Avian influenza viruses in humans: lessons from past outbreaks.

BACKGROUND: Human infections with avian influenza viruses (AIV) represent a persistent public health threat. The principal risk factor governing human infection with AIV is from direct contact with infected poultry and is primarily observed in Asia and Egypt where live-bird markets are common. AREAS OF AGREEMENT: Changing patterns of virus transmission and a lack of obvious disease manifestations in avian species hampers early detection and efficient control of potentially zoonotic AIV. AREAS OF CONTROVERSY: Despite extensive studies on biological and environmental risk factors, the exact conditions required for cross-species transmission from avian species to humans remain largely unknown. GROWING POINTS: The development of a universal ('across-subtype') influenza vaccine and effective antiviral therapeutics are a priority. AREAS TIMELY FOR DEVELOPING RESEARCH: Sustained virus surveillance and collection of ecological and physiological parameters from birds in different environments is required to better understand influenza virus ecology and identify risk factors for human infection.

Emergence and evolution of avian H5N2 influenza viruses in chickens in Taiwan.

UNLABELLED: Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available information suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multiple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the prevalence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003. IMPORTANCE: H5N2 avian influenza viruses first appeared in chickens in Taiwan in 2003 and caused a series of outbreaks afterwards. Phylogenetic analyses show that the chicken H5N2 viruses have H5 and N2 genes that are closely related to those of a vaccine strain originating from Mexico in 1994, while the contemporary duck H5N2 viruses in Taiwan belong to the Eurasian gene pool. The unusually high similarity of the chicken H5N2 viruses to the Mexican vaccine strain suggests that these viruses might have been introduced to Taiwan by using inadequately inactivated or attenuated vaccines. These chicken H5N2 viruses are developing varying levels of pathogenicity that could lead to significant consequences for the local poultry industry. These findings emphasize the need for strict quality control and competent oversight in the manufacture and usage of avian influenza virus vaccines and indicate that alternatives to widespread vaccination may be desirable.

Optimization and Improvement of qPCR Detection Sensitivity of SARS-CoV-2 in Saliva.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major public health threat globally, especially during the beginning of the pandemic in 2020. Reverse transcription-quantitative PCR (RT-qPCR) is utilized for viral RNA detection as part of control measures to limit the spread of COVID-19. Collecting nasopharyngeal swabs for RT-qPCR is a routine diagnostic method for COVID-19 in clinical settings, but its large-scale implementation is hindered by a shortage of trained health professionals. Despite concerns over its sensitivity, saliva has been suggested as a practical alternative sampling approach to the nasopharyngeal swab for viral RNA detection. In this study, we spiked saliva from healthy donors with inactivated SARS-CoV-2 from an international standard to evaluate the effect of saliva on viral RNA detection. On average, the saliva increased the cycle threshold (CT) values of the SARS-CoV-2 RNA samples by 2.64 compared to the viral RNA in viral transport medium. Despite substantial variation among different donors in the effect of saliva on RNA quantification, the outcome of the RT-qPCR diagnosis was largely unaffected for viral RNA samples with CT values of <35 (1.55 log10 IU/mL). The saliva-treated viral RNA remained stable for up to 6 h at room temperature and 24 h at 4°C. Further supplementing protease and RNase inhibitors improved the detection of viral RNA in the saliva samples. Our data provide practical information on the storage conditions of saliva samples and suggest optimized sampling procedures for SARS-CoV-2 diagnosis. IMPORTANCE The primary method for detection of SARS-CoV-2 is using nasopharyngeal swabs, but a shortage of trained health professionals has hindered its large-scale implementation. Saliva-based nucleic acid detection is a widely adopted alternative, due to its convenience and minimally invasive nature, but the detection limit and direct impact of saliva on viral RNA remain poorly understood. To address this gap in knowledge, we used a WHO international standard to evaluate the effect of saliva on SARS-CoV-2 RNA detection. We describe the detection profile of saliva-treated SARS-CoV-2 samples under different storage temperatures and incubation periods. We also found that adding protease and RNase inhibitors could improve viral RNA detection in saliva. Our research provides practical recommendations for the optimal storage conditions and sampling procedures for saliva-based testing, which can improve the efficiency of COVID-19 testing and enhance public health responses to the pandemic.

Lineage BA.2 dominated the Omicron SARS-CoV-2 epidemic wave in the Philippines.

The Omicron severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant led to a dramatic global epidemic wave following detection in South Africa in November 2021. The BA.1 Omicron lineage was dominant and responsible for most SARS-CoV-2 outbreaks in countries around the world during December 2021-January 2022, while other Omicron lineages, including BA.2, accounted for the minority of global isolates. Here, we describe the Omicron wave in the Philippines by analysing genomic data. Our results identify the presence of both BA.1 and BA.2 lineages in the Philippines in December 2021, before cases surged in January 2022. We infer that only the BA.2 lineage underwent sustained transmission in the country, with an estimated emergence around 18 November 2021 (95 per cent highest posterior density: 6-28 November), while despite multiple introductions, BA.1 transmission remained limited. These results suggest that the Philippines was one of the earliest areas affected by BA.2 and reiterate the importance of whole genome sequencing for monitoring outbreaks.

H5Nx Viruses Emerged during the Suppression of H5N1 Virus Populations in Poultry.

Highly pathogenic avian influenza (HPAI) H5 viruses have posed a substantial pandemic threat through repeated human infection since their emergence in China in 1996. Nationwide control measures, including vaccination of poultry, were implemented in 2005, leading to a sharp reduction in H5N1 virus outbreaks. In 2008, novel non-N1 subtype (H5Nx) viruses emerged, gradually replacing the dominant H5N1 subtype and causing global outbreaks. The cause of this major shift in the ecology of HPAI H5 viruses remains unknown. Here, we show that major H5N1 virus lineages underwent population bottlenecks in 2006, followed by a recovery in virus populations between 2007 and 2009. Our analyses indicate that control measures, not competition from H5Nx viruses, were responsible for the H5N1 decline, with an H5N1 lineage capable of infecting poultry and wild birds experiencing a less severe population bottleneck due to circulation in unaffected wild birds. We show that H5Nx viruses emerged during the successful suppression of H5N1 virus populations in poultry, providing an opportunity for antigenically distinct H5Nx viruses to propagate. Avian influenza vaccination programs would benefit from universal vaccines targeting a wider diversity of influenza viruses to prevent the emergence of novel subtypes. IMPORTANCE A major shift in the ecology of highly pathogenic avian influenza (HPAI) H5 viruses occurred from 2008 to 2014, when viruses with non-N1 neuraminidase genes (termed H5Nx viruses) emerged and caused global H5 virus outbreaks. Here, we demonstrate that nationwide control measures, including vaccination in China, successfully suppressed H5N1 populations in poultry, providing an opportunity for antigenically distinct H5Nx viruses to emerge. In particular, we show that the widespread use of H5N1 vaccines likely conferred a fitness advantage to H5Nx viruses due to the antigenic mismatch of the neuraminidase genes. These results indicate that avian influenza vaccination programs would benefit from universal vaccines that target a wider diversity of influenza viruses to prevent potential emergence of novel subtypes.

Genomic Evidence for Sequestration of Influenza A Virus Lineages in Sea Duck Host Species.

Wild birds are considered the natural reservoir of influenza A viruses (IAVs) making them critical for IAV surveillance efforts. While sea ducks have played a role in novel IAV emergence events that threatened food security and public health, very few surveillance samples have been collected from sea duck hosts. From 2014-2018, we conducted surveillance focused in the Mississippi flyway, USA at locations where sea duck harvest has been relatively successful compared to our other sampling locations. Our surveillance yielded 1662 samples from sea ducks, from which we recovered 77 IAV isolates. Our analyses identified persistence of sea duck specific IAV lineages across multiple years. We also recovered sea duck origin IAVs containing an H4 gene highly divergent from the majority of North American H4-HA with clade node age of over 65 years. Identification of IAVs with long branch lengths is indicative of substantial genomic change consistent with persistence without detection by surveillance efforts. Sea ducks play a role in the movement and long-term persistence of IAVs and are likely harboring more undetected IAV diversity. Sea ducks should be a point of emphasis for future North American wild bird IAV surveillance efforts.

Co-circulation of both low and highly pathogenic avian influenza H5 viruses in current poultry epidemics in Taiwan.

Highly pathogenic avian influenza (HPAI) A(H5) viruses belonging to clade 2.3.4.4c of the A/goose/Guangdong/1/96-like (Gs/GD) lineage caused severe global outbreaks in domestic birds from 2014 to 2015, that also represented the first incursions of Gs/GD viruses into Taiwan and the USA. However, few studies have investigated the circulation of clade 2.3.4.4c viruses after 2015. Here, we describe Gs/GD clade 2.3.4.4c and Mexican-like H5N2 viruses that were isolated in Taiwan during active surveillance conducted in chicken farms from February to March 2019. Phylogenetic analysis demonstrated two distinct genome constellations of the clade 2.3.4.4c H5 viruses, with the internal genes of one of the new genotypes closely related to a virus isolated from a pintail (Anas acuta) in Taiwan, providing the first direct evidence that migratory birds play a role in importing viruses into Taiwan. Our study also confirmed the co-circulation of Gs/GD clade 2.3.4.4c and Mexican-like H5 lineage viruses in Taiwan, presenting a rare case where Gs/GD viruses developed sustained transmission alongside another enzootic H5 lineage, raising the possibility that homosubtypic immunity may mask virus transmission, potentially frustrating detection, and the implementation of appropriate control measures. To eradicate H5 viruses from poultry in Taiwan, further studies on the effect of co-circulation in poultry of low pathogenic avian influenza and HPAI viruses are needed. Furthermore, only with continued surveillance efforts globally can we fully discern dispersal patterns and risk factors of virus transmission both to and within Taiwan.

Emerging lethal infectious bronchitis coronavirus variants with multiorgan tropism.

Infectious bronchitis virus (IBV) causes respiratory diseases in chickens and poses an economic threat to the poultry industry worldwide. Despite vaccine use, there have been field outbreaks of IBV in Taiwan. This study aimed to characterize the emerging IBV variants circulating in Taiwan. The analysis of the structural protein genes showed that these variants emerged through frequent recombination events among Taiwan strains, China strains, Japan strains and vaccine strains. Cross-neutralization tests revealed that two of the variants exhibited novel serotypes. Clinicopathological assessment showed that two of the variants caused high fatality rates of 67% and 20% in one-day-old SPF chicks, and all the variants possessed multiorgan tropisms, including trachea, proventriculus and urogenital tissues. Furthermore, the commercial live-attenuated Mass-type vaccine conferred poor protection against these variants. This study identified novel genotypes, serotypes and pathotypes of emerging IBV variants circulating in Taiwan. There is an urgent need for effective countermeasures against these variant strains.

Ecological factors associated with persistent circulation of multiple highly pathogenic avian influenza viruses among poultry farms in Taiwan during 2015-17.

Emergence and intercontinental spread of highly pathogenic avian influenza A (HPAI) H5Nx virus clade 2.3.4.4 has resulted in substantial economic losses to the poultry industry in Asia, Europe, and North America. The long-distance migratory birds have been suggested to play a major role in the global spread of avian influenza viruses during this wave of panzootic outbreaks since 2013. Poultry farm epidemics caused by multiple introduction of different HPAI novel subtypes of clade 2.3.4.4 viruses also occurred in Taiwan between 2015 and 2017. The mandatory and active surveillance detected H5N3 and H5N6 circulation in 2015 and 2017, respectively, while H5N2 and H5N8 were persistently identified in poultry farms since their first arrival in 2015. This study intended to assess the importance of various ecological factors contributed to the persistence of HPAI during three consecutive years. We used satellite technology to identify the location of waterfowl flocks. Four risk factors consistently showed strong association with the spatial clustering of H5N2 and H5N8 circulations during 2015 and 2017, including high poultry farm density (aOR:17.46, 95%CI: 5.91-74.86 and 8.23, 95% CI: 2.12-54.86 in 2015 and 2017, respectively), poultry heterogeneity index (aOR of 12.28, 95%CI: 5.02-31.14 and 2.79, 95%CI: 1.00-7.69, in 2015 and 2017, respectively), non-registered waterfowl flock density (aOR: 6.8, 95%CI: 3.41-14.46 and 9.17, 95%CI: 3.73-26.20, in 2015 and 2017, respectively) and higher percentage of cropping land coverage (aOR of 1.36, 95%CI: 1.10-1.69 and 1.04, 95%CI: 1.02-1.07, in 2015 and 2017, respectively). Our study highlights the application of remote sensing and clustering analysis for the identification and characterization of environmental factors in facilitating and contributing to the persistent circulation of certain subtypes of H5Nx in poultry farms in Taiwan.

Inter- and intra-host sequence diversity reveal the emergence of viral variants during an overwintering epidemic caused by dengue virus serotype 2 in southern Taiwan.

Purifying selection during dengue viral infection has been suggested as the driving force of viral evolution and the higher complexity of the intra-host quasi-species is thought to offer an adaptive advantage for arboviruses as they cycle between arthropod and vertebrate hosts. However, very few studies have been performed to investigate the viral genetic changes within (intra-host) and between (inter-host) humans in a spatio-temporal scale. Viruses of different serotypes from various countries imported to Taiwan cause annual outbreaks. During 2001-2003, two consecutive outbreaks were caused by dengue virus serotype 2 (DENV-2) and resulted in a larger-scale epidemic with more severe dengue cases in the following year. Phylogenetic analyses showed that the viruses from both events were similar and related to the 2001 DENV-2 isolate from the Philippines. We comprehensively analyzed viral sequences from representative dengue patients and identified three consensus genetic variants, group Ia, Ib and II, with different spatio-temporal population dynamics. The phylodynamic analysis suggested group Ib variants, characterized by lower genetic diversity, transmission rate, and intra-host variant numbers, might play the role of maintenance variants. The residential locations among the patients infected by group Ib variants were in the outer rim of case clusters throughout the 2001-2003 period whereas group Ia and II variants were located in the centers of case clusters, suggesting that group Ib viruses might serve as "sheltered overwintering" variants in an undefined ecological niche. Further deep sequencing of the viral envelope (E) gene directly from individual patient serum samples confirmed the emergence of variants belonging to three quasi-species (group Ia, Ib, and II) and the ancestral role of the viral variants in the latter phase of the 2001 outbreak contributed to the later, larger-scale epidemic beginning in 2002. These findings enhanced our understanding of increasing epidemic severity over time in the same epidemic area. It also highlights the importance of combining phylodynamic and deep sequencing analysis as surveillance tools for detecting dynamic changes in viral variants, particularly searching for and monitoring any specific viral subpopulation. Such subpopulations might have selection advantages in both fitness and transmissibility leading to increased epidemic severity.

Identification of an infectious bronchitis coronavirus strain exhibiting a classical genotype but altered antigenicity, pathogenicity, and innate immunity profile.

Avian coronavirus infectious bronchitis virus (IBV) poses economic threat to the poultry industry worldwide. Pathogenic IBV 3575/08 was isolated from broilers vaccinated with the attenuated viral vaccine derived from a Taiwan strain 2575/98. In this study, extensive investigations were conducted on the genome sequences, antigenicity, pathogenicity, and host immune responses of several IBV strains in specific-pathogen-free chickens. Sequence analyses revealed that 3575/08 and 2575/98 shared high homology in their structural genes, but not in non-structural accessory proteins such as 3a, 3b and 5b. Despite a high degree of homology in their spike protein genes, cross neutralization test showed low cross protection between 3575/08 and 2575/98, suggesting distinct antigenicity for the two strains. Animal challenge experiments exhibited strong respiratory and renal pathogenicity for 3575/08. In addition, early and prolonged viral shedding and rapid viral dissemination were observed. Immune gene expression profiling by PCR array showed chickens infected with 3575/08 had delayed expression of a subset of early innate immune genes, whereas chickens infected with the wild-type or attenuated-type 2575/08 revealed quick gene induction and efficient virus control. In summary, this study reveals a new IBV strain, which harbors a known local genotype but displays remarkably altered antigenicity, pathogenicity and host defenses.

Lessons from the Largest Epidemic of Avian Influenza Viruses in Taiwan, 2015.

The largest epidemic of avian influenza (AI) in history attacked poultry and wild birds throughout Taiwan starting January 6, 2015. This study analyzed surveillance results, epidemiologic characteristics, and viral sequences by using government-released information, with the intention to provide recommendations to minimize future pandemic influenza. The H5 clade 2.3.4.4 highly pathogenic AI viruses (HPAIVs) had not been detected in Taiwan before 2015. During this epidemic, four types of etiologic agents were identified: the three novel subtypes H5N2, H5N8, and H5N3 clade 2.3.4.4 HPAIVs and one endemic chicken H5N2 subtype (Mexican-like lineage) of low pathogenic AI viruses. Cocirculation of mixed subtypes also occurred, with H5N2 clade 2.3.4.4 HPAIVs accompanied by the H5N8 and H5N3 subtypes or old H5N2 viruses in the same farm. More than 90% of domestic geese died from this AI epidemic; geese were affected the most at the early outbreaks. The epidemic peaked in mid-January for all three novel H5 subtypes. Spatial epidemiology found that most affected areas were located in southwestern coastal areas. In terrestrial poultry (mostly chickens), different geographic distributions of AI virus subtypes were detected, with hot spots of H5N2 clade 2.3.4.4 vs. past-endemic old H5N2 viruses in Changhwa (P = 0.03) and Yunlin (P = 0.007) counties, respectively, of central Taiwan. Phylogenetic and sequence analyses of all the early 10 Taiwan H5 clade 2.3.4.4 isolates covering the three subtypes showed that they were very different from the HA of the past local H5 viruses from domestic ducks (75%-80%) and chickens (70%-75%). However, they had the highest sequence identity percentages (99.53%-100%), with the HA of A/crane/Kagoshima/KU13/2014(H5N8) isolated on December 7, 2014, in Japan being higher than those of recent American and Korean H5 HPAIVs [A/Northern pintail/Washington/40964/2014 (H5N2) and A/gyrfalcon/Washington/41088-6/2014 (H5N8): 99.02%-99.54% and A/Baikal teal/Korea/Donglim3/2014 (H5N8): 98.61%-99.08%], implying a likely common ancestor of these H5 clade 2.3.4.4 viruses. The multiple subtypes of H5 clade 2.3.4.4 HPAIVs imply high viral reassortment. We recommend establishing an integrated surveillance system, involving clinical, virologic, and serologic surveillance in poultry and wild birds, swine and other mammals prevalent on multiple-animal mixed-type traditional farms, and high-risk human populations, as a crucially important step to minimize future pandemic influenza.

Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan.

BACKGROUND: Human infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past. METHODS: Two duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved. RESULTS: The DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells. CONCLUSION: These results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission.