Molecular diagnosis and characterization of Cryptosporidium spp. in turkeys and chickens in Germany reveals evidence for previously undetected parasite species.

A total of 256 fecal specimens were randomly collected from farmed poultry in Germany and screened for the presence of Cryptosporidium spp. by PCR and further characterized by direct automated DNA sequencing. Using a nested PCR amplifying approximately 830 bp 18S rDNA fragment, 7.03% (n = 18) of the samples were Cryptosporidium-positive. In detail, Cryptosporidium was detected in 9.3% (8/86) of turkeys, 5.7% (9/158) of broilers and 8.3% (1/12) of layers. After DNA sequencing, Cryptosporidium parvum the most frequently observed species was identified in 5.1% (13/256) of all poultry species, including 8.1% (7/86) of turkeys, 3.2% (5/158) of broilers and 8.3% (1/12) of layers. Cryptosporidium baileyi was detected in 1.3% (2/256) of the broilers only. Three novel unclassified Cryptosporidium spp. were detected in 1.2% (1/86) of turkeys and 1.3% (2/158) of broilers. The infection rate was high in 13-20 week old turkeys, 1-6 weeks old broilers and >20 weeks old layers but differences between age groups were not significant. This is the first study in Germany uses molecular methods for the detection of Cryptosporidium in poultry. The results indicate that Cryptosporidium parasites are common among broilers and turkeys in Germany. Considering the large size of the poultry industry, the large amount of poultry meat that is consumed and the fact that C. parvum is also the most common Cryptosporidium parasite in humans, poultry might also be a source of human infections.

Progressive glycosylation of the haemagglutinin of avian influenza H5N1 modulates virus replication, virulence and chicken-to-chicken transmission without significant impact on antigenic drift.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and continues to pose a pandemic threat. Studying the progressive genetic changes in A/H5N1 after long-term circulation in poultry may help us to better understand A/H5N1 biology in birds. A/H5N1 clade 2.2.1.1 antigenic drift viruses have been isolated from vaccinated commercial poultry in Egypt. They exhibit a peculiar stepwise accumulation of glycosylation sites (GS) in the haemagglutinin (HA) with viruses carrying, beyond the conserved 5 GS, additional GS at amino acid residues 72, 154, 236 and 273 resulting in 6, 7, 8 or 9 GS in the HA. Available information about the impact of glycosylation on virus fitness and pathobiology is mostly derived from mammalian models. Here, we generated recombinant viruses imitating the progressive acquisition of GS in HA and investigated their biological relevance in vitro and in vivo. Our in vitro results indicated that the accumulation of GS correlated with increased glycosylation, increased virus replication, neuraminidase activity, cell-to-cell spread and thermostability, however, strikingly, without significant impact on virus escape from neutralizing antibodies. In vivo, glycosylation modulated virus virulence, tissue tropism, replication and chicken-to-chicken transmission. Predominance in the field was towards viruses with hyperglycosylated HA. Together, progressive glycosylation of the HA may foster persistence of A/H5N1 by increasing replication, stability and bird-to-bird transmission without significant impact on antigenic drift.

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens.

Highly pathogenic (HP) avian influenza viruses (AIV) evolve from low pathogenic (LP) precursors after circulation in poultry by reassortment and/or single mutations in different gene segments including that encoding NS1. The carboxyl terminal end (CTE) of NS1 exhibits deletions between amino acid 202 and 230 with still unknown impact on virulence of AIV in chickens. In this study, NS1 protein sequences of all AIV subtypes in birds from 1902 to 2015 were analyzed to study the prevalence and distribution of CTE truncation (ΔCTE). Thirteen different ΔCTE forms were observed in NS1 proteins from 11 HA and 8 NA subtypes with high prevalences in H9, H7, H6 and H10 and N9, N2, N6 and N1 subtypes particularly in chickens and minor poultry species. With 88% NS217 lacking amino acids 218-230 was the most common ΔCTE form followed by NS224 (3.6%). NS217 was found in 10 and 8 different HA and NA subtypes, respectively, whereas NS224 was detected exclusively in the Italian HPAIV H7N1 suggesting relevance for virulence. To test this assumption, 3 recombinant HPAIV H7N1 were constructed carrying wild-type HP NS1 (Hp-NS224), NS1 with extended CTE (Hp-NS230) or NS1 from LPAIV H7N1 (Hp-NSLp), and tested in-vitro and in-vivo. Extension of CTE in Hp NS1 significantly decreased virus replication in chicken embryo kidney cells. Truncation in the NS1 decreased the tropism of Hp-NS224 to the endothelium, central nervous system and respiratory tract epithelium without significant difference in virulence in chickens. This study described the variable forms of ΔCTE in NS1 and indicated that CTE is not an essential virulence determinant particularly for the Italian HPAIV H7N1 but may be a host-adaptation marker required for efficient virus replication.

Phylogenetic analysis of human influenza A/H3N2 viruses isolated in 2015 in Germany indicates significant genetic divergence from vaccine strains.

Infections by H3N2-type influenza A viruses (IAV) resulted in significant numbers of hospitalization in several countries in 2014-2015, causing disease also in vaccinated individuals and, in some cases, fatal outcomes. In this study, sequence analysis of H3N2 viruses isolated in Germany from 1998 to 2015, including eleven H3N2 isolates collected early in 2015, was performed. Compared to the vaccine strain A/Texas/50/2012 (H3N2), the 2015 strains from Germany showed up to 4.5 % sequence diversity in their HA1 protein, indicating substantial genetic drift. The data further suggest that two distinct phylogroups, 3C.2 and 3C.3, with 1.6-2.3 % and 0.3-2.4 % HA1 nucleotide and amino acid sequence diversity, respectively, co-circulated in Germany in the 2014/2015 season. Distinct glycosylation patterns and amino acid substitutions in the hemagglutinin and neuraminidase proteins were identified, possibly contributing to the unusually high number of H3N2 infections in this season and providing important information for developing vaccines that are effective against both genotypes.

A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens.

UNLABELLED: In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE: In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

Improved universal cloning of influenza A virus genes by LacZα-mediated blue/white selection.

Reverse genetics of influenza A viruses facilitates both basic research and vaccine development. However, efficient cloning of virus gene segments was cumbersome in established systems due to the necessary cleavage of amplicons with outside cutter restriction enzymes followed by ligation. Occasionally, virus genes may contain cleavage sites for those enzymes. To circumvent that problem, we previously established target-primed plasmid amplification using the negative selection marker ccdB cloned into the plasmid pHW2000, flanked by the highly conserved gene segment termini. Here, we further introduced the LacZα fragment downstream of the ccdB region for additional ad-hoc selection of transformed bacteria by blue/white pre-screening. For comparison, we cloned three gene segments (PA, HA, and NS) from the influenza strain A/Swine/Belgium/1/1979 (H1N1) (SwBelg79) into plasmid vectors pHWSccdB and pHWSccdB-LacZα and observed same cloning efficiency. Furthermore, the plasmid pHWSccdB-LacZα allows easy elimination of bacterial colonies containing empty plasmid clones. Using this improved plasmid, we obtained the complete genomic set of eight functional plasmids for SwBelg79.

The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken.

Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant.

Genetic changes that accompanied shifts of low pathogenic avian influenza viruses toward higher pathogenicity in poultry.

Avian influenza viruses (AIV) of H5 and H7 subtypes exhibit two different pathotypes in poultry: infection with low pathogenic (LP) strains results in minimal, if any, health disturbances, whereas highly pathogenic (HP) strains cause severe morbidity and mortality. LPAIV of H5 and H7 subtypes can spontaneously mutate into HPAIV. Ten outbreaks caused by HPAIV are known to have been preceded by circulation of a predecessor LPAIV in poultry. Three of them were caused by H5N2 subtype and seven involved H7 subtype in combination with N1, N3, or N7. Here, we review those outbreaks and summarize the genetic changes which resulted in the transformation of LPAIV to HPAIV under natural conditions. Mutations that were found directly in those outbreaks are more likely to be linked to virulence, pathogenesis, and early adaptation of AIV.

Highly pathogenic avian influenza virus H5N1 from Egypt escapes vaccine-induced immunity but confers clinical protection against a heterologous clade 2.2.1 Egyptian isolate.

The poultry populations of Egypt are endemically infected by highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1. Vaccination was chosen as an auxiliary tool to control HPAIV in poultry. Potency of commercial vaccines regarding emerging variants is under discussion. In the current study efficacy of four different inactivated whole H5 virus vaccines representing different sublineages of HPAIV H5N1 were tested in chickens against challenge viruses currently co-circulating in Egypt and representing two antigenically widely distinct HPAIV H5N1 lineages, i.e., "variant" (clade 2.2.1var) and "proper" (clade 2.2.1pro) viruses. All vaccines induced clinical protection against challenge with 2.2.1pro Egyptian strains. In contrast, when challenged with a variant strain, only chickens vaccinated with the homologous Egyptian clade 2.2.1var virus or an inactivated re-assorted H5N1 strain (Re-5, clade 2.3) were protected. However, only the homologous virus induced sterile immunity whereas chickens clinically protected after Re-5 vaccination shed virus at day two after infection indistinguishable to H5N2 vaccines. In conclusion, monitoring vaccine-driven evolution of HPAIV H5N1 by surveillance, antigenic characterization, and challenge studies is essential to assess efficacy of AIV vaccination campaigns.

Simultaneous detection and differentiation by multiplex real time RT-PCR of highly pathogenic avian influenza subtype H5N1 classic (clade 2.2.1 proper) and escape mutant (clade 2.2.1 variant) lineages in Egypt.

BACKGROUND: The endemic status of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1 v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1 v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR) protocols due to mismatches in the primers/probe binding sites. RESULTS: We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV). A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt. CONCLUSIONS: The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt.

Modified H5 real-time reverse transcriptase-PCR oligonucleotides for detection of divergent avian influenza H5N1 viruses in Egypt.

The efforts exerted to prevent circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in birds are the best way to prevent the emergence of a new virus subtype with pandemic potential. Despite the blanket vaccination strategy against HPAI H5N1 in Egypt, continuous circulation of the virus in poultry has increased since late 2007 as a result of the presence of genetic and antigenic distinct variant strains that have escaped during the immune response of vaccinated birds. Although the suspected poultry flocks have had signs and lesions commonly seen in HPAI H5N1-infected birds, escape of variant strains from detection by real-time reverse transcriptase-PCR (RRT-PCR) was observed. Sequence analysis of these variants revealed multiple single nucleotide substitutions in the primers and probe target sequences of the H5 gene by real-time RT-PCR. This study describes the results of RRT-PCR, modified from an existing protocol with regard to the detection of the partial H5 gene segment of the Egyptian H5N1 divergent viruses and applied to nationwide surveillance. The modified RRT-PCR assay was more sensitive than the original one in the detection of Egyptian isolates, with 104% amplification efficiency. Sixty-one field samples were found to be positive in our assay, but only 51 samples tested positive by the original protocol and were more sensitive than matrix gene RRT-PCR detection assay. A detection limit of 10 mean embryo infective dose (EID50) with the updated oligonucleotides primers and probe set was found. For the foreseeable future, mutation of H5N1 viruses and the endemic situation in developing countries require continuous improvement of current diagnostics to aid in the containment of the H5N1 virus in poultry sectors and to lower the threat of influenza virus spread.