Migratory birds play a critical role in the rapid spread of highly pathogenic avian influenza (HPAI) H5N8 virus clade 2.3.4.4 across Eurasia. Elucidating the timing and pattern of virus transmission is essential therefore for understanding the spatial dissemination of these viruses. In this study, we surveyed >27,000 wild birds in China, tracked the year-round migration patterns of 20 bird species across China since 2006, and generated new HPAI H5N8 virus genomic data. Using this new data set, we investigated the seasonal transmission dynamics of HPAI H5N8 viruses across Eurasia. We found that introductions of HPAI H5N8 viruses to different Eurasian regions were associated with the seasonal migration of wild birds. Moreover, we report a backflow of HPAI H5N8 virus lineages from Europe to Asia, suggesting that Europe acts as both a source and a sink in the global HPAI virus transmission network.

The spatial spread of H5N1 avian influenza, significant ongoing mutations, and long-term persistence of the virus in some geographic regions has had an enormous impact on the poultry industry and presents a serious threat to human health. We applied phylogenetic analysis, geospatial techniques, and time series models to investigate the spatiotemporal pattern of H5N1 outbreaks in China and the effect of vaccination on virus evolution. Results showed obvious spatial and temporal clusters of H5N1 outbreaks on different scales, which may have been associated with poultry and wild-bird transmission modes of H5N1 viruses. Lead–lag relationships were found among poultry and wild-bird outbreaks and human cases. Human cases were preceded by poultry outbreaks, and wild-bird outbreaks were led by human cases. Each clade has gained its own unique spatiotemporal and genetic dominance. Genetic diversity of the H5N1 virus decreased significantly between 1996 and 2011; presumably under strong selective pressure of vaccination. Mean evolutionary rates of H5N1 virus increased after vaccination was adopted in China. A clear signature of positively selected sites in the clade 2.3.2 virus was discovered and this may have resulted in the emergence of clade 2.3.2.1. Our study revealed two different transmission modes of H5N1 viruses in China, and indicated a significant role of poultry in virus dissemination. Furthermore, selective pressure posed by vaccination was found in virus evolution in the country.

Rabies is an important but underestimated threat to public health, with most cases reported in Asia. Since 2000, a new epidemic wave of rabies has emerged in Yunnan Province, southwestern China, which borders three countries in Southeast Asia. We estimated gene-specific evolutionary rates for rabies virus using available data in GenBank, then used this information to calibrate the timescale of rabies virus (RABV) spread in Asia. We used 452 publicly available geo-referenced complete nucleoprotein (N) gene sequences, including 52 RABV sequences that were recently generated from samples collected in Yunnan between 2008 and 2012. The RABV N gene evolutionary rate was estimated to be 1.88 × 10−4 (1.37–2.41 × 10−4, 95% Bayesian credible interval, BCI) substitutions per site per year. Phylogenetic reconstructions show that the currently circulating RABV lineages in Yunnan result from at least seven independent introductions (95% BCI: 6–9 introductions) and represent each of the three main Asian RABV lineages, SEA-1, -2 and -3. We find that Yunnan is a sink location for the domestic spread of RABV and connects RABV epidemics in North China, South China, and Southeast Asia. Cross-border spread from southeast Asia (SEA) into South China, and intermixing of the North and South China epidemics is also well supported. The influx of RABV into Yunnan from SEA was not well-supported, likely due to the poor sampling of SEA RABV diversity. We found evidence for a lineage displacement of the Yunnan SEA-2 and -3 lineages by Yunnan SEA-1 strains, and considered whether this could be attributed to fitness differences. Overall, our study contributes to a better understanding of the spread of RABV that could facilitate future rabies virus control and prevention efforts.

Background: Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories. Methods: Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays. Results: This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10⁻⁵ (i.e. one parasite copy in 10⁵ host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence (R² = 0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians. Conclusions: We developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories.

Avian blood parasites have been preliminarily studied in East Asia, but no data are available from long-term monitoring. The aim of this study was to evaluate the prevalence, genetic diversity, and temporal dynamics of Plasmodium, Haemoproteus, and Leucocytozoon in two passerine communities (one forest and one urban) in north China from 2008 to 2013, as well as the association between infected lineages and host specificities. Out of 633 birds from 40 species, 157 individuals (24.8 %) were infected; overall prevalence was 26.7 % and 16.8 % in two sites, respectively. The dominant avian blood parasite genus in the forest park changed yearly between Plasmodium and Haemoproteus, while the Leucocytozoon maintained a low infection level. Forty-four haplotypes were identified by sequencing a 432-bp fragment of the cytochrome b (cyt b) gene; more than 70 % were novel (six Plasmodium lineages, 16 Haemoproteus lineages, and nine Leucocytozoon lineages). Based on our data gathered over consecutive years, we found that the highly observed lineages of Haemoproteus showed higher host diversities than those of Plasmodium, and the most infected lineage EMEL01 (100 % identity with SGS1) take on the highest host diversity but low temporal diversity of the two genera, implying that this lineage infected a great diversity of species in certain years, but maintained a lower infection level or even disappeared in other years. The results suggest that genetic diversity of avian blood parasites in East Asia is high and provides scope for further research. In addition, compared with overall analysis, yearly prevalence monitoring is important in uncovering the temporal dynamic and host specificity variations over time.