Isolation of Avian Influenza virus from Polymerase Chain Reaction–Negative Cloacal Samples of Waterfowl

Reverse transcription polymerase chain reaction (RT-PCR) was used routinely to detect the avian influenza virus (AIV) nucleoprotein (NP) gene. The purpose of the present study was to compare the correctness of a nested RT-PCR (nRT-PCR), one conventional RT-PCR with its outer primer (oRT-PCR) and the other conventional RT-PCR with its inner primer (iRT-PCR) to detect AIV NP gene. A total of 365 AI-free fecal swabs (73 pools), 7 tracheal swabs and anllantoic fluid from 25 chicken embryos were used to determine the analytic specificities of those tests. Compared with the iRT-PCR, the nRT-PCR was more sensitive for AIV detection. However, the specificities of nRT-PCR, oRT-PCR and iRT-PCR were 48.6% (35/72), 100% (67/67) and 91.3% (84/92), respectively. The amplifying band was sequenced and confirmed to be the AIV NP gene as the positive control. The specificity of this nRT-PCR is too low to be used for the AIV screening test.

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Pathological and Molecular Characterization of H5 Avian Influenza Virus in Poultry Flocks from Egypt over a Ten-Year Period (2009–2019)

Animals ◽

10.3390/ani10061010 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1010

Author(s):

Samah Mosad Mosad ◽

Fatma A. El-Gohary ◽

Hanaa Said Ali ◽

Hanem El-Sharkawy ◽

Ehab Kotb Elmahallawy

Keyword(s):

Polymerase Chain Reaction ◽

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Blood Vessels ◽

Molecular Characterization ◽

Histopathological Examination ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain

Avian influenza virus (AIV) remains one of the enzootic zoonotic diseases that challenges the poultry industry in Egypt. In the present study, a total of 500 tissue samples were collected from 100 chicken farms (broilers and layers) suspected to be infected with AIV through the period from 2009 to 2019 from Dakahlia governorate, Egypt. These samples were pooled in 100 working samples and screened for AIV then the positive samples were subjected to histopathological examination combined with real time-polymerase chain reaction (RRT-PCR). RRT-PCR positive samples were also subjected to conventional reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of H5 AIV and some of these resulting positive samples were sequenced for detection of the molecular nature of the studied virus. Interestingly, the histopathological examination revealed necrotic liver with leukocytic infiltration with degenerative changes with necrotic pancreatitis, edema, and intense lymphoid depletion of splenic tissue and hyperplastic tracheal epithelium. Likewise, edema and congested sub mucosal blood vessels and intense bronchial necrosis with hyalinized wall vascular wall and heterophils infiltration were reported. Pneumonic areas with intense leukocytic aggregation mainly and vasculitis of the pulmonary blood vessels were also detected in lung. Collectively, these significant pathological changes in examined tissues cohered with AIV infection. Regarding the molecular characterization, 66 samples were positive for AIV by RRT-PCR and 52 of them were positive for H5 AIV by RT-PCR. The phylogenetic analysis revealed that the H5 viruses identified in this study were aligned with other Egyptian H5N1 AIVs in the Egyptian sub clade 2.2.1, while some of the identified strains were aligned with other Egyptian H5N8 strains in the new Egyptian sub clade 2.3.4.4. Taken together, our present findings emphasize the wide spread of AIV in Egypt and the importance of developing an efficient surveillance and periodical screening program for controlling such disease of public health concern.

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Development of multiplex reverse transcription polymerase chain reaction (RT-PCR) for simultaneous detection of matrix, haemagglutinin and neuraminidase genes of H5N1 avian influenza virus

Bangladesh Veterinarian ◽

10.3329/bvet.v28i2.10653 ◽

2012 ◽

Vol 28 (2) ◽

pp. 55-59 ◽

Cited By ~ 2

Author(s):

R. Mojumder ◽

E. H. Chowdhury ◽

R Parvin ◽

J. A. Begum ◽

M. Giasuddin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Influenza Virus ◽

Avian Influenza ◽

Matrix Protein ◽

Simultaneous Detection ◽

High Fidelity ◽

Rt Pcr ◽

Chain Reaction ◽

One Step ◽

Polymerase Chain

Influenza A virus, subtype H5N1 causes a fatal disease in domestic poultry and could spread directly from poultry to humans. The aim of this study was to develop a multiplex reverse transcription polymerase chain reaction (mRT-PCR) for simultaneous detection of Type A influenza virus-specific matrix protein (M) gene as well as H5 and N1 genes of highly pathogenic avian influenza (HPAI) viruses. Finnzymes Phusion-Flash High- Fidelity PCR Master Mix (Finnzymes Oy, Finland) and Qiagen one-step RT-PCR enzyme mix (Qiagen, Germany) were used in a one-step RT-PCR. RNA was extracted from two known positive samples using Qiagen RNA extraction kit. RT-PCR was carried out with a mixture of primers specific for the Type A influenza virus matrix protein (M), and H5 and N1 genes of H5N1 HPAI viruses in a single reaction system. The mRT-PCR cDNA products were visualized by gel electrophoresis. The mRT-PCR yielded fragments of 245 bp for M, 545 bp for H5 and 343 bp for N1 genes of HPAI virus, which were clearly distinguishable. The mRT-PCR using the Finnzymes Phusion-Flash High-Fidelity PCR Master Mix (Finnzymes Oy, Finland) with Qiagen one-step RT-PCR Enzyme Mix (Qiagen, Germany) required only one hour and 20 minutes. (Bangl. vet. 2011. Vol. 28, No. 2, 55 59)DOI: http://dx.doi.org/10.3329/bvet.v28i2.10653

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