Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt, 2014/15.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks.

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.

Isolation of highly pathogenic avian influenza H5N1 from table eggs after vaccinal break in commercial layer flock.

In May 2009, during routine monitoring of a commercial layer flock of about 87,000 birds kept in cages in 4 different houses that had been vaccinated 3 times with an inactivated H5N1 vaccine at weeks 1, 7, and 16, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was isolated and detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tracheal and cloacal swabs collected from houses 3 and 4; 7 days after onset of clinical signs, there was an increase in mortality accompanied by a decrease in egg production and egg quality. In addition, using RT-PCR, the viral RNA could be detected from albumin and eggshell as well. Seven days after the onset of the clinical signs, the hemagglutination inhibition (HI) titers in the affected houses were 3.2 and 1.9 log2. In the other two houses, there were no clinical signs, and all tested samples were negative using virus isolation and real-time RT-PCR. The HI titers were 6.6 and 7.0 log2 in nonaffected houses. The isolated virus from egg albumin showed high nucleotides and amino-acid identities and clustered with viruses from recently H5N1-confirmed human infections and poultry from different places in Egypt. Moreover, several amino-acid substitutions of viral H5 protein were observed. The vaccinal break seems to be associated with immune escape mutants and/or improper vaccination. The role of contaminated eggs as a source of infection and as a vehicle for spread of the virus should be considered in area with avian influenza outbreaks.

Circulation of avian influenza H5N1 in live bird markets in Egypt.

The poultry meat trade in Egypt depends mainly on live bird markets (LBMs) because of insufficient slaughterhouses, lack of marketing infrastructure, and cultural preference for consumption of freshly slaughtered poultry. There are two types of LBMs in Egypt: retail shops and traditional LBMs where minimal, if any, food safety standards or veterinary inspection are implemented. Before January 2009, LBMs were considered to be a missing link in the epidemiology of avian influenza in Egypt. This incited us to initiate this surveillance to better understand the perpetuation of H5N1 and the risk of infection in poultry markets. Seventy-one out of 573 (12.4%) examined LBMs were positive for the H5N1 subtype by real-time--quantitative polymerase chain reaction (RT-qPCR) from January to April 2009. Where a 70.4% detection rate from LBMs had waterfowl only as a solitary sold species, a 26.8% detection rate from LBMs had waterfowl mixed with chicken and/or turkey, and 2.8% from LBMs had only turkey. Higher incidence, 40.8%, of positive LBMs was recorded during the cold month of February and concentrated mainly in the highly populated Nile Delta. These findings revealed wide circulation of H5N1 avian influenza virus in LBMs in Egypt, which poses a threat to public health and the poultry industry. Long-term control measures are required, and routine surveillance of bird markets should be conducted year-round.

Genetic characterization of variant strains of highly pathogenic avian influenza H5N1 that escaped detection by real-time reverse transcriptase-PCR diagnostic tests.

The highly pathogenic avian influenza virus of subtype H5N1 that caused serious outbreaks in Egypt in 2006 was efficiently detected using a commercially available real-time reverse transcriptase-PCR (RRT-PCR) for the type A specific matrix (M) gene in field samples of cloacal and tracheal swabs. RRT-PCR was also used for subtyping and confirmation of H5 subtype. During late 2007 the National Laboratory for Veterinary Quality Control on Poultry Production detected five field cases that were positive for avian influenza virus (AIV) based on the M gene RRT-PCR. Three different commercial H5 RRT-PCRs were used for identification of the H5 subtype, as well as a published World Organization for Animal Health (OIE) H5 RRT-PCR that had been previously carefully validated. The five cases had positive results for the H5 gene using the published OIE H5 RRT-PCR, but the three commercial H5 RRT-PCRs tests only returned two to four positive results out of the five positive cases. The hemagglutinin gene (HA) sequencing analysis of these five isolates showed multiple nucleotide substitution mutations, suggesting genetic variation that could affect the H5 primer and/or probe binding sequences. These data highlight the importance of continued monitoring of RRT-PCR primers and probes to ensure that sensitivity and specificity are maintained. The use of conventional methods in national and reference AIV laboratories, including virus isolation, serologic subtyping, and alternative RRT-PCR primers, is necessary to detect the newly emerging variant H5N1 strains that affect diagnostic performance.

384-channel parallel microfluidic cytometer for rare-cell screening.

We have constructed a 384-channel parallel microfluidic cytometer (PMC). The multichannel architecture allows 384 unique samples for a cell-based screen to be read out in approximately 6-10 min, about 30-times the speed of a conventional fluorescence-activated cytometer system (FACS). This architecture also allows the signal integration time to be varied over a larger range than is practical in single-channel FACS and is suitable for detection of rare-cells in a high background of negatives. The signal-to-noise advantages have been confirmed by using the system to count rare clonal osteocytes in the most difficult early stages of an expression-cloning screen for the carboxy-terminal parathyroid hormone receptor (CPTHR). This problem requires finding several dozen positive cells in a background of one million negatives. The system is automated around a scanning laser confocal detector and a 96-tip robotic pipettor and can maintain in vitro cultures on-system in 384-well plates. It is therefore directly practical for biology applications using existing high-throughput culture facilities. The PMC system lends itself to high-sample-number cytometry with an unusual capability for time synchronization and rare-cell sensitivity. A limited ability to handle large sample numbers has restricted applications of single-channel FACS in combinatorial cell assays; therefore the PMC could have a significant application in high-throughput screening.

Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling.

Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1-84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca(2+)](i)) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca(2+)](i) was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca(2+)](i) was observed in OC59 cells in response to the CPTH fragment hPTH-(53-84) (250 nM). No [Ca(2+)](i) signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1-34) nor a mutant CPTH analog, [Ala(55-57)]hPTH-(53-84), that does not to bind to CPTHRs, increased [Ca(2+)](i) in OC59 cells. The [Ca(2+)](i) response to hPTH-(53-84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), omega-agatoxin IVA (10 nM), and omega-conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs.

Immunohistochemical localization of gross cystic disease fluid protein-15, -24 and -44 in ductal carcinoma in situ of the breast: relationship to the degree of differentiation.

AIMS: Three major proteins present in breast gross cystic disease fluid and expressed by the cyst lining apocrine epithelium are gross cystic disease fluid protein-15 (GCDFP-15), apolipoprotein-D (APO-D; GCDFP-24) and zinc alpha2-glycoprotein (ZnGP; GCDFP-44). The aim of this study was to investigate the expression of these proteins in ductal carcinoma in situ (DCIS) of the breast and to relate their expression with the degree of differentiation of DCIS. METHODS AND RESULTS: An immunohistochemical study of these proteins was performed in 57 cases of DCIS and nine cases of morphologically apocrine DCIS. Positivity was seen in 24/57 (42.1%) cases with anti-GCDFP-15, 20/57 (35.1%) cases with anti-GCDFP-24 and 22/57 (38.6%) cases with anti-GCDFP-44. GCDFP-15 positivity was noted in 5/13 (38.5%) of the well-differentiated, 11/19 (57.9%) intermediately differentiated and 8/25 (32.0%) of the poorly differentiated cases (P=0.217). GCDFP-24 positivity was seen in 3/13 (23.0%) well-differentiated, 9/19 (47.4%) intermediately differentiated and 8/25 (32.0%) poorly differentiated cases (P=0.336). GCDFP-44 was detected in 5/13 (38.5%) of well-differentiated cases, 11/19 (57.9%) intermediately differentiated and 6/25 (24.0%) poorly differentiated cases (P=0.074). In the nine cases of apocrine DCIS, GCDFP-15 positivity was detected in seven (77.8%), while five (55.6%) and six (66.7%) cases were positive for GCDFP-24 and GCDFP-44, respectively. CONCLUSIONS: The results indicate that there is no significant association between the expression of the studied proteins and the degree of differentiation of DCIS of the breast. Moreover, some morphologically apocrine DCIS cases appear to lose expression of these proteins.

Keratoconus-tetany-menopause: the new association.

This report describes the unusual case of 46-year-old white European woman who presented spontaneous keratoconus. This case suggests a new association: keratoconus-tetany-menopause. This can be explained by Thalasselis' syndrome, a syndrome showing a relation between keratoconus, magnesium (Mg) deficiency, type A behavior, and allergy. Furthermore, Thalasselis' syndrome integrates old and new theories. This report also suggests that the alteration induced by severe but reversible Mg deficiency at the intra- and extracellular levels, associated with metabolic and hormonal imbalances, may trigger the development of keratoconus. Clinical data which support this thesis are reported.