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The circulation of avian influenza A viruses in poultry is a public health concern due to the potential transmissibility and severity of these viral infections. Monitoring the susceptibility of these viruses to antivirals is important for developing measures to strengthen the level of preparedness against influenza pandemics. However, drug susceptibility information on these viruses is limited. Here, we determined the susceptibilities of avian influenza A(H5N1), A(H5N2), A(H5N8), A(H7N7), A(H7N9), A(H9N1), and A(H9N2) viruses isolated in Japan to the antivirals approved for use there: an M2 inhibitor (amantadine), neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir) and RNA polymerase inhibitors (baloxavir and favipiravir). Genotypic methods that detect amino acid substitutions associated with antiviral resistance and phenotypic methods that assess phenotypic viral susceptibility to drugs have revealed that these avian influenza A viruses are susceptible to neuraminidase and RNA polymerase inhibitors. These results suggest that neuraminidase and RNA polymerase inhibitors currently approved in Japan could be a treatment option against influenza A virus infections in humans.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P–GI.2) or an RHDV G1-GI.1b variant (GI.1bP–GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP–GI.2 recombinant virus detected in Ehime prefecture and the GI.3P–GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

Ubiquitin-specific protease 18 (USP18) is a well-established innate immune factor in vertebrates. Although Anatidae birds rarely exhibit distinctive clinical signs during high pathogenicity avian influenza virus (HPAIV) infections, some virus strains cause deadly diseases. Here, we investigated the association between USP18 expression and pathogenicity during HPAIV infections in the Anatidae mallard Anas platyrhynchos. First, mallard USP18 gene (duUSP18) was cloned, and its transcriptional variants, with three different open reading frames, were characterized. Experimental infections with two different pathogenic strains, Miyazaki and Takeo, demonstrated an early induction of duUSP18 mRNA upon HPAIV infection in a bird's whole body in vivo and in primary duck cells in vitro, which was positively associated with pathogenicity in mallards. In addition, duUSP18 knockdown under interferon-β stimulation attenuated viral replication, regardless of pathogenicity. These results indicate a role for duUSP18 in favoring viral replication and virus resistance to type I interferon immunity in mallards.

In winter 2020-2021, Japan experienced multiple serious outbreaks of H5N8 high pathogenicity avian influenza (HPAI)-52 outbreaks at poultry farms and 58 cases in wild birds or the environment-that occurred simultaneously with outbreaks in Europe. Here, we examined how the H5N8 HPAI viruses (HPAIVs) emerged and spread through Japan and across the Eurasian continent. Phylogenetic and phylogeographic analyses were performed using full genetic sequences of the viruses that caused 52 outbreaks at poultry farms or were isolated from 11 infected wild birds. Genetically, the viruses showed five genotypes (E1, E2, E3, E5 and E7) that have already been reported in Korea. The viruses showing the E3 genotype were found to have caused most of the HPAI outbreaks at poultry farms and were detected over the longest period of time. The internal genes of the viruses were genetically related to those of AIVs isolated through avian influenza surveillance activities in regions of Siberia including Buryatia, Yakutia and Amur regions, suggesting that the Japanese viruses emerged via reassortment events with AIVs genetically related to Siberian AIVs. In addition, H5N2 and H5N8 HPAIVs were isolated from wild birds during surveillance activities conducted in the Novosibirsk region of Siberia in summer 2020. Phylogenetic analyses revealed that these viruses possessed haemagglutinin genes that were related to those of H5N8 HPAIVs that were circulating in Europe in winter 2020-2021. These results suggest that the viruses in wild birds during summer in Siberia most likely spread in both Asia and Europe the following winter. Together, the present results emphasize the importance of continual monitoring of AIVs in Siberia for forecasting outbreaks not only in Asia but also further away in Europe.

The Goose/Guangdong-lineage (Gs/Gd) H5 high pathogenicity avian influenza viruses (HPAIVs) spread among poultry and wild birds worldwide; an association has been identified between the migration of wild birds and spread of HPAIVs. Every autumn-spring season, the mallard (Anas platyrhynchos) migrates to Japan in substantial numbers for overwintering; however, to the best of our knowledge, no virological studies have focused on mallards' susceptibility to the HPAIVs in Japan. To evaluate the susceptibility of mallards to infection with Gs/Gd H5 HPAIVs isolated during previous outbreaks in Japan, we experimentally infected the birds with various virus strains: A/chicken/Yamaguchi/7/2004 (H5N1) (clade 2.5), A/chicken/Miyazaki/K11/2007 (H5N1) (clade 2.2), A/whooper swan/Akita/1/2008 (H5N1) (clade 2.3.2), A/mandarin duck/Miyazaki/22M-765/2011 (H5N1) (clade 2.3.2.1c), A/duck/Chiba/26-372-48/2014 (H5N8) (clade 2.3.4.4c), A/duck/Hyogo/1/2016 (H5N6) (clade 2.3.4.4e) and A/mute swan/Shimane/3211A002/2017 (H5N6) (clade 2.3.4.4b). The birds exhibited high tracheal shedding for a prolonged period, particularly those infected with A/duck/Hyogo/1/2016 (H5N6). Various clinical manifestations ranging from asymptomatic to mild (corneal opacity) infections to neurological disorders accompanied by mortality were noted depending on the virus strain. Furthermore, virus-infected mallards contaminated both cohoused mallards and water in their surroundings. Thus, mallards may disseminate viruses in the environment, thereby influencing HPAI outbreaks in Japan. Therefore, mallards represent an important migratory bird species that spread HPAIVs in Japan.

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is also a public concern due to sporadic human infections being reported in China. In the current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were most closely related (99.6%-99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4%-99.9% homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to 2018. This indicated that AI470 was introduced into Vietnam from China. In addition, molecular characteristics related to mammalian adaptation among the recent human H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are cause for concern since H5N6 HPAIV strains that possess a risk of human infection have crossed the Chinese border.

An H5N6 highly pathogenic avian influenza virus (HPAIV) outbreak occurred in poultry in Japan during January 2018, and H5N6 HPAIVs killed several wild birds in 3 prefectures during Winter 2017-2018. Time-measured phylogenetic analyses demonstrated that the Hemagglutinin (HA) and internal genes of these isolates were genetically similar to clade 2.3.4.4.B H5N8 HPAIVs in Europe during Winter 2016-2017, and Neuraminidase (NA) genes of the poultry and wild bird isolates were gained through distinct reassortments with AIVs that were estimated to have circulated possibly in Siberia during Summer 2017 and Summer 2016, respectively. Lethal infectious dose to chickens was similar between the poultry and wild-bird isolates. H5N6 HPAIVs during Winter 2017-2018 in Japan had higher 50% chicken lethal doses and lower transmission efficiency than the H5Nx HPAIVs that caused previous outbreaks in Japan, thus explaining in part why cases during the 2017-2018 outbreak were sporadic.

Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.

The aim of this study was to investigate the sensitivity of three breeds of Japanese native chickens, commercial broilers, and specific-pathogen-free (SPF) white leghorns to three strains of the H5 subtype of highly pathogenic avian influenza viruses (HPAIVs). Chickens were experimentally inoculated with doses of 102, 104, and 106 50% egg infective dose of A/mandarin duck/Miyazaki/22M-765/2011 (duck-11), A/chicken/Miyazaki/7/2014 (chicken-14), and A/chicken/Kumamoto/1-2C/2016 (chicken-16). The 50% chicken lethal dose of each virus, mean death time, and viral shedding patterns were compared. The Japanese native chickens showed varied susceptibility to the three H5 HPAIV isolates. Although two of the breeds showed some degree of resistance to duck-11 and chicken-14, all three were more sensitive to chicken-16 than commercial broiler chickens. We have shown that Japanese native chickens do not necessarily have resistance to HPAIV and that the pathogenic characteristics of HPAIVs are quite different between native and commercial chickens.

To determine the influence of media composition on Salmonella exclusion of Nurmi-type cultures, two and four types of cultures in the first and second trial, respectively, were prepared from the cecal contents of conventional laying hens, and Salmonella exclusion was assessed in newly hatched chicks. In the first trial, modified Viande Levure (VL) broth or nutrient broth (NB) were used to prepare Nurmi-type cultures (N-VL and N-NB), which were administered to the newly hatched chicks. Twenty-four hours later, the chicks were challenged with Salmonella enterica Typhimurium EF85-9 (ST). ST recoveries (log10 colony forming units/g of cecal contents) from the N-VL-, N-NB-, and control-treated groups 5 days after the challenge were 7.6±0.6, 0.9±1.9, and 7.7±0.4, respectively. The results suggested the influence of l-cysteine (Cys) present in the VL broth. Thus, we determined the effect of Cys in the second trial. We prepared two other cultures using VL broth without Cys (N-VL−Cys) and NB with Cys (NNB−Cys). ST recoveries from the cecal contents of the N-VL-, N-VL−Cys-, and control-treated groups were 6.3±0.9, 2.1±2.5, and 9.2±0.8, respectively. ST was not recovered from the N-NB- and N-NB−Cys-treated groups. To identify bacteria with Salmonella exclusion activity, we isolated 41 bacterial strains from the ceca of N-NB-treated chicks without Salmonella challenge. Most isolates were identified as Enterococcus faecalis or E. mundtii based on 16S rRNA gene sequencing, and only four cultures excluded Salmonella. Therefore, VL broth containing Cys was not always required for preparing Nurmi-type cultures. The use of media prepared with Cys at the lowest possible concentration or without Cys would promote to enhance Salmonella exclusion from Nurmi-type cultures.

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.

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.

Background We have previously developed a recombinant avian avulavirus serotype 10 (rAAvV-10/HA) expressing the hemagglutinin (HA) gene of a highly pathogenic avian influenza virus (HPAIV) as an emergency vaccine for poultry. rAAvV-10/HA can overcome the activity of the anti-AAvV-1 (Newcastle disease virus) antibody acquired by commercial chickens upon routine vaccination. Most chickens do not have the anti-AAvV-10 antibody, which could interfere with the vaccine efficacy. However, the vaccine efficacy of rAAvV-10/HA is not satisfactory in chickens even though it affords protection against an HPAIV challenge. In the present study, we improved the rAAvV-10/HA vaccine by enhancing the expression of the exogenous HA protein. Methods The 5′ and 3′ untranslated regions (UTR) of each AAvV-10 gene were flanked with the exogenous HA gene cassette to modify rAAvV-10/HA, yielding different rAAv10-UTRs. As a control, rAAv10-nonUTR that did not contain any UTRs was generated. The effects of UTRs on mRNA transcription, HA protein expression, and vaccine efficacy were then examined using embryonated chicken eggs and white leghorn chickens. Results The proportion of the HA gene mRNA among the vector-derived mRNAs (1.55–1.84-fold increase vs. the control) and HA protein levels (148–1151-fold increase vs. the control) in cells infected with rAAv10-UTRs were higher than in those infected with rAAv10-nonUTR. In vivo, vaccination of chickens with rAAv10-UTRs resulted in 100% protection against an HPAIV challenge. No chickens vaccinated with rAAv10-NP-UTR, rAAv10-F-UTR, or rAAv10-HN-UTR shed the virus in the throat and cloaca swabs. By contrast, rAAv10-nonUTR vaccination offered 70% protection, with 50% of chickens shedding the virus in the cloaca or throat swabs after the challenge. We conclude that the AAvV-10 UTRs can enhance the expression of the exogenous HA gene, resulting in improved efficacy of the rAAvV-10/HA vector vaccine. This improvement aids in the protection of flocks worldwide from the highly pathogenic avian influenza.

To determine the influence of media composition on Salmonella exclusion of Nurmi-type cultures, two and four types of cultures in the first and second trial, respectively, were prepared from the cecal contents of conventional laying hens, and Salmonella exclusion was assessed in newly hatched chicks. In the first trial, modified Viande Levure (VL) broth or nutrient broth (NB) were used to prepare Nurmi-type cultures (N-VL and N-NB), which were administered to the newly hatched chicks. Twenty-four hours later, the chicks were challenged with Salmonella enterica Typhimurium EF85-9 (ST). ST recoveries (log10 colony forming units/g of cecal contents) from the N-VL-, N-NB-, and control-treated groups 5 days after the challenge were 7.6±0.6, 0.9±1.9, and 7.7±0.4, respectively. The results suggested the influence of l-cysteine (Cys) present in the VL broth. Thus, we determined the effect of Cys in the second trial. We prepared two other cultures using VL broth without Cys (N-VL-Cys) and NB with Cys (NNB-Cys). ST recoveries from the cecal contents of the N-VL-, N-VL-Cys-, and control-treated groups were 6.3±0.9, 2.1±2.5, and 9.2±0.8, respectively. ST was not recovered from the N-NB- and N-NB-Cys-treated groups. To identify bacteria with Salmonella exclusion activity, we isolated 41 bacterial strains from the ceca of N-NB-treated chicks without Salmonella challenge. Most isolates were identified as Enterococcus faecalis or E. mundtii based on 16S rRNA gene sequencing, and only four cultures excluded Salmonella. Therefore, VL broth containing Cys was not always required for preparing Nurmi-type cultures. The use of media prepared with Cys at the lowest possible concentration or without Cys would promote to enhance Salmonella exclusion from Nurmi-type cultures.

Surveillance studies of influenza A virus of swine (IAV-S) have accumulated information regarding IAVs-S circulating in Thailand, but how IAVs-S evolve within a farm remains unclear. In the present study, we isolated 82 A(H1N1)pdm09 and 87 H3N2 viruses from four farms from 2011 through 2017. We then phylogenetically and antigenically analyzed the isolates to elucidate their evolution within each farm. Phylogenetic analysis demonstrated multiple introductions of A(H1N1)pdm09 viruses that resembled epidemic A(H1N1)pdm09 strains in humans in Thailand, and they reassorted with H3N2 viruses as well as other A(H1N1)pdm09 viruses. Antigenic analysis revealed that the viruses had acquired antigenic diversity either by accumulating substitutions in the hemagglutinin protein or through the introduction of IAV-S strains with different antigenicity. Our results, obtained through continuous longitudinal surveillance, revealed that IAV-S can be maintained on a pig farm over several years through the generation of antigenic diversity due to the accumulation of mutations, introduction of new strains, and reassortment events.

In Japan during the 2016-2017 winter season, clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) of the H5N6 subtype caused 12 outbreaks in chicken and Muscovy duck farms. These viruses have been circulating in Vietnam and China since 2014. In this study, we evaluated the susceptibility of chicken, Pekin duck (Anas platyrhynchos domesticus) and Muscovy duck (Cairina moschata) to H5N6 HPAIVs that originated in Japan, Vietnam and China. The H5N6 HPAIVs examined in this study were highly lethal to chickens compared with their pathogenicity in Pekin duck and Muscovy duck. One of five chickens infected with A/Muscovy duck/Aomori/1-3T/2016 (MusDk/Aomori) survived despite viral shedding, although all of the chickens infected with the other viruses died. The 50% chicken lethal dose differed among the Japanese strains that shared the same gene constellation indicating that gene constellation was not a major determinant of pathogenicity in chicken. MusDk/Aomori, A/chicken/Niigata/1-1T/2016 (Ck/Niigata) and A/duck/Hyogo/1/2016 (Dk/Hyogo) infected all Muscovy ducks inoculated; Ck/Niigata killed 50% of the ducks it infected whereas the other two did not kill any ducks. A/chicken/Japan/AnimalQuarantine-HE144/2016 (HE144) isolated from chicken meat that originated in China was highly pathogenic to Pekin duck: all of the ducks died within 3.75 days of inoculation. This study shows that the pathogenicity of the clade 2.3.4.4 H5N6 HPAIVs differs not only between hosts but also within the same host species.

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.

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.

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high-agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O(2)-depleted 8% CO(2) condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid-deficient rhlA mutant TR failed to exhibit spreading growth on a high-agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard-agar medium was unique to P. aeruginosa under CO(2)-rich anaerobic conditions.

Poultry outbreaks caused by H5N8 highly pathogenic avian influenza viruses (HPAIVs) occurred in Japan between December 2014 and January 2015. During the same period; H5N8 HPAIVs were isolated from wild birds and the environment in Japan. The hemagglutinin (HA) genes of these isolates were found to belong to clade 2.3.4.4 and three sub-groups were distinguishable within this clade. All of the Japanese isolates from poultry outbreaks belonged to the same sub-group; whereas wild bird isolates belonged to the other sub-groups. To examine whether the difference in pathogenicity to chickens between isolates of different HA sub-groups of clade 2.3.4.4 could explain why the Japanese poultry outbreaks were only caused by a particular sub-group; pathogenicities of A/chicken/Miyazaki/7/2014 (Miyazaki2014; sub-group C) and A/duck/Chiba/26-372-48/2014 (Chiba2014; sub-group A) to chickens were compared and it was found that the lethality of Miyazaki2014 in chickens was lower than that of Chiba2014; according to the 50% chicken lethal dose. This indicated that differences in pathogenicity may not explain why the Japanese poultry outbreaks only involved group C isolates.

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.

Serratia marcescens has been known as a temperature-dependent producer of two chemically different exolipids (red pigment prodigiosin and biosurfactant serrawettin W1) in parallel. During genetic investigation of such control mechanisms, mini-Tn 5 insertional mutant Tan1 overproducing these exolipids was isolated. The gene concerning such disregulation was identified as hexS by DNA cloning followed by sequencing and homology analysis of the presumed product with 314 amino-acids. The product HexS was the homologue of HexA of Erwinia carotovora ssp. carotovora and classified as a transcriptional regulator belonging to LysR family. By RT-PCR analysis, the hexS mutant was shown to over-transcribe the pigA gene (the first gene of the pig cluster involved in prodigiosin synthesis) and the swrW gene encoding serrawettin W1 synthetase belonging to the nonribosomal peptide synthetase family. In contrast, transcription of the pswP gene encoding phosphopantetheinyl transferase in Tan1 was in the level of parent strain 274. Purified protein encoded in his(6)-hexS demonstrated binding activity to DNA fragments of the upstream region of pigA and swrW genes and not to that of the pswP gene. S. marcescens strain 274 transformed with a low-copy plasmid carrying hexS demonstrated reduced production of prodigiosin and serrawettin W1, and reduced activity of exoenzymes (protease, chitinase, and DNase) except phospholipase C. Possible generation of virulent S. marcescens by derepression or mutation of the hexS gene in infected tissues or ex vivo environments was suggested.

Serrawettin W1 produced by Serratia marcescens is a surface active exolipid having various functions supporting behaviors of bacteria on surface environments. Through the genetic analyses of serrawettin-less mutants of S. marcescens 274, the swrW gene encoding putative serrawettin W1 synthetase was identified. Homology analysis of the putative SwrW demonstrated the presence of condensation, adenylation, thiolation, and thioesterase domains which are characteristic for nonribosomal peptide synthetase (NRPS). NRPSs have been known as multi-modular enzymes. Linear alignment of these modules specifying respective amino acids will enable peptide bond formation resulting in a specific amino acid sequence. Putative SwrW was uni-modular NRPS specifying only L-serine. Possible steps in this simple unimodular NRPS for biosynthesis of serrawettin W1 [ cyclo-(D-3-hydroxydecanoyl-L-seryl) (2) ] were predicted by referring to the ingenious enzymatic activity of gramicidin S synthetase (multi-modular NRPS) of Brevibacillus brevis.

Serratia marcescens produces the red pigment prodigiosin and biosurfactant serrawettin at 30°C but not at 37°C. In an analytic study of the thermoregulation of S. marcescens 274, we noticed another temperature-dependent activity that was bacteriostatic to gram-negative and -positive bacterial species, at 37°C but not at 30°C. The thermo-insensitive isogenic mutant strain N0075 (a producer of prodigiosin and serrawettin even at 37°C) was devoid of such growth inhibitory activity. The revertant of strain N0075 made by transformation again exhibited the temperature-dependent mode of prodigiosin and serrawettin production, and bacteriostatic activity. This novel activity was correlated to the thermo-responsive acidification of the medium by S. marcescens. In response to higher environmental temperatures, S. marcescens seems to suppress its own growth and the growth of other bacteria.

Serrawettins are nonionic biosurfactants produced by Serratia marcescens. Three molecular species, serrawettin W1, cyclo(d-3-hydroxydecanoyl-l-seryl)2; W2, d-3-hydroxydecanoyl-d-leucyl-l-seryl-l-threonyl-d-phenylalanyl-l-isoleucyl lactone; and W3, cyclodepsipeptide composed of five amino acids and one dodecanoic acid, have been reported. Serratia rubidaea produces rubiwettin R1, linked d-3-hydroxy fatty acids and RG1, β-glucopyranosyl linked d-3-hydroxy fatty acids. These biosurfactants are produced mainly at 30°C, but not at 37°C, and secreted through extracellular vesicles on solid media. The contribution of the biosurfactants to spreading growth in surface environments has been determined, and it is prominent under nutrient-poor conditions. Analyses of S. marcescens mutants revealed the involvement of three novel genes for serrawettin W1 production. The gene pswP encodes a phosphopantetheinyl transferase group enzyme, swrW encodes a unimodular synthetase belonging to the nonribosomal peptide synthetase (NRPS) family, and hexS encodes a LysR-type transcriptional regulator working as a downregulator of Serratia exolipids and some exoenzymes. Autoinducer-dependent serrawettin W2 production has been elucidated by the finding of SwrI/SwrR (homolog of LuxI/LuxR) and N-acyl homoserine lactones in the study on quorum-sensing controlled-swarming of S. marcescens.

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.