Avian flu: «for whom the bell tolls¼?

The family Orthomyxoviridae consists of 9 genera, including Alphainfluenza virus, which contains avian influenza viruses. In two subtypes H5 and H7 besides common low-virulent strains, a specific type of highly virulent avian virus have been described to cause more than 60% mortality among domestic birds. These variants of influenza virus are usually referred to as «avian influenza virus¼. The difference between high (HPAI) and low (LPAI) virulent influenza viruses is due to the structure of the arginine-containing proteolytic activation site in the hemagglutinin (HA) protein. The highly virulent avian influenza virus H5 was identified more than 100 years ago and during this time they cause outbreaks among wild and domestic birds on all continents and only a few local episodes of the disease in humans have been identified in XXI century. Currently, a sharp increase in the incidence of highly virulent virus of the H5N1 subtype (clade h2.3.4.4b) has been registered in birds on all continents, accompanied by the transmission of the virus to various species of mammals. The recorded global mortality rate among wild, domestic and agricultural birds from H5 subtype is approaching to the level of 1 billion cases. A dangerous epidemic factor is becoming more frequent outbreaks of avian influenza with high mortality among mammals, in particular seals and marine lions in North and South America, minks and fur-bearing animals in Spain and Finland, domestic and street cats in Poland. H5N1 avian influenza clade h2.3.4.4b strains isolated from mammals have genetic signatures of partial adaptation to the human body in the PB2, NP, HA, NA genes, which play a major role in regulating the aerosol transmission and the host range of the virus. The current situation poses a real threat of pre-adaptation of the virus in mammals as intermediate hosts, followed by the transition of the pre-adapted virus into the human population with catastrophic consequences.

An algorithm for the characterization of influenza A viruses from various host species and environments.

Due to the extensive host range of influenza A viruses, it is difficult to determine the best diagnostic algorithm to efficiently screen samples from a variety of host species for influenza A viruses. While there are some influenza diagnostic algorithms that are specific to host species, to our knowledge, no single algorithm exists for the characterization of influenza A viruses across multiple host species. In this paper, we propose an algorithm that can serve as a guide for screening human, animal, and environmental samples for influenza A viruses of high human and animal health importance.

Accumulated metagenomic studies reveal recent migration, whole genome evolution, and undiscovered diversity of orthomyxoviruses.

IMPORTANCE: The number of known virus species has increased dramatically through metagenomic studies, which search genetic material sampled from a host for non-host genes. Here, we focus on an important viral family that includes influenza viruses, the Orthomyxoviridae, with over 100 recently discovered viruses infecting hosts from humans to fish. We find that one virus called Wǔhàn mosquito virus 6, discovered in mosquitoes in China, has spread across the globe very recently. Surface proteins used to enter cells show signs of rapid evolution in Wǔhàn mosquito virus 6 and its relatives which suggests an ability to infect vertebrate animals. We compute the rate at which new orthomyxovirus species discovered add evolutionary history to the tree of life, predict that many viruses remain to be discovered, and discuss what appropriately designed future studies can teach us about how diseases cross between continents and species.

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota).

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Bourbon virus, a newly discovered zoonotic thogotovirus.

The recent discovery of Bourbon virus (BRBV) put a new focus on the genus of thogotoviruses as zoonotic, tick-transmitted pathogens within the orthomyxovirus family. Since 2014, BRBV has been linked to several human cases in the Midwest United States with severe acute febrile illness and a history of tick bites. The detection of the virus in the Lone Star tick, Amblyomma americanum, and a high sero-prevalence in wild animals suggest widespread circulation of BRBV. Phylogenetic analysis of the viral RNA genome classified BRBV into the subgroup of Dhori-like thogotoviruses. Strikingly, BRBV is apathogenic in mice, contrasting not only with the fatal disease in affected patients but also with the severe disease in mice caused by other members of the thogotovirus genus. To gain insights into this intriguing discrepancy, we will review the molecular biology and pathology of BRBV and its unique position within the thogotovirus genus. Lastly, we will discuss the zoonotic threat posed by this newly discovered pathogen.

Validation of a TaqMan one-step real-time RT-PCR assay targeting ISAV segment 7 spliced mRNA.

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98 % (R2 = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108 % (R2 = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 % (± 0.7), 1.7 % (± 0.3), and 1.5 % (± 0.1) for the low virulent and 9.4 % (± 2.2), 4.7 % (± 0.8), and 6.2 % (± 0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics.

Continuous surveillance of potentially zoonotic avian pathogens detects contemporaneous occurrence of highly pathogenic avian influenza viruses (HPAIV H5) and flaviviruses (USUV, WNV) in several wild and captive birds.

Three avian viral pathogens circulate in Germany with particular importance for animal disease surveillance due to their zoonotic potential, their impact on wild bird populations and/or poultry farms: Highly pathogenic (HP) avian influenza virus (AIV) of subtype H5 (HPAIV H5), Usutu virus (USUV), and West Nile virus (WNV). Whereas HPAIV H5 has been mainly related to epizootic outbreaks in winter, the arthropod-borne viruses USUV and WNV have been detected more frequently during summer months corresponding to peak mosquito activity. Since 2021, tendencies of a potentially year-round, i.e. enzootic, status of HPAIV in Germany have raised concerns that Orthomyxoviruses (AIV) and Flaviviruses (USUV, WNV) may not only circulate in the same region, but also at the same time and in the same avian host range. In search of a host species group suitable for a combined surveillance approach for all mentioned pathogens, we retrospectively screened and summarized case reports, mainly provided by the respective German National Reference Laboratories (NRLs) from 2006 to 2021. Our dataset revealed an overlap of reported infections among nine avian genera. We identified raptors as a particularly affected host group, as the genera Accipiter, Bubo, Buteo, Falco, and Strix represented five of the nine genera, and highlighted their role in passive surveillance. This study may provide a basis for broader, pan-European studies that could deepen our understanding of reservoir and vector species, as HPAIV, USUV, and WNV are expected to further become established and/or spread in Europe in the future and thus improved surveillance measures are of high importance.

The infectious salmon anemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition.

Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease.

Identification of RNA Virus-Derived RdRp Sequences in Publicly Available Transcriptomic Data Sets.

RNA viruses are abundant and highly diverse and infect all or most eukaryotic organisms. However, only a tiny fraction of the number and diversity of RNA virus species have been catalogued. To cost-effectively expand the diversity of known RNA virus sequences, we mined publicly available transcriptomic data sets. We developed 77 family-level Hidden Markov Model profiles for the viral RNA-dependent RNA polymerase (RdRp)-the only universal "hallmark" gene of RNA viruses. By using these to search the National Center for Biotechnology Information Transcriptome Shotgun Assembly database, we identified 5,867 contigs encoding RNA virus RdRps or fragments thereof and analyzed their diversity, taxonomic classification, phylogeny, and host associations. Our study expands the known diversity of RNA viruses, and the 77 curated RdRp Profile Hidden Markov Models provide a useful resource for the virus discovery community.

Chest computed tomography analysis of lung sparing morphology: differentiation of COVID-19 pneumonia from influenza pneumonia and bacterial pneumonia using the arched bridge and vacuole signs.

INTRODUCTION: This study evaluated the arched bridge and vacuole signs, which constitute morphological patterns of lung sparing in coronavirus disease 2019 (COVID-19), then examined whether these signs could be used to differentiate COVID-19 pneumonia from influenza pneumonia or bacterial pneumonia. METHODS: In total, 187 patients were included: 66 patients with COVID-19 pneumonia, 50 patients with influenza pneumonia and positive computed tomography findings, and 71 patients with bacterial pneumonia and positive computed tomography findings. Images were independently reviewed by two radiologists. The incidences of the arched bridge sign and/or vacuole sign were compared among the COVID-19 pneumonia, influenza pneumonia, and bacterial pneumonia groups. RESULTS: The arched bridge sign was much more common among patients with COVID-19 pneumonia (42/66, 63.6%) than among patients with influenza pneumonia (4/50, 8.0%; P<0.001) or bacterial pneumonia (4/71, 5.6%; P<0.001). The vacuole sign was also much more common among patients with COVID-19 pneumonia (14/66, 21.2%) than among patients with influenza pneumonia (1/50, 2.0%; P=0.005) or bacterial pneumonia (1/71, 1.4%; P<0.001). The signs occurred together in 11 (16.7%) patients with COVID-19 pneumonia, but they did not occur together in patients with influenza pneumonia or bacterial pneumonia. The arched bridge and vacuole signs predicted COVID-19 pneumonia with respective specificities of 93.4% and 98.4%. CONCLUSION: The arched bridge and vacuole signs are much more common in patients with COVID-19 pneumonia and can help differentiate COVID-19 pneumonia from influenza and bacterial pneumonia.

Severe Human Case of Zoonotic Infection with Swine-Origin Influenza A Virus, Denmark, 2021.

During routine surveillance at the National Influenza Center, Denmark, we detected a zoonotic swine influenza A virus in a patient who became severely ill. We describe the clinical picture and the genetic characterization of this variant virus, which is distinct from another variant found previously in Denmark.

Possible bidirectional human-swine and subsequent human-human transmission of influenza virus A(H1N1)/2009 in Japan.

In 2019, sows at a swine farm in Japan showed influenza-like illness (ILI) shortly after contact with an employee that exhibited ILI. Subsequently, a veterinarian became sick shortly after examining the sows and was diagnosed with influenza A virus (IAV) infection. Then, her family also contracted the infection. Subsequently, Pandemic A(H1N1)2009 viruses were isolated from all samples obtained from the sows, veterinarian and her family. Whole-genome analysis of the isolates confirmed that the viruses belonged to the same lineage (6B.1A) and the genome sequences obtained from all of the isolates were almost identical to each other. Furthermore, an epidemiological survey revealed no contact between veterinarians or their families and influenza patients prior to the onset of illness. These results strongly indicated a case of bidirectional infection between humans and sows. At the same time, we found a few unique mutations in the IAV genomes corresponding to the host species. The mutations that occurred in the virus after it was transferred from the farm worker to the sows were not observed in the humans infected from the sows, probably as a result of the mutations reverting to the original nucleotides. These results demonstrate that the bidirectional transmission of IAV is a potential risk for the next pandemic outbreak due to the emergence of new mutant strains.

Epidemiology of influenza virus reinfection in Guangxi, China: a retrospective analysis of a nine-year influenza surveillance data: Characteristics of influenza virus reinfection.

BACKGROUND: Epidemiological characteristic profile of the reinfection of the influenza virus has not been well described. METHODS: This study included all influenza cases of Guangxi, China from January 2011-December 2019 that were recorded in the National Notifiable Infectious Disease Reporting Information System (NIDRIS) within 24 hours after diagnosis. RESULTS: A total of 53,605.6 person-months and the median time of 8.7 months were observed for reinfection. The median age at the first influenza virus infection was 4.5 (interquartile range=2.0-7.5) years. The cumulative reinfection incidence was 2% at 6 months, 4% at 12 months, 5% at 24 months, and 7% after 59 months. Living in the rural area (hazard ratio [HR]=1.37 [95% confidence interval (CI), 1.29-1.45]), age ≤6 years (HR=11.43 [95% CI, 9.47-13.80]) were independent risk factors associated with influenza reinfection. Among 49 patients experiencing two laboratory tests, 32 patients (65.3%) were found to be infected with different virus types. The interval between two consecutive laboratory-confirmed episodes of the four groups differed (p=0.148): the maximum was 72.9 months and the minimum was 1.2 months. CONCLUSIONS: The reinfection of the influenza virus in Guangxi was independently and positively associated with living the rural area and younger age. The unusually high frequency of reinfection points to a need for further prospective longitudinal studies to better investigate the sufficient impact on different subtypes.

No Evidence of the Vertical Transmission of Non-Virulent Infectious Salmon Anaemia Virus (ISAV-HPR0) in Farmed Atlantic Salmon.

The nonvirulent infectious salmon anaemia virus (ISAV-HPR0) is the putative progenitor for virulent-ISAV, and a potential risk factor for the development of infectious salmon anaemia (ISA). Understanding the transmission dynamics of ISAV-HPR0 is fundamental to proper management and mitigation strategies. Here, we demonstrate that ISAV-HPR0 causes prevalent and transient infections in all three production stages of Atlantic salmon in the Faroe Islands. Phylogenetic analysis of the haemagglutinin-esterase gene from 247 salmon showed a clear geographical structuring into two significantly distinct HPR0-subgroups, which were designated G2 and G4. Whereas G2 and G4 co-circulated in marine farms, Faroese broodfish were predominantly infected by G2, and smolt were predominantly infected by G4. This infection pattern was confirmed by our G2- and G4-specific RT-qPCR assays. Moreover, the HPR0 variants detected in Icelandic and Norwegian broodfish were never detected in the Faroe Islands, despite the extensive import of ova from both countries. Accordingly, the vertical transmission of HPR0 from broodfish to progeny is uncommon. Phylogenetic and statistical analysis suggest that HPR0 persists in the smolt farms as "house-strains", and that new HPR0 variants are occasionally introduced from the marine environment, probably by HPR0-contaminated sea-spray. Thus, high biosecurity-including water and air intake-is required to avoid the introduction of pathogens to the smolt farms.

Reassortant Influenza A(H1N1)pdm09 Virus in Elderly Woman, Denmark, January 2021.

A case of human infection with influenza A(H1N1)pdm09 virus containing a nonstructural gene highly similar to Eurasian avian-like H1Nx swine influenza virus was detected in Denmark in January 2021. We describe the clinical case and report testing results of the genetic and antigenic characterizations of the virus.

Use of hemagglutinin and neuraminidase amplicon-based high-throughput sequencing with variant analysis to detect co-infection and resolve identical consensus sequences of seasonal influenza in a university setting.

BACKGROUND: Local transmission of seasonal influenza viruses (IVs) can be difficult to resolve. Here, we study if coupling high-throughput sequencing (HTS) of hemagglutinin (HA) and neuraminidase (NA) genes with variant analysis can resolve strains from local transmission that have identical consensus genome. We analyzed 24 samples collected over four days in January 2020 at a large university in the US. We amplified complete hemagglutinin (HA) and neuraminidase (NA) genomic segments followed by Illumina sequencing. We identified consensus complete HA and NA segments using BLASTn and performed variant analysis on strains whose HA and NA segments were 100% similar. RESULTS: Twelve of the 24 samples were PCR positive, and we detected complete HA and/or NA segments by de novo assembly in 83.33% (10/12) of them. Similarity and phylogenetic analysis showed that 70% (7/10) of the strains were distinct while the remaining 30% had identical consensus sequences. These three samples also had IAV and IBV co-infection. However, subsequent variant analysis showed that they had distinct variant profiles. While the IAV HA of one sample had no variant, another had a T663C mutation and another had both C1379T and C1589A. CONCLUSION: In this study, we showed that HTS coupled with variant analysis of only HA and NA genes can help resolve variants that are closely related. We also provide evidence that during a short time period in the 2019-2020 season, co-infection of IAV and IBV occurred on the university campus and both 2020/2021 and 2021/2022 WHO recommended H1N1 vaccine strains were co-circulating.

Novel quaranjavirus and other viral sequences identified from ticks parasitizing hunted wildlife in Trinidad and Tobago.

Hunters are at a higher risk for exposure to zoonotic pathogens due to their close interactions with wildlife and arthropod vectors. In this study, high throughput sequencing was used to explore the viromes of two tick species, Amblyomma dissimile and Haemaphysalis juxtakochi, removed from hunted wildlife in Trinidad and Tobago. We identified sequences from 3 new viral species, from the viral families Orthomyxoviridae, Chuviridae and Tetraviridae in A. dissimile.

Avian Influenza Viruses Detected in Birds in Sub-Saharan Africa: A Systematic Review.

In the recent past, sub-Saharan Africa has not escaped the devastating effects of avian influenza virus (AIV) in poultry and wild birds. This systematic review describes the prevalence, spatiotemporal distribution, and virus subtypes detected in domestic and wild birds for the past two decades (2000-2019). We collected data from three electronic databases, PubMed, SpringerLink electronic journals and African Journals Online, using the Preferred Reporting Items for Systematic reviews and Meta-Analyses protocol. A total of 1656 articles were reviewed, from which 68 were selected. An overall prevalence of 3.0% AIV in birds was observed. The prevalence varied between regions and ranged from 1.1% to 7.1%. The Kruskal-Wallis and Wilcoxon signed-rank sum test showed no significant difference in the prevalence of AIV across regions, χ2(3) = 5.237, p = 0.1553 and seasons, T = 820, z = -1.244, p = 0.2136. Nineteen hemagglutinin/neuraminidase subtype combinations were detected during the reviewed period, with southern Africa recording more diverse AIV subtypes than other regions. The most detected subtype was H5N1, followed by H9N2, H5N2, H5N8 and H6N2. Whilst these predominant subtypes were mostly detected in domestic poultry, H1N6, H3N6, H4N6, H4N8, H9N1 and H11N9 were exclusively detected in wild birds. Meanwhile, H5N1, H5N2 and H5N8 were detected in both wild and domestic birds suggesting circulation of these subtypes among wild and domestic birds. Our findings provide critical information on the eco-epidemiology of AIVs that can be used to improve surveillance strategies for the prevention and control of avian influenza in sub-Saharan Africa.

Rapid sequence modification in the highly polymorphic region (HPR) of the hemagglutinin gene of the infectious salmon anaemia virus (ISAV) suggests intra-segmental template switching recombination.

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

The Power of First Impressions: Can Influenza Imprinting during Infancy Inform Vaccine Design?

Influenza virus infection causes severe respiratory illness in people worldwide, disproportionately affecting infants. The immature respiratory tract coupled with the developing immune system, and lack of previous exposure to the virus is thought to synergistically play a role in the increased disease severity in younger age groups. No influenza vaccines are available for those under six months, although maternal influenza immunization is recommended. In children aged six months to two years, vaccine immunogenicity is dampened compared to older children and adults. Unlike older children and adults, the infant immune system has fewer antigen-presenting cells and soluble immune factors. Paradoxically, we know that a person's first infection with the influenza virus during infancy or childhood leads to the establishment of life-long immunity toward that particular virus strain. This is called influenza imprinting. We contend that by understanding the influenza imprinting event in the context of the infant immune system, we will be able to design more effective influenza vaccines for both infants and adults. Working through the lens of imprinting, using infant influenza animal models such as mice and ferrets which have proven useful for infant immunity studies, we will gain a better understanding of imprinting and its implications regarding vaccine design. This review examines literature regarding infant immune and respiratory development, current vaccine strategies, and highlights the importance of research into the imprinting event in infant animal models to develop more effective and protective vaccines for all including young children.