonging to the Coronaviridae family have a single stranded positive sense RNA genome of 26–31 kb. Members of this family include both human pathogens, such as severe acute respiratory syndrome virus (SARS-CoV)1, and animal pathogens, such as porcine epidemic diarrhea virus2. Currently, the International Committee on the Taxonomy of Viruses (ICTV) recognizes four genera in the Coronaviridae family: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. While the reservoirs of the Alphacoronavirus and Betacoronavirus genera are believed to be bats, the Gammacoronavirus and Deltacoronavirus genera have been shown to spread primarily through birds3. The first three species of the Deltacoronavirus genus were discovered in 20094 and recent work has vastly expanded the Deltacoronavirus genus, adding seven additional species3.

By contrast relatively few species within the Gammacoronavirus genus have been identified. There are currently two recognized species in the Gammacoronavirus genus: avian coronavirus (ACoV) and beluga whale coronavirus SW1 (SW1). ACoVs infect multiple avian hosts and include several important poultry pathogens, such as infectious bronchitis virus (IBV) and turkey coronavirus (TCoV)5. IBV was first described in the United States6 but has since been described around the globe7. Turkey Coronavirus is the cause of acute enteritis in domestic turkeys8. The second species in the Gammacornavirus genus SW1 was first discovered in beluga whales9 but has since been detected in other cetaceans, such as Indo-Pacific bottlenose dolphins10. Despite IBV being the first discovered coronavirus and the impact it has on the poultry industry11, the number of identified species within the Gammacoronavirus genus remains small in comparison to the other coronavirus genera. Coronaviruses from several other avian hosts for which partial sequences are available suggest relatedness to IBV and TCoV. These viruses, which include goose coronavirus (GCoV), were tentatively classified as part of the ACoV species. An approximately 3 kb region, including the nucleocapsid gene and several accessory genes, of GCoV were previously sequenced from a greylag goose in Norway12.

Here we present the full genome of Canada goose coronavirus (CGCoV) sequenced directly from the cloacal swab of a Canada goose, which expired in a mass die-off in a remote region near the arctic in Nunavut, Canada. Our analyses demonstrate that it should be classified as a novel species in the Gammacoronavirus genus.

Avian infectious bronchitis (IB) and Newcastle disease (ND) are both common, highly contagious, and acute avian diseases and have been causing heavy losses in the poultry industry. Infectious bronchitis virus (IBV), the pathogen of IB, is a member of Gammacoronavirus of the Coronaviridae family (http://www.ictv.global). The genome of IBV is about 27.6 kb in length, encoding fifteen non-structural proteins and four structural proteins: Spike glycoprotein (S), small membrane protein (E), membrane glycoprotein (M), and phosphorylated nucleocapsid protein (N). The M glycoprotein, the most abundant protein in the viral envelope, performs core functions in the process of coronavirus assembly and budding. Furthermore, the M protein is generally considered to be an essential component in the formation of coronavirus-like particles (CoVLPs). The S glycoprotein, which is post-translationally cleaved into two distinct functional subunits (S1 and S2), forms the distinctive spikes on the surface of IBV. The S protein is responsible for receptor binding and determining host tropisms; in particular, the S1 subunit can elicit virus-neutralizing antibodies, while the S2 subunit anchors the S protein in the surface of the virion through the C-terminal trans-membrane domain (TM, aa1093-1136 of S protein of IBV) inlaid into the envelope and the carboxy-terminal domain (CT, aa1137-1162 of S protein of IBV) noncovalently interacting with the M glycoprotein. ND is caused by virulent strains of Newcastle disease virus (NDV), which is a member of the genus Avulavirus under family Paramyxoviridae (http://www.ictv.global). NDV has a 15kb long genome comprising six genes which individually encode the nucleocapsid (N), matrix protein (M), phosphoprotein (P), fusion protein (F), haemagglutinin-neuraminidase protein (HN), and large polymerase protein (L). F and HN are two glycoproteins displayed on the virion surface. The F protein mediates virus fusion with the host cell membrane and plays the major role in the virulence of NDV strains. The HN protein assists the F protein in its function. The F protein consists of an ectodomain (Fecto, aa1-499 of F protein of NDV) displayed on the viral envelope, a hydrophobic transmembrane domain (TM, aa500-523 of F protein of NDV), and a cytoplasmic domain (CT, aa524-553 of F protein of NDV) near the carboxyl terminus; similar to the IBV S protein, the TM/CT domain anchors the F protein in the surface of the virion. F is thought to be the predominant antigen in NDV vaccine studies. Antibodies elicited by the F protein can protect chickens from lethal NDV challenge.

Nowadays, IBV and NDV are controlled using live-attenuated vaccines and inactivated vaccines. Live-attenuated vaccines are thought to be the most effective vaccines. However, safety is the main concern about live vaccines. Live vaccines may cause diseases in immunocompromised individuals. Mutations in the genome of live vaccine strains could cause a reversion to virulence and further result in diseases in vaccinated individuals. Novel IBV strains arising from genome recombination have been reported in recent years. Genome recombination events between NDV vaccine strains and NDV wild strains were also observed. In addition, NDV-attenuated vaccines may cause mild respiratory or gastrointestinal disease, resulting in weight loss, reduced egg production, and increased sensitivity to other pathogens. Compared with live vaccines, inactivated vaccines are safer but induce weaker and shorter-lived immune responses; most notably, inactivated vaccines could not effectively stimulate cellular immune responses. Furthermore, the potential for the incomplete inactivation of a virus can also result in disease in vaccinated individuals. According to these facts, developing novel effective vaccines against IBV and NDV is badly needed. Virus-like particles (VLPs) are empty shells composed of virus structural proteins (and a viral envelope in enveloped viruses), possessing similar morphology with the native viruses. Due to the absence of virus genome, VLPs are noninfectious. These qualities contribute to the effectiveness and safety of VLPs as vaccines. In addition, antigens or epitopes from different pathogens can be simultaneously exhibited on the surface of VLPs through either recombinant DNA technology or chemical conjugation, making the chimeric VLPs a multi-antigenicity vaccine platform. The first VLPs were constructed with the surface antigen of hepatitis B virus in 1982. To date, VLPs of various viruses had been constructed and applied as nonreplicating subunit vaccines against viral infection. IBV VLPs have been shown to be a promising vaccine approach, and they possess the potential to carry other virus antigens to form multivalent vaccines.

In this study, Fecto and the IBV S1 protein were fused to the TM and CT domain of the IBV S protein (STMCT), forming the recombinant F (rF) and recombinant S (rS) protein, respectively, and chimeric infectious bronchitis-Newcastle disease (IB-ND) VLPs were constructed with these two recombinant proteins and IBV M proteins through the baculovirus system. Subsequently, the immunogenicity of the chimeric VLPs were evaluated as a vaccine in specific-pathogen-free (SPF) chickens.