ClassyFlu: Classification of Influenza A Viruses with Discriminatively Trained Profile-HMMs

<div><p>Accurate and rapid characterization of influenza A virus (IAV) hemagglutinin (HA) and neuraminidase (NA) sequences with respect to subtype and clade is at the basis of extended diagnostic services and implicit to molecular epidemiologic studies. ClassyFlu is a new tool and web service for the classification of IAV sequences of the HA and NA gene into subtypes and phylogenetic clades using discriminatively trained profile hidden Markov models (HMMs), one for each subtype or clade. ClassyFlu merely requires as input unaligned, full-length or partial HA or NA DNA sequences. It enables rapid and highly accurate assignment of HA sequences to subtypes H1–H17 but particularly focusses on the finer grained assignment of sequences of highly pathogenic avian influenza viruses of subtype H5N1 according to the cladistics proposed by the H5N1 Evolution Working Group. NA sequences are classified into subtypes N1–N10. ClassyFlu was compared to semiautomatic classification approaches using BLAST and phylogenetics and additionally for H5 sequences to the new “Highly Pathogenic H5N1 Clade Classification Tool” (IRD-CT) proposed by the Influenza Research Database. Our results show that both web tools (ClassyFlu and IRD-CT), although based on different methods, are nearly equivalent in performance and both are more accurate and faster than semiautomatic classification. A retraining of ClassyFlu to altered cladistics as well as an extension of ClassyFlu to other IAV genome segments or fragments thereof is undemanding. This is exemplified by unambiguous assignment to a distinct cluster within subtype H7 of sequences of H7N9 viruses which emerged in China early in 2013 and caused more than 130 human infections. <a href="http://bioinf.uni-greifswald.de/ClassyFlu" target="_blank">http://bioinf.uni-greifswald.de/ClassyFlu</a> is a free web service. For local execution, the ClassyFlu source code in PERL is freely available.</p></div

Pairwise comparison of the H5 HA classification of full sequences.

<p>Pairwise comparison of the H5 HA classification of full sequences.</p

Pairwise comparison of the H5 HA classification of PanHA fragments.

<p>Pairwise comparison of the H5 HA classification of PanHA fragments.</p

Avian Influenza Virus H3 Hemagglutinin May Enable High Fitness of Novel Human Virus Reassortants

<div><p>Reassortment of influenza A virus genes enables antigenic shift resulting in the emergence of pandemic viruses with novel hemagglutinins (HA) acquired from avian strains. Here, we investigated whether historic and contemporary avian strains with different replication capacity in human cells can donate their hemagglutinin to a pandemic human virus. We performed double-infections with two avian H3 strains as HA donors and a human acceptor strain, and determined gene compositions and replication of HA reassortants in mammalian cells. To enforce selection for the avian virus HA, we generated a strictly elastase-dependent HA cleavage site mutant from A/Hong Kong/1/68 (H3N2) (Hk68-Ela). This mutant was used for co-infections of human cells with A/Duck/Ukraine/1/63 (H3N8) (DkUkr63) or the more recent A/Mallard/Germany/Wv64-67/05 (H3N2) (MallGer05) in the absence of elastase but presence of trypsin. Among 21 plaques analyzed from each assay, we found 12 HA reassortants with DkUkr63 (4 genotypes) and 14 with MallGer05 (10 genotypes) that replicated in human cells comparable to the parental human virus. Although DkUkr63 replicated in mammalian cells at a reduced level compared to MallGer05 and Hk68, it transmitted its HA to the human virus, indicating that lower replication efficiency of an avian virus in a mammalian host may not constrain the emergence of viable HA reassortants. The finding that HA and HA/NA reassortants replicated efficiently like the human virus suggests that further HA adaptation remains a relevant barrier for emergence of novel HA reassortants.</p></div

Gene composition of DkUkr63 reassortants.

<p>Gene segments originating from DkUkr63 (D) and Hk68 (H) are shown in dark and light grey, respectively (HD indicates a mixed population). Co-infections with Hk68-Ela and DkUkr63 were performed either in the presence (+) or absence (−) of TPCK-treated trypsin. Supernatants were harvested either at eight or 24 hours after co-infection, the RNA isolated and subjected to RT-PCR for genotyping. Reassortants with no detectable HA titer in the supernatant after co-infection were propagated in embryonated chicken eggs and are indicated by a super-scripted ^e.</p

Growth curves of DkUkr63 HA reassortants.

<p>The gene segment constellation of each reassortant is given next to each chart: Hk68 genes (black) and DkUkr63 genes (red). The presence of diverse additional DkUkr63 segments is indicated by the abbreviation ‘div’. From supernatants of A549 cells infected with a MOI of 10⁻³, viral titers were determined by plaque assays on MDCK cells. The growth curves of the parental viruses Hk68 (black filled circles), Hk68-Ela (black empty circles) and DkUkr63 (red filled circles) in each chart represent identical data and were drawn in for comparison. Growth curves were determined by plaque titrations of duplicate or quadruplicate infected cell cultures (in the latter case indicated by a diamond in the chart legend). Reassortants reaching titers at 48 h not less than one magnitude below Hk68 are highlighted by grey rectangles.</p

Growth curves of MallGer05 HA reassortants.

<p>The gene segment composition of the each reassortant is given next to each chart: Hk68 genes (black) and MallGer05 genes (red). The presence of diverse additional MallGer05 segments is indicated by the abbreviation ‘div’. From supernatants of A549 cells infected with a MOI of 10⁻³, viral titers were determined by plaque assays on MDCK cells. The growth curves of the parental viruses Hk68 (black filled circles), Hk68-Ela (black empty circles) and MallGer05 (red filled circles) in each chart represent identical data and were drawn in for comparison. Growth curves were determined by plaque titrations of duplicate or quadruplicate infected cell cultures (in the latter case indicated by a diamond in the chart legend). Reassortants reaching titers at 48 h not less than one magnitude below Hk68 are highlighted by grey rectangles.</p

Experimental set up for alveolar breath sampling in goats.

<p>A—sampling mask; B—CO₂-sensor; C—Needle Trap Device (NTD); D—CO₂ triggered flow valve; E—Capnogard for time resolved CO₂-monitoring; F—sampling box; dashed arrows represent air flows; continuous arrows represent electronic signals.</p

PCA-scatterplots based on VOC-analysis of headspace over feces.

<p>PCA (a) was done for substances (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123980#pone.0123980.t003" target="_blank">Table 3</a>) having significantly different (p < 0.05) concentrations in inoculated and non-inoculated animals. The 3D-scatterplot (b) is derived from the same PCA with respect to weeks after inoculation on the third axis. The loading plot referring to this PCA analysis is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123980#pone.0123980.s003" target="_blank">S3 Fig</a>. Blue dots represent the non-inoculated group. Red dots represent the inoculated group. PC-01 explains 31% and PC-02 explains 16% of variance.</p

Virus shedding after vaccination.

<p>Swab samples taken on the indicated days from oropharynx of MDA- chickens (A) immunized with chNDVFHN _PMV8H5 at three weeks of age and swabs combined from oropharynx and cloaca of MDA+ chickens (B) immunized on day 1 (group a) or day 7 (group b) after hatch were analyzed for the presence of NDV NP gene-specific RNA by RT-qPCR. Values were transformed to genome equivalents (GEQ) using calibration curves of defined RNA standards that were included with each RT-qPCR run. The number of positive swabs by RT-qPCR is given below the box plots. Significant differences (P < 0.0125, Bonferroni correction) between vaccinated groups and controls are indicated (*).</p