The cDNA was synthesized using AccuPowder RT PreMix kit (BioNeer Corporation, Republic of Korea) according to the manufacturer's instruction. The primers were specific for 3′ UTR gene. Five μL of total RNA and one μL of each 10 pmol primer were used for cDNA preparation. PCR was performed to amplify a 276-bp fragment of the 3′ UTR gene of avian infectious bronchitis virus. The same primers were used in the PCR master mix containing: 2.5 μL PCR buffer 10X, 0.75 μL MgCl2 (50 mM), 0.5 μL dNTPs (10 mM), 1 μL of each 10 pmol primer (UTR1 and UTR2), 3 μL cDNA, 15.75 μL water, and, at the end, 0.5 μL Taq DNA polymerase (5 IU/μL) was added. The program in Ependorf thermal cycler was 95°C for 3 min and 35 cycles including 95°C for 45 sec., 55.6°C for 45 sec, 72°C for 50 sec, and a postpolymerization step at 72°C for 7 min. The products were analyzed in 1% agarose gel containing ethidium bromide, using an ultraviolet transilluminator.

Amplification was performed on original swab samples and allantoic fluids. None of these samples was positive for the 3’-UTR of the avian coronavirus (UTR11−/41+). Three out of 10 swab samples were positive for the N gene coronavirus, but none of them was positive for the S1 IBV gene. However, four out of 10 samples of allantoic fluid tested were positive for the coronavirus N gene. Of the two that tested positive for the S1 gene of IBV, only one of them, which was designated as parrot/Indonesia/BX9/16, was able to be further sequenced. These results are presented in Table-3.

All tissue samples were immediately stored at −70°C until used. RNA of the samples was extracted using the Accuzol Userś Manual (BioNeer Corporation, Republic of Korea) according to the manufacturer's protocol. Briefly, appropriate tissue (50–100 mg of tissue) was homogenized with 1 mL of Accuzol, and then 200 μL chloroform was added into the mixture and the mixture was centrifuged at 12000 rpm at 4°C for 15 min. The upper phase was added to an equal volume of isopropyl alcohol and stored at −20°C for 10 min then centrifuged at 12000 rpm at 4°C for 10 min. After the washing step, by using 80% ethanol and centrifuging at 12000 rpm at 4°C for 5 min, the pellet was dissolved in a final volume of 50 μL distilled water (DW) and stored at −70°C until used.

Statistical analysis was performed using GraphPad Prism 6.0. Correlation of C. psittaci bacterial load and adenovirus viral load was determined using linear regression analysis. For statistical calculation, 50% of the lower detection limit (6,000 copies/ml for C. psittaci and 600 copies/ml for adenovirus) was assigned to specimens that tested negative. Log-transformed viral loads were used for statistical analysis.

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

The patient was a 55-year-old male artisan working at the NTNAMC. He presented after four days of dyspnea and two days of hemoptysis. Chest radiograph showed bilateral patchy consolidation. Blood test showed neutrophilia and elevated liver enzymes with alanine transaminase of 51 U/L. He was put on non-invasive positive pressure ventilation for respiratory failure. Bronchoalveolar lavage tested positive for C. psittaci and rhinovirus by PCR and reverse transcriptase-PCR respectively. Direct fluorescent antigen detection and viral culture did not reveal other viral co-pathogens. Paired serology, collected 18 days apart (on days 2 and 20 after hospitalization), showed a rise of C. psittaci IgG titer from <32 to 128 by microimmunofluorescence assay, but there was no increase in adenovirus or other respiratory virus antibody titer. The patient recovered with oral doxycycline. He had contact with birds, monkeys, iguanas and snakes at the NTNAMC within one month of symptom onset.

Developing RT-PCR using vaccinal and reference strains of IBV and NDV. The specificity of duplex-RT-PCR was shown using IB88 and 793/B strains of IBV and two standard strains of NDV. The duplex-RT-PCR products visualize by gel electrophoresis was 433 bp for IBV and 121 bp for NDV (Fig. 1).

Application of developed duplex-RT-PCR for detection and differentiation of IBV and NDV in clinical samples. The applicability of developed duplex-RT-PCR assay for detection and differentiation of IBV and NDV in the diagnosis was validated examining 12 clinical samples as showed in Fig. 2. Among five positive clinical samples belonged to five different broiler farms, three farms were infected with only one virus and two farms were co-infected with IBV and NDV.

Viral respiratory diseases are common causes of economic losses in poultry industry. These diseases cause reduction of growth rate and production, high rate of death, prevention and treatment costs. Quick detection and differentiation of causative viruses can play an important role in controlling these viruses.20 IBV and NDV are the viruses that frequently affect the respiratory tract of chickens.21 There are several clinically similar viral diseases that can occur in intensive poultry production and require laboratory differential diagnosis. Infectious bronchitis is a global and highly infectious viral disease,22 and Newcastle disease is also an economically important viral disease in poultry industry.23 Several studies have shown the circulating of different viral respiratory disease including IBV,24-27 NDV28 and avian influenza in Iranian poultry farms.29-31

The duplex RT-PCR assay which can be able to quickly identify IBV and NDV will be of great importance in the epidemiology of these viruses especially for controlling of disease transmission among poultry farms and reduction of the economic losses in poultry industries.32, 33 Because of high sensitivity and specificity that PCR offers, since its introduction researchers use it extensively as an indispensable diagnostic method to detect viruses. Using single PCR takes up much time. Therefore using duplex PCR can solve this restriction of PCR.34

In the present study, developed duplex RT-PCR was able to detect and differentiate two important viral respiratory diseases of poultry and more importantly the technique was able to simultaneously detect infected birds with both viruses. Since the rapid detection of viral infectious agents in intensive poultry production system is very important, this procedure will be useful to detect more than one infectious agent in the infected farms reducing the time and also costs involved.

Because of the importance of avian respiratory pathogens, many researches have undertaken the detection and differentiation of these pathogens especially AIV, IBV and NDV.35-37 A duplex RT-PCR was developed to detect class І and class ІІ strains of NDV. It was shown that this method had high specificity and high sensitivity.38 In another study, Chaharaein et al. used duplex RT-PCR for detecting H5, H7 and H9 subtypes of avian influenza viruses.39

In the present study two farms were co-infected with IBV and NDV viruses. In was concluded that the developed duplex RT-PCR could be a rapid and economic procedure for detection of IBV and NDV in poultry farms. Using this procedure for detecting these viruses in wild birds is also recommended.

The present study has revealed that the chickens infected with the three tested strains showed the specific respiratory signs with high clinical signs. These infected chicks were sero-converted at 14 dpi and confirmed the spread of the inoculated virus. In addition, the macroscopic lesions and histopathological examination revealed specific lesions of trachea and lung, without renal manifestations. The histopathology of respiratory organs demonstrated a high lesional score of IBV/TU strain which correlate with the clinical score of 140. Moreover, virus re-isolation was performed successfully for all the three tested strains. Results obtained with real time PCR in organs sampled from infected birds (trachea, lung and kidney) and from fluid allantoic in the first passage in SPF eggs, showed a significant abundance of the virus in the respiratory tract. In other words, the findings of molecular analysis of virus re-isolation are strongly correlated with the histopathological tests.

Finally, this current report justify that Italy 02 genotype isolated from different regions of Morocco is capable to induce a severe respiratory disease with a wide distribution of respiratory system. However, this genotype do not cause any kidney damage and without causing mortality.

Currently, most methods of AIV, NDV, IBV, IBDV and other avian viral agents detection are adapted to specific detection of one agent in a sample. Multiplex RT-PCR is successfully used for detection of AIV and its subtypes [19, 20] and for diagnosing double infections such as combination of NDV and AIV. Also methods with use of multiplex real-time RT-PCR for AIV, NDV and IBV subtypes differentiation have been developed [22–24]. At present development of a test based on microarray technology for simultaneous detection of AIV, NDV, IBV and IBDV in one sample is important for poultry industry in the Republic of Kazakhstan.

Use of microarray improves quality and shortens the analysis duration in molecular diagnosis of infectious diseases and therefore is employed as an independent method in screening for several genes of large numbers of pathology samples [25–27]. There are biochips for influenza diagnosis that allow screening not only for HA and NA, but for M and NP genes of influenza A virus [25, 28]. In identification of NDV molecular methods with use of oligonucleotides specific to conservative regions of NP-gene of NDV were used. Recently VP2 gene region of IBDV is successfully used in synthesis of oligonucleotide primers and probes from highly conservative regions for molecular diagnosis [30–33]. Molecular methods for IBV diagnosis are oriented at using more conservative sequences located in S1 and S2 genes of IBV [34, 35].

In the proposed microarray probes were developed on the basis of conservative regions of gene fragments encoding NP and M (AIV), NP (NDV), VP2 (IBDV), S1 (IBV) array proteins from Genbank Database. All viral gene fragments demonstrated high rate of conservatism and therefore the test is universal for detecting AIV, NDV, IBV and IBDV strains. So, high homology of nucleotide sequences of gene regions encoding AIV, NDV, IBV and IBDV array proteins compared to GenBank data confirms specificity of the developed microarray for rapid diagnosis of avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease.

Total analysis duration without time required for the viral RNA extraction is 5–6 h, and 16 specimens can be simultaneously assayed. Duration of the assay with use of the proposed microarray is not longer than in other molecular methods and simultaneous testing of samples for AIV, NDV, IBV and IBDV provides its advantage over other methods.

Various methods have been developed for the diagnosis of bird infection, such as virus isolation in cell culture, embryonated chicken eggs, or young specific-pathogen-free (SPF) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry. All these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. These methods lack the ability to detect low levels of antigens in tissues [36–40].

In the present study field samples (122 in total) were used to test effectiveness and reliability of the microarray. Nevertheless, positive result of using molecular and biological methods, being very important in emergency cases, should always be confirmed by the method of virus isolation.

The results of the study show that diagnostic sensitivity (99.16%) and diagnostic specificity (100%) of the DNA microarray are comparable with the same of the real-time RT-PCR (99.15 and 100%, respectively).

Diagnostic effectiveness as percentage ratio of true results to the total number of obtained results for the developed DNA microarray and real-time RT-PCR was 99.18%.

Analysis of the obtained data shows that the microarray test for rapid diagnosis of avian infections demonstrates the effectiveness comparable to that of the molecular method real-time RT-PCR and is more rapid and less resource-consuming owing to its ability to detect simultaneously AIV, NDV, IBV IBDV positive samples in the course of one experiment. Universality of the test makes it suitable for wide use in veterinary laboratories for prompt detection of avian infections.

Different strains of AIV, NDV, IBV and IBDV were used to test the oligonucleotide microarray. Testing was carried out in comparison with real-time RT-PCR (Table 5).

Fifteen different strains of AIV, NDV, IBV and IBDV, diverse in their origin, epidemiological and biological characteristic, were identified correctly with use of DNA microarray. Diagnostic results of testing DNA microarray with use of virus strains from the RIBSP microbial collection were comparable to the results of the real-time RT-PCR. Sensitivity of the microarray was comparable to the sensitivity of real-time RT-PCR.

This approach uses viral RNA, amplified either directly (one-step RT-PCR) or following cDNA synthesis (two-step RT-PCR). An RT-PCR assay was designed and introduced in 1991 for detecting the IBV-S2 gene. Subsequently, general and serotype-specific RT-PCR assays were designed to target different regions and/or fragments (Figure 10) in the IBV viral genome [71–73]. The UTR and N-gene-based RT-PCR are used for universal detection, because of the conserved nature of the target region in many IBV serotypes [68, 71]. A pan-coronavirus primer, targeting a conserved region of different coronavirus isolates, could also be used in one-step RT-PCR amplification of IBV strains. However, amplification and sequencing of the S1 gene provide a reliable means for genotypic classification of new IBV strains. A serotype-specific PCR assay has been designed to enable differentiation of Massachusetts, Connecticut, Arkansas, and Delaware field isolates.

Several parts of tracheal rings were prepared from each infected bird at 5dpi that coincides with the pic of the clinical signs. At 12 dpi, the ciliary activity was also evaluated from all the infected birds that have recovered. The prepared tracheal rings were immersed immediately in cell culture medium (D-MEM supplemented with 10 % of foetal calf serum), were microscopically analyzed for estimating the ciliary movement in tracheal infected and in tracheal control, and were scored on a scale from 0 (100 % activity) to 4 (no activity). The mean score of each strain was then compared to evaluate the respiratory pathogenicity.

In the past, serological assays such as virus neutralization (VN) and haemagglutination inhibition (HI) were used widely for detecting and serotyping IBV strains. These tests also have been used to measure flock protection following vaccination [50, 51]. Serotype-specific antibodies usually are detected using HI, even though the HI test is less reliable. On the other hand, ELISA assays are more sensitive and easily applied for field use and in monitoring antibody response following vaccination or exposure. However, emergence of different IBV serotypes that do not cross-react with commonly available antisera generally made serological tests less applicable and nonconclusive in classifying new or emerging IBV isolates [52, 53].

The samples were collected as part of a national active and passive surveillance program, coordinated by the Finnish Food Safety Authority Evira and University of Helsinki. The sample panels included (1) hunted clinically healthy birds (active surveillance), (2) hunted clinically diseased birds (active surveillance), and (3) birds found dead (passive surveillance) or (4) clinically diseased (passive surveillance) (Table S1). The sample material used for RNA detection was cloacal swab, tracheal swab, oropharyngeal swab, or tissue. The swab samples were collected using nylon swabs and stored and transported in Universal Transport Medium (both by Copan International). Prior to RNA isolation from the swab specimen (stored at −80°C), the samples were centrifuged to remove solid particles and supplemented with additional antibiotics (streptomycin-penicillin). The tissue samples were homogenized and separated by centrifugation and supplied with antibiotics (streptomycin-penicillin). The sample panel from 2010 consisted of 343 samples, the 2011 panel of 171 samples, the 2012 panel of 287 samples, and the 2013 panel of 138 samples. The samples were also screened for influenza A virus RNA.

Tissue samples from the trachea, lungs, spleen, glandular stomach, kidneys, and bursa of Fabricius were fixed in 10% neutral formalin for 48 h at room temperature. These samples were processed routinely, embedded in paraffin wax, and cut into 5 μm sections. The sections were stained with hematoxylin and eosin (H&E) and examined for lesions with light microscopy.

Tear IBV-specific IgA was detected using a commercial IgG ELISA IBV antibody test kit (IDEXX, Westbrook, ME, USA). Briefly, tears were serially diluted two-fold in PBS and incubated in duplicate in wells overnight at 4 °C. All wash steps were performed using PBS-Tween 20 (0.05% Tween 20). Plates were incubated at 23 °C for 2 h in monoclonal mouse anti-chicken IgA-BIOT (1:1000, clone A-1, Southern Biotech, Birmingham, AL, USA), followed by 1hr in Streptavidin-HRP (1:4000, Southern Biotech, Birmingham, AL, USA). Final antibody detection steps were completed according to the manufacturer’s instructions. Endpoint titers were determined by reporting the lowest dilution at which the optical density (OD), recorded at 650 nm wavelength, was at least three standard deviations above the mean of 12 control wells incubated with no tear samples. Data from wells with a pinpoint color change due to residual substrate or air bubbles were excluded from analysis, and results were reported as log2 of the endpoint titer.

To evaluate tracheal ciliostasis, three sections of the upper, middle, and lower parts of the trachea (nine rings per bird) were analyzed. The rings were placed in 96-well plates with Eagle’s culture medium containing 10% fetal bovine serum. They were then examined by inverted light microscopy at a magnification of 400× to determine the degree of integrity and the preservation of ciliary movement in the tracheal epithelial cells. A score of 0 was given if the cilia in the complete tracheal section showed movement; a score of 1 was given if 75–100% of the cilia in the tracheal section showed movement; a score of 2 was given if 50–75% of the cilia in the tracheal section showed movement; a score of 3 was given if 25–50% of the cilia in the tracheal section showed movement; and a score of 4 was given if <25% of the cilia in the tracheal section showed movement or there was no movement at all. The average ciliostasis score was calculated for each group.

IBV-specific IgG titers were detected using a commercial IgG enzyme-linked immunosorbent assay (ELISA) IBV antibody test kit (IDEXX, Westbrook, ME, USA). Briefly, serum samples (stored at −20 °C) were diluted 1:500, and the procedure was performed according to the manufacturer’s protocol.

Frequent human-animal contact is the major cause for viral cross-species transmission. Next-generation sequencing is a highly efficient method for rapid identification of microorganisms and for surveillance of pathogens for infectious diseases. Animal models and other laboratory tests would be needed to pinpoint the causative agents. The novel coronaviruses in Wuhan likely had a bat origin, but how the human-infecting viruses evolved from bats requires further study. The human-infecting virus may become more infectious but less virulent as it continues to (co-)evolve and adapt to human hosts. Since Wuhan is one of the largest inland transportation hubs in China and the city has been closed off, it is urgently necessary to step up molecular surveillance and restrict the movement of people in and out of the affected areas promptly, in addition to closing the seafood markets. To prevent human-to-human transmission events, close monitoring of at-risk humans, including medical professionals in contact with infected patients, should also be enforced. Finally, virome projects should be encouraged to help identify animal viral threats before viral spillover or becoming pandemics.

Anti-viral drugs are thought to be backbone of a management plan of an avian flu pandemic. Only two anti-viral drugs have shown promise in treating avian influenza: oseltamivir (Tamiflu®) and zanamivir (Relenza®). A treatment of Tamiflu® includes 10 pills taken over five days while Relenza® is administered by oral inhalation. The US Food and Drug Administration has approved both anti-viral drugs for treating influenza but only Tamiflu® has been approved to prevent influenza infection. Because antivirals can be stored without refrigeration and for longer periods than vaccines, developing a stockpile of antivirals has advantages as part of an overall strategy to control a flu epidemic. However, there are limitations to the use of antivirals: Tamiflu® needs to be taken within 2 days of initial flu symptoms for it to be effective, but many people may not be aware that they have the flu early in the disease. Some research in animals and recent experience in the use of the drug to treat human cases have also found that Tamiflu may be less effective against the recent strains for the current H5N1 virus than the 1997 strain. Improper compliance to antivirals by irresponsible individuals during an outbreak may results in the emergence of a drug-resistant strain. Lastly, there are current concerns about the safety of Tamiflu® which has been associated with increased psychiatric symptoms among Japanese adolescents.

The HACCP framework enables the identification of risks within a system and the design of control methods. It does not contain the scope for monitoring or ensuring compliance of the control points identified; such control should be applied via other means. Given that EIDs are appearing with increasing frequency, often in countries where they place additional strain on already over-burdened public health and healthcare systems, being able to rapidly identify and design strategies for control has valuable application in responding to emerging health threats such as the Middle East Respiratory Syndrome (MERS) virus which first appeared in Saudi Arabia in late 2012 or the rapidly spreading outbreak of a novel avian influenza A H7H9 in China since March 2013. Conducting detailed, timely and comprehensive field investigations into HPAI H5N1 outbreaks is hampered by the majority of cases occurring in developing countries. Advantages to such a framework are that it requires minimal resources and can be implemented by local health officials and international expertise, if required, can be provided remotely. It also complements recently developed diagnostic statistical models for known pathogens. Subsequent detailed and time-consuming experimental analyses can then be conducted if required. Whereas in-depth epidemiological studies can take weeks or months to produce results and recommendations the HACCP framework may provide a means of producing a response within days of an outbreak occurring.

South Eastern of Brazil is classified as being tropical wet and dry or savannah (Aw) type according to Köpper climate classification system, since the area presents an extend dry season during winter (end of May to September) temperature winter month > 20ºC and rainy season during summer (October to March approximately) precipitation in the driest summer month < 30 mm (19). The meteorological data (rainfall and relative humidity) for this region during the period of study presented monthly average temperature ranging from 39.3 and 31.2ºC, the precipitation in rainy season varied from 40 to 270.3 mm/month, and there was no rain at all during dry season (results not shown).

Descriptive statistics were used to determine the frequency of positive poults for TAstV-2 and TCoV in all samples tested and the two RT-PCR assays applied. The results of all samples were also used to estimate the apparent positivity during summer and winter. The apparent prevalence was adjusted for specificity and sensitivity of each test to obtain the true prevalence (19). A primary screening test to identify climatic characteristic significantly related to TAstV-2 and TCoV positive results was performed using χ 2-test. To calculate the specificity and sensitivity of each RT-PCR, the accuracy of two conditionally independent tests in the absence of a gold standard was used (10). Variable analysis was performed using the odds ratios (OR) to quantify the association between climatic conditions (summer and winter) and positively results for TAstV-2 and TCoV from CS and faeces; from TAstV-2 and TCoV in tissues and sera; from TAstV-2 and TCoV RT-PCRs. The Microsoft Access for Windows and statistical comparisons were performed using the Microsoft Excel 8.0 and Epi-Info 3.3 version. Differences were considered significant when p values were less than 0.05.

There is no available vaccine against COVID-19, while previous vaccines or strategies used to develop a vaccine against SARS-CoV can be effective. Recombinant protein from the Urbani (AY278741) strain of SARS-CoV was administered to mice and hamsters, resulted in the production of neutralizing antibodies and protection against SARS-CoV,. The DNA fragment, inactivated whole virus or live-vectored strain of SARS-CoV (AY278741), significantly reduced the viral infection in various animal models,,,,,. Different other strains of SARS-CoV were also used to produce inactivated or live-vectored vaccines which efficiently reduced the viral load in animal models. These strains include, Tor2 (AY274119),, Utah (AY714217), FRA (AY310120), HKU-39849 (AY278491),, BJ01 (AY278488),, NS1 (AY508724), ZJ01 (AY297028), GD01 (AY278489) and GZ50 (AY304495). However, there are few vaccines in the pipeline against SARS-CoV-2. The mRNA based vaccine prepared by the US National Institute of Allergy and Infectious Diseases against SARS-CoV-2 is under phase 1 trial. INO-4800-DNA based vaccine will be soon available for human testing. Chinese Centre for Disease Control and Prevention (CDC) working on the development of an inactivated virus vaccine,. Soon mRNA based vaccine’s sample (prepared by Stermirna Therapeutics) will be available. GeoVax-BravoVax is working to develop a Modified Vaccina Ankara (MVA) based vaccine. While Clover Biopharmaceuticals is developing a recombinant 2019-nCoV S protein subunit-trimer based vaccine.

Although research teams all over the world are working to investigate the key features, pathogenesis and treatment options, it is deemed necessary to focus on competitive therapeutic options and cross-resistance of other vaccines. For instance, there is a possibility that vaccines for other diseases such as rubella or measles can create cross-resistance for SARS-CoV-2. This statement of cross-resistance is based on the observations that children in china were found less vulnerable to infection as compared to the elder population, while children are being largely vaccinated for measles in China.