http://europepmc.org/articles/PMC7126491?pdf=render

Interspecies transmission and emergence of novel viruses: lessons from bats and birds.

Avian influenza virus infections in humans.

H5N1 avian influenza is a highly fatal infectious disease that could cause a potentially devastating pandemic if the H5N1 virus mutates into a form that spreads efficiently among humans. Recent findings have led to a basic understanding of cell and organ histopathology caused by the H5N1 virus. Here we review the pathology of H5N1 avian influenza reported in postmortem and clinical studies and discuss the key pathogenetic mechanisms. Specifically, the virus infects isolated pulmonary epithelial cells and causes diffuse alveolar damage and hemorrhage in the lungs of infected patients. In addition, the virus may infect other organs, including the trachea, the intestines, and the brain, and it may penetrate the placental barrier and infect the fetus. Dysregulation of cytokines and chemokines is likely to be one of the key mechanisms in the pathogenesis of H5N1 influenza. We also review the various molecular determinants of increased pathogenicity that have been identified in recent years and the role of avian and human influenza virus receptors in relation to the transmissibility of the H5N1 virus. A comprehensive appreciation of H5N1 influenza pathogenetic mechanisms should aid in the design of effective strategies for prevention, diagnosis, and treatment of this emerging disease.

Pathology, molecular biology, and pathogenesis of avian influenza A (H5N1) infection in humans.

Avian influenza H5N1 virus is known to cross the species barrier and infect humans and felines. We report a fatal H5N1 infection in a dog following ingestion of an H5N1-infected duck during an outbreak in Thailand in 2004. With new reports of H5N1 virus continuing across Asia, Europe, and Africa, this finding highlights the need for monitoring of domestic animals during outbreaks.

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Fatal avian influenza A H5N1 in a dog.

We report H5N1 virus infection in a domestic cat infected by eating a pigeon carcass. The virus isolated from the pigeon and the cat showed the same cluster as the viruses obtained during the outbreak in Thailand. Since cats are common house pets, concern regarding disease transmission to humans exists.

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Avian Influenza H5N1 in Naturally Infected Domestic Cat

Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection.

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

Genetic characterization of influenza A viruses (H5N1) isolated from 3rd wave of Thailand AI outbreaks

To the Editor: To thoroughly investigate avian influenza outbreaks, identifying highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) is essential. Currently, determination of inserted basic amino acids within the hemagglutinin cleavage site of HPAI relies on nucleotide sequencing (1–3). Direct sequencing is relatively time-consuming and laborious and thus is not suitable for local and regional diagnostic laboratories that receive large numbers of samples that may contain HPAI or LPAI subtype H5N1. Therefore, a rapid diagnostic assay was developed to discriminate between HPAI and LPAI subtype H5 viruses by 1-step real-time reverse transcription–polymerase chain reaction (RT-PCR) with melting curve analysis.

H5 primers flanking the cleavage site were designed from conserved regions among HPAI and LPAI strains by using nucleotide sequences obtained from GenBank and our previous studies (4–6). The primers consisted of a forward primer H5F3+ (nucleotides 1001–1021: 5´-AACAGATTAGTCCTTGCGACTG-3´) and a reverse primer H5R2+ (nucleotides 1124–1103: 5´-CATCTACCATTCCCTGCCATCC-3´), which yielded products of ≈124 bp and ≈112 bp, corresponding to HPAI and LPAI, respectively. Consequently, the sizes of the amplicons and percentage guanine-cytosine content were different, allowing discrimination between HPAI and LPAI by melting curve analysis (7).

One-step, real-time RT-PCR with melting curve analysis was performed in an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). In each reaction, 3.0 μL of RNA sample was combined with a reaction mixture containing 10 μL 2× SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μL 40× MultiScribe (Applied Biosystems) and RNase inhibitor, each primer (at final concentration of 0.5 μmol/L), 1.5 mmol/L MgCl2, and RNase-free water in a final volume of 20 μL. The thermal profile began with incubation at 48°C for 45 min (reverse transcription), then incubation at 95°C for 10 min (predenaturation), followed by 40 cycles of amplification alternating between 94°C for 15 s (denaturation) and 68°C for 40 s (annealing/extension). The SYBR Green I fluorescent signal was obtained once per cycle at the end of the extension step. After amplification, melting curve analysis was performed by heating the sample to 95°C for 15 s, then cooling it to 70°C for 1 min, followed by a linear temperature increase to 95°C at a rate of 0.5°C/s, while continuously monitoring the fluorescent signal. Data were analyzed by the 7500 System SDS Software version 1.2 (Applied Biosystems).

To develop the assay, samples previously identified as influenza A/chicken/Nakorn-Patom/Thailand/CU-K2/04 (H5N1) served as a control for HPAI, and A/duck/Hong Kong/308/78 (H5N3) served as a control for LPAI. The H5 genes (nucleotides 914–1728) of each strain were inserted into pGEM-T Easy Vector and then transcribed in vitro by using RiboMAX Large Scale RNA Production System-T7 (Promega, Madison, WI, USA). Serial 10-fold dilutions of the standard H5 RNA were subjected to a sensitivity test (8). The fluorescent signal can be detected at RNA dilutions as low as 102 copies/μL. To assess the specificity, viral RNA extracted from other subtypes of influenza A viruses (H1–H4 and H6–H15) was tested. The assay was specific for the H5 subtype, since no amplification was detected from other subtypes.

Three preliminary melting curve analyses showed that this assay was effective in discriminating between the melting peaks of HPAI and LPAI (Figure). The variations of melting temperature (Tm) between runs were experimentally determined. The mean (standard deviation) of Tm values for HPAI and LPAI were 77.43°C (0.21°C) and 79.57°C (0.23°C), respectively. This assay provided high reliability and reproducibility, since the coefficients of variation were <0.30.



Figure

Discrimination between highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) by melting curve analysis based on real-time reverse transcription–polymerase chain reaction of the H5 HA gene with SYBR Green I fluorescent ...



Seventy-eight specimens of influenza A virus were used to validate the assay. The 75 HPAI samples were isolated during the 2004 outbreak in Thailand; 3 LPAI samples, including A/avian/NY/01 (H5N2), A/Chicken/Mexico/31381-3/94 (H5N2), and A/shoveler/Egypt/03 (H5N2), were provided by the Centers for Disease Control and Prevention. The viruses were isolated in embryonated chicken eggs as described previously (9). RNA was extracted from 140 μL of allantoic fluid, and RT-PCR was performed as described above. Melting curve analysis showed that all H5N1 isolates from the 2004 outbreak in Thailand were interpreted as HPAI, whereas the 3 samples of H5N2 subtype were classified as LPAI. The Tm varied from 77.2°C to 78.1°C for HPAI and 78.75°C to 79.5°C for LPAI. The melting curve analysis results were completely concordant with the results of direct sequence analysis of the H5 gene for all samples tested.

The melting curve analysis described here provides a rapid, accurate, and high-throughput approach to discriminate between HPAI and LPAI. This assay is particularly attractive for large-scale screening of suspected subtype H5 influenza A virus during outbreaks to identify candidate LPAI that could be used as vaccine strains.

Sudarat Damrongwantanapokin

Papers

Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection.

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

Genetic characterization of influenza A viruses (H5N1) isolated from 3rd wave of Thailand AI outbreaks

Discrimination between Highly Pathogenic and Low Pathogenic H5 Avian Influenza A Viruses