Characterization of Highly Pathogenic Avian Influenza Virus A(H5N6), Japan, November 2016.

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016-2017 in Northern Japan.

On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs.

Sequencing methods for HA and NA genes of avian influenza viruses from wild bird feces using Oxford Nanopore sequencing.

We developed a method to determine the sequences of hemagglutinin (HA) and neuraminidase (NA) from RNA extracted directly from wild bird fecal samples, using Nanopore Flongle. We determined the nucleotide sequences and subtypes of HA and NA in 16 and 15 samples respectively, using Flongle. The results of HA and NA subtyping determined by the conventional method were consistent with their subtypes determined by our method, thereby the applicability of this method in the identification of HA and NA subtypes. In addition, the homology between the HA fragments in this and the Sanger methods ranged from 98.5 % to 100 %. Compared with conventional PCR with the Sanger method, this method can easily determine HA and NA subtypes and sequences directly from the fecal samples. It is easier to implement and has lower running costs (USD100$) than other NGS-based methods, making it a useful tool for avian influenza surveillance in wild birds.

Current situation regarding lead exposure in birds in Japan (2015-2018); lead exposure is still occurring.

Birds of a number of species have died as a result of lead (Pb) poisoning, including many Steller's sea eagles (Haliaeetus pelagicus) and white-tailed sea eagles (Haliaeetus albicilla) in Hokkaido, the northernmost island of Japan. To address this issue, the use of any type of Pb ammunition for hunting of large animals was prohibited in Hokkaido in 2004. However, Pb poisoning is still being reported in this area, and there are few regulations regarding the use of Pb ammunition in other parts of Japan, where it has been reported that eagles and water birds have been exposed to Pb. This study was performed to accurately determine the current level of Pb exposure of birds found dead in the field or dead in the wild bird centers in Japan (June 2015-May 2018) and to identify the sources of Pb. Pb exposure was found to still be occurring in raptors and water birds in various parts of Japan. Twenty-six point five % and 5.9% of the recorded deaths of Steller's sea eagles and white-tailed sea eagles, respectively, were found to have been poisoned by Pb. In addition, Pb isotope ratio analysis showed that both Pb rifle bullets and Pb shot pellets cause Pb exposure in birds, and these endangered eagles are also exposed to Pb in Hokkaido due to the illegal use of Pb ammunition. Changing to Pb-free ammunition, such as copper (Cu) rifle bullets, steel shot pellets, or bismuth shot pellets, will be essential for the conservation of avian species in Japan.

Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds.

The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop-mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required.

Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in the Ryukyu Islands tree rat (Diplothrix legata).

Angiostrongylus cantonensis is a zoonotic nematode with rodents serving as natural definitive hosts. We report A. cantonensis in the Ryukyu Islands tree rat (Diplothrix legata, Thomas, 1906), a native endangered species in Japan. Adult and larvae of A. cantonensis were macroscopically, histologically, and genetically detected in three tree rats collected between August 2011 and January 2012 in the Yambaru area of Okinawa Prefecture, Japan. Pathologic observations of the lungs of rats showed that infection may be lethal. We also conducted a retrospective genetic survey of helminths parasitic in lung in cryopreserved lung samples of Ryukyu Islands tree rats collected between 2007 and 2011 in the Yambaru area and found A. cantonensis DNA in one of 29 samples, which was collected in December 2010.