Taichiro Tanikawa

Intracontinental and intercontinental dissemination of Asian H5 highly pathogenic avian influenza virus (clade 2.3.4.4) in the winter of 2014-2015.

Asian H5 highly pathogenic avian influenza viruses (HPAIVs) that possess the clade 2.3.4.4 HA gene have been identified in wild birds and poultry since late 2014 in both Europe and North America (N. America). Clade 2.3.4.4 H5 HPAIVs of the H5N8 subtype have been isolated in both regions, whereas reassortment viruses with NA N1 and N2 subtypes of the North American (N. American). avian lineage have only been identified in N. America. The HA genes of those isolates were closely related to genes of the HPAIVs that have caused massive outbreaks in poultry in Korea since January 2014. The outbreaks caused by those viruses and the genetic relatedness of their HA and NA genes are reviewed in this study. Although the illegal movement of poultry and poultry products cannot be ruled out as a cause of intercontinental and intracontinental dissemination of clade 2.3.4.4 H5 HPAIVs during the winter of 2014–2015, transmission of the viruses by infected migratory birds appears to be a more plausible mechanism for their dissemination. In particular, the involvement of migratory birds in HPAIV transmission between Asia and N. America is highly likely because of the reassortments between H5N8 HPAIV and the N. American lineage avian influenza viruses. Copyright

Chicken MDA5 senses short double-stranded RNA with implications for antiviral response against avian influenza viruses in chicken.

Mammalian melanoma differentiation-associated gene-5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) selectively sense double-stranded RNA (dsRNA) according to length, as well as various RNA viruses to induce an antiviral response. RIG-I, which plays a predominant role in the induction of antiviral responses against influenza virus infection, has been considered to be lacking in chicken, putting the function of chicken MDA5 (chMDA5) under the spotlight. Here, we show that chMDA5, unlike mammalian MDA5, preferentially senses shorter dsRNA synthetic analogues, poly(I:C), in chicken DF-1 fibroblasts. A requirement for caspase activation and recruitment domains for chMDA5-mediated chicken interferon beta (chIFNβ) induction and its interaction with mitochondrial antiviral signaling proteins were demonstrated. We also found that chMDA5 is involved in chIFNβ induction against avian influenza virus infection. Our findings imply that chMDA5 compensates in part the function of RIG-I in chicken, and highlights the importance of chMDA5 in the innate immune response in chicken.

Genetics and infectivity of H5N1 highly pathogenic avian influenza viruses isolated from chickens and wild birds in Japan during 2010-11.

Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds.

Five distinct reassortants of H5N6 highly pathogenic avian influenza A viruses affected Japan during the winter of 2016–2017

To elucidate the evolutionary pathway, we sequenced the entire genomes of 89 H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated in Japan during winter 2016-2017 and 117 AIV/HPAIVs isolated in Japan and Russia. Phylogenetic analysis showed that at least 5 distinct genotypes of H5N6 HPAIVs affected poultry and wild birds during that period. Japanese H5N6 isolates shared a common genetic ancestor in 6 of 8 genomic segments, and the PA and NS genes demonstrated 4 and 2 genetic origins, respectively. Six gene segments originated from a putative ancestral clade 2.3.4.4 H5N6 virus that was a possible genetic reassortant among Chinese clade 2.3.4.4 H5N6 HPAIVs. In addition, 2 NS clusters and a PA cluster in Japanese H5N6 HPAIVs originated from Chinese HPAIVs, whereas 3 distinct AIV-derived PA clusters were evident. These results suggest that migratory birds were important in the spread and genetic diversification of clade 2.3.4.4 H5 HPAIVs.

Amino acid substitutions in PB1 of avian influenza viruses influence pathogenicity and transmissibility in chickens

ABSTRACT Amino acid substitutions were introduced into avian influenza virus PB1 in order to characterize the interaction between polymerase activity and pathogenicity. Previously, we used recombinant viruses containing the hemagglutinin (HA) and neuraminidase (NA) genes from the highly pathogenic avian influenza virus (HPAIV) H5N1 strain and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken and wild-bird hosts (LP and WB, respectively) to demonstrate that the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in chickens is regulated by the PB1 gene (Y. Uchida et al., J. Virol. 86:2686–2695, 2012, doi:http://dx.doi.org/10.1128/JVI.06374-11). In the present study, we introduced a C38Y substitution into WB PB1 and demonstrated that this substitution increased both polymerase activity in DF-1 cells in vitro and the pathogenicity of the recombinant viruses in chickens. The V14A substitution in LP PB1 reduced polymerase activity but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increased polymerase activity correlates directly with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis. IMPORTANCE We identified 2 novel amino acid substitutions in the avian influenza virus PB1 gene that affect the characteristics of highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype, such as viral replication and polymerase activity in vitro and pathogenicity and transmissibly in chickens. An amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens and was associated with changes in polymerase activity in vitro. A substitution at residue 14 reduced polymerase activity in vitro, while its effects on pathogenicity and transmissibility depended on the constellation of internal genes.

Co-infection of influenza A viruses of swine contributes to effective shuffling of gene segments in a naturally reared pig

Following the 2009 H1N1 pandemic, surveillance activities have been accelerated globally to monitor the emergence of novel reassortant viruses. However, the mechanism by which influenza A viruses of swine (IAV-S) acquire novel gene constellations through reassortment events in natural settings remains poorly understood. To explore the mechanism, we collected 785 nasal swabs from pigs in a farm in Thailand from 2011 to 2014. H3N2, H3N1, H1N1 and H1N2 IAVs-S were isolated from a single co-infected sample by plaque purification and showed a high degree of diversity of the genome. In particular, the H1N1 isolates, possessing a novel gene constellation previously unreported in Thailand, exhibited greater variation in internal genes than H3N2 IAVs-S. A pair of isolates, designated H3N2-B and H1N1-D, was determined to have been initially introduced to the farm. These results demonstrate that numerous IAVs-S with various gene constellations can be created in a single co-infected pig via reassortment.

Recombinant Avian Paramyxovirus Serotypes 2, 6, and 10 as Vaccine Vectors for Highly Pathogenic Avian Influenza in Chickens with Antibodies Against Newcastle Disease Virus

SUMMARY Recombinant Newcastle disease virus (rNDV) expressing the hemagglutinin of highly pathogenic avian influenza virus (HPAIV HA) induces protective immunity against HPAIV in chickens. However, the efficacy of rNDV vectors is hampered when chickens are pre-immune to NDV, and most commercial chickens are routinely vaccinated against NDV. We recently showed that avian paramyxovirus serotypes 2, 6, and 10 (APMV-2, APMV-6, and APMV-10), which belong to the same genus as NDV, have low cross-reactivity with anti-NDV antisera. Here, we used reverse genetics to generate recombinant APMV-2, APMV-6, and APMV-10 (rAPMV-2/HA, rAPMV-6/HA, and rAPMV-10/HA) that expressed an HA protein derived of subtype H5N1 HPAIV, A/chicken/Yamaguchi/7/2004. Chickens pre-immunized against NDV (age, 7 wk) were vaccinated with rAPMV/HAs; 14 days after vaccination, chickens were challenged with a lethal dose of HPAIV. Immunization of chickens pre-immunized against NDV with rAPMV-2/HA, rAPMV-6/HA, or rAPMV-10/HA protected 50%, 50%, and 25%, respectively, in groups of chickens given an rAPMV/HA with 106 median embryo infectious dose (EID50) or 50%, 50%, and 90%, respectively, in those with 107 EID50; in contrast, rNDV/HA protected none of the chicken vaccinated with 106 EID50 and induced only partial protection even with 107 EID50. Therefore, the presence of anti-NDV antibodies did not hamper the efficacy of rAPMV-2/HA, rAPMV-6/HA, or rAPMV-10/HA. These results suggest that rAPMV-2, rAPMV-6, and rAPMV-10 are potential vaccine vectors, especially for commercial chickens, which are routinely vaccinated against NDV.

Spatial transmission of H5N6 highly pathogenic avian influenza viruses among wild birds in Ibaraki Prefecture, Japan, 2016–2017

From 29 November 2016 to 24 January 2017, sixty-three cases of H5N6 highly pathogenic avian influenza virus (HPAIV) infections were detected in wild birds in Ibaraki Prefecture, Japan. Here, we analyzed the genetic, temporal, and geographic correlations of these 63 HPAIVs to elucidate their dissemination throughout the prefecture. Full-genome sequence analysis of the Ibaraki isolates showed that 7 segments (PB2, PB1, PA, HA, NP, NA, NS) were derived from G1.1.9 strains while the M segment was from G1.1 strains; both groups of strains circulated in south China. Pathological studies revealed severe systemic infection in dead swans (the majority of dead birds and the only species necropsied), thus indicating high susceptibility to H5N6 HPAIVs. Coalescent phylogenetic analysis using the 7 G1.1.9-derived segments enabled detailed analysis of the short-term evolution of these highly homologous HPAIVs. This analysis revealed that the H5N6 HPAIVs isolated from wild birds in Ibaraki Prefecture were divided into 7 groups. Spatial analysis demonstrated that most of the cases concentrated around Senba Lake originated from a single source, and progeny viruses were transmitted to other locations after the infection expanded in mute swans. In contrast, within just a 5-km radius of the area in which cases were concentrated, three different intrusions of H5N6 HPAIVs were evident. Multi-segment analysis of short-term evolution showed that not only was the invading virus spread throughout Ibaraki Prefecture but also that, despite the small size of this region, multiple invasions had occurred during winter 2016–2017.

Replication of a low-pathogenic avian influenza virus is enhanced by chicken ubiquitin-specific protease 18

Previous research revealed the induction of chicken USP18 (chUSP18) in the lungs of chickens infected with highly pathogenic avian influenza viruses (HPAIVs). This activity was correlated with the degree of pathogenicity of the viruses to chickens. As mammalian ubiquitin-specific protease (USP18) is known to remove type I interferon (IFN I)-inducible ubiquitin-like molecules from conjugated proteins and block IFN I signalling, we explored the function of the chicken homologue of USP18 during avian influenza virus infection. With this aim, we cloned chUSP18 from cultured chicken cells and revealed that the putative chUSP18 ORF comprises 1137 bp. Comparative analysis of the predicted aa sequence of chUSP18 with those of human and mouse USP18 revealed relatively high sequence similarity among the sequences, including domains specific for the ubiquitin-specific processing protease family. Furthermore, we found that chUSP18 expression was induced by chicken IFN I, as observed for mammalian USP18. Experiments based on chUSP18 over-expression and depletion demonstrated that chUSP18 significantly enhanced the replication of a low-pathogenic avian influenza virus (LPAIV), but not an HPAIV. Our findings suggest that chUSP18, being similar to mammalian USP18, acts as a pro-viral factor during LPAIV replication in vitro.

Transmission of an H5N8-Subtype Highly Pathogenic Avian Influenza Virus from Infected Hens to Laid Eggs

SUMMARY. We showed here that an H5N8-subtype highly pathogenic avian influenza virus (HPAIV) was transmitted to both the internal contents and shells of eggs laid by white leghorn hens experimentally infected with the virus. Seven of eight HPAIV-infected hens laid eggs until 4 days postinoculation (dpi). The mean number of eggs laid per head daily decreased significantly from 0.58 before inoculation to 0.18 after viral inoculation. The virus was detected in the eggs laid by three of the seven hens. Viral transmission was detectable beginning on 3 dpi, and virus titers in tracheal and cloacal swabs from the hens that laid the contaminated eggs exceeded 2.9 log10 EID50. The level of viral replication and its timing when virus replicates enough to be detected in oviduct after virus inoculation appear to be key factors in the transmission of H5N8 HPAIV from infected hens to laid eggs.