Molecular identification of adenoviruses associated with respiratory infection in Egypt from 2003 to 2010.

BACKGROUND: Human adenoviruses of species B, C, and E (HAdV-B, -C, -E) are frequent causative agents of acute respiratory infections worldwide. As part of a surveillance program aimed at identifying the etiology of influenza-like illness (ILI) in Egypt, we characterized 105 adenovirus isolates from clinical samples collected between 2003 and 2010. METHODS: Identification of the isolates as HAdV was accomplished by an immunofluorescence assay (IFA) and confirmed by a set of species and type specific polymerase chain reactions (PCR). RESULTS: Of the 105 isolates, 42% were identified as belonging to HAdV-B, 60% as HAdV-C, and 1% as HAdV-E. We identified a total of six co-infections by PCR, of which five were HAdV-B/HAdV-C co-infections, and one was a co-infection of two HAdV-C types: HAdV-5/HAdV-6. Molecular typing by PCR enabled the identification of eight genotypes of human adenoviruses; HAdV-3 (n = 22), HAdV-7 (n = 14), HAdV-11 (n = 8), HAdV-1 (n = 22), HAdV-2 (20), HAdV-5 (n = 15), HAdV-6 (n = 3) and HAdV-4 (n = 1). The most abundant species in the characterized collection of isolates was HAdV-C, which is concordant with existing data for worldwide epidemiology of HAdV respiratory infections. CONCLUSIONS: We identified three species, HAdV-B, -C and -E, among patients with ILI over the course of 7 years in Egypt, with at least eight diverse types circulating.

A high diversity of Eurasian lineage low pathogenicity avian influenza A viruses circulate among wild birds sampled in Egypt.

Surveillance for influenza A viruses in wild birds has increased substantially as part of efforts to control the global movement of highly pathogenic avian influenza A (H5N1) virus. Studies conducted in Egypt from 2003 to 2007 to monitor birds for H5N1 identified multiple subtypes of low pathogenicity avian influenza A viruses isolated primarily from migratory waterfowl collected in the Nile Delta. Phylogenetic analysis of 28 viral genomes was performed to estimate their nearest ancestors and identify possible reassortants. Migratory flyway patterns were included in the analysis to assess gene flow between overlapping flyways. Overall, the viruses were most closely related to Eurasian, African and/or Central Asian lineage low pathogenicity viruses and belonged to 15 different subtypes. A subset of the internal genes seemed to originate from specific flyways (Black Sea-Mediterranean, East African-West Asian). The remaining genes were derived from a mixture of viruses broadly distributed across as many as 4 different flyways suggesting the importance of the Nile Delta for virus dispersal. Molecular clock date estimates suggested that the time to the nearest common ancestor of all viruses analyzed ranged from 5 to 10 years, indicating frequent genetic exchange with viruses sampled elsewhere. The intersection of multiple migratory bird flyways and the resulting diversity of influenza virus gene lineages in the Nile Delta create conditions favoring reassortment, as evident from the gene constellations identified by this study. In conclusion, we present for the first time a comprehensive phylogenetic analysis of full genome sequences from low pathogenic avian influenza viruses circulating in Egypt, underscoring the significance of the region for viral reassortment and the potential emergence of novel avian influenza A viruses, as well as representing a highly diverse influenza A virus gene pool that merits continued monitoring.

Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007-2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

Delayed 2009 pandemic influenza A virus subtype H1N1 circulation in West Africa, May 2009-April 2010.

To understand 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) circulation in West Africa, we collected influenza surveillance data from ministries of health and influenza laboratories in 10 countries, including Cameroon, from 4 May 2009 through 3 April 2010. A total of 10,203 respiratory specimens were tested, of which 25% were positive for influenza virus. Until the end of December 2009, only 14% of all detected strains were A(H1N1)pdm09, but the frequency increased to 89% from January through 3 April 2010. Five West African countries did not report their first A(H1N1)pdm09 case until 6 months after the emergence of the pandemic in North America, in April 2009. The time from first detection of A(H1N1)pdm09 in a country to the time of A(H1N1)pdm09 predominance varied from 0 to 37 weeks. Seven countries did not report A(H1N1)pdm09 predominance until 2010. Introduction and transmission of A(H1N1)pdm09 were delayed in this region.

Surveillance of avian influenza viruses in migratory birds in Egypt, 2003-09.

Migratory (particularly aquatic) birds are the major natural reservoirs for type A influenza viruses. However, their role in transmitting highly pathogenic avian influenza (HPAI) viruses is unclear. Egypt is a "funnel" zone of wild bird migration pathways from Central Asia and Europe to Eastern and Central Africa ending in South Africa. We sought to detect and isolate avian influenza viruses in migratory birds in Egypt. During September 2003-February 2009, the US Naval Medical Research Unit Number 3, Cairo, Egypt, in collaboration with the Egyptian Ministry of Environment, obtained cloacal swabs from 7,894 migratory birds captured or shot by hunters in different geographic areas in Egypt. Samples were processed by real-time reverse transcriptase PCR for detection of the influenza A matrix gene. Positive samples were processed for virus isolation in specific-pathogen-free embryonated eggs and isolates were subtyped by PCR and partial sequencing. Ninety-five species of birds were collected. Predominant species were Green-Winged Teal (Anas carolinensis; 32.0%, n=2,528), Northern Shoveler (Anas clypeata; 21.4%, n=1,686), and Northern Pintail (Anas acuta; 11.1%, n=877). Of the 7,894 samples, 745 (9.4%) were positive for the influenza A matrix gene (mainly from the above predominant species). Thirteen of the 745 (1.7%) were H5-positive by PCR (11 were low-pathogenic avian influenza and two were HPAI H5N1). The prevalences of influenza A was among regions were 10-15%, except in Middle Egypt (4%). Thirty-nine influenza isolates were obtained from PCR-positive samples. Seventeen subtypes of avian influenza viruses (including H5N1 and H7N7) were classified from 39 isolates using PCR and partial sequencing. Only one HPAI H5N1 was isolated in February 2006, from a wild resident Great Egret (Ardea alba). No major die-offs or sick migratory birds were detected during the study. We identified avian influenza virus subtypes not previously reported in Egypt. The HPAI H5N1 isolated or detected indicates that migratory birds may play a role in the dispersal of HPAI virus, but a detailed mechanism of this role needs to be elucidated.

Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry.

Highly pathogenic avian influenza (HPAI) H5N1 (clade 2.2) was introduced into Egypt in early 2006. Despite the control measures taken, including mass vaccination of poultry, the virus rapidly spread among commercial and backyard flocks. Since the initial outbreaks, the virus in Egypt has evolved into a third order clade (clade 2.2.1) and diverged into antigenically and genetically distinct subclades. To better understand the dynamics of HPAI H5N1 evolution in countries that differ in vaccination policy, we undertook an in-depth analysis of those virus strains circulating in Egypt between 2006 and 2010, and compared countries where vaccination was adopted (Egypt and Indonesia) to those where it was not (Nigeria, Turkey and Thailand). This study incorporated 751 sequences (Egypt n=309, Indonesia n=149, Nigeria n=106, Turkey n=87, Thailand n=100) of the complete haemagglutinin (HA) open reading frame, the major antigenic determinant of influenza A virus. Our analysis revealed that two main Egyptian subclades (termed A and B) have co-circulated in domestic poultry since late 2007 and exhibit different profiles of positively selected codons and rates of nucleotide substitution. The mean evolutionary rate of subclade A H5N1 viruses was 4.07×10(-3) nucleotide substitutions per site, per year (HPD 95%, 3.23-4.91), whereas subclade B possessed a markedly higher substitution rate (8.87×10(-3); 95% HPD 7.0-10.72×10(-3)) and a stronger signature of positive selection. Although the direct association between H5N1 vaccination and virus evolution is difficult to establish, we found evidence for a difference in the evolutionary dynamics of H5N1 viruses among countries where vaccination was or was not adopted. In particular, both evolutionary rates and the number of positively selected sites were higher in virus populations circulating in countries applying avian influenza vaccination for H5N1, compared to viruses circulating in countries which had never used vaccination. We therefore urge a greater consideration of the potential consequences of inadequate vaccination on viral evolution.

Yersinia pestis IS1541 transposition provides for escape from plague immunity.

Yersinia pestis is perhaps the most feared infectious agent due to its ability to cause epidemic outbreaks of plague disease in animals and humans with high mortality. Plague infections elicit strong humoral immune responses against the capsular antigen (fraction 1 [F1]) of Y. pestis, and F1-specific antibodies provide protective immunity. Here we asked whether Y. pestis generates mutations that enable bacterial escape from protective immunity and isolated a variant with an IS1541 insertion in caf1A encoding the F1 outer membrane usher. The caf1A::IS1541 insertion prevented assembly of F1 pili and provided escape from plague immunity via F1-specific antibodies without a reduction in virulence in mouse models of bubonic or pneumonic plague. F1-specific antibodies interfere with Y. pestis type III transport of effector proteins into host cells, an inhibitory effect that was overcome by the caf1A::IS1541 insertion. These findings suggest a model in which IS1541 insertion into caf1A provides for reversible changes in envelope structure, enabling Y. pestis to escape from adaptive immune responses and plague immunity.

Immunization with recombinant V10 protects cynomolgus macaques from lethal pneumonic plague.

Vaccine and therapeutic strategies that prevent infections with Yersinia pestis have been sought for over a century. Immunization with live attenuated (nonpigmented) strains and immunization with subunit vaccines containing recombinant low-calcium-response V antigen (rLcrV) and recombinant F1 (rF1) antigens are considered effective in animal models. Current antiplague subunit vaccines in development for utilization in humans contain both antigens, either as equal concentrations of the two components (rF1 plus rLcrV) or as a fusion protein (rF1-rLcrV). Here, we show that immunization with either purified rLcrV (a protein at the tip of type III needles) or a variant of this protein, recombinant V10 (rV10) (lacking amino acid residues 271 to 300), alone or in combination with rF1, prevented pneumonic lesions and disease pathogenesis. In addition, passive immunization studies showed that specific antibodies of macaques immunized with rLcrV, rV10, or rF1, either alone or in combination, conferred protection against bubonic plague challenge in mice. Finally, we found that when we compared the reactivities of anti-rLcrV and anti-rV10 immune sera from cynomolgus macaques, BALB/c mice, and brown Norway rats with LcrV-derived peptides, rV10, but not rLcrV immune sera, lacked antibodies recognizing linear LcrV oligopeptides.

Yersinia pestis caf1 variants and the limits of plague vaccine protection.

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.

Protective immunity against plague.

Plague, an infectious disease that reached catastrophic proportions during three pandemics, continues to be a legitimate public health concern worldwide. Although antibiotic therapy for the causative agent Yersinia pestis is available, pharmaceutical supply limitations, multi-drug resistance from natural selection as well as malicious bioengineering are a reality. Consequently, plague vaccinology is a priority for biodefense research. Development of a multi-subunit vaccine with Fraction 1 and LcrV as protective antigens seems to be receiving the most attention. However, LcrV has been shown to cause immune suppression and Y. pestis mutants lacking F1 expression are thought to be fully virulent in nature and in animal experiments. The LcrV variant, rV10, retains the well documented protective antigenic properties of LcrV but with diminished inhibitory effects on the immune system. More research is required to examine the molecular mechanisms of vaccine protection afforded by surface protein antigens and to decipher the host mechanisms responsible for vaccine success.