Influenza Surveillance Efforts Research Articles

H5N1 clade 2.3.4.4b avian influenza viruses replicate in differentiated bovine airway epithelial cells cultured at air-liquid interface.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.

Genetic Variation and Phylogenetic Analysis of Influenza A/H3N2 Hemagglutinin Gene in Shiraz, Southern Iran: Implications for Influenza Control and Vaccine Development

Background: Variation in the hemagglutinin (HA) of influenza A/H3N2 can affect antigenicity and virulence, potentially leading to the emergence of vaccine escape variants and antigenic shift viruses. This study aimed to evaluate the genetic variation of the HA gene to enhance global influenza virus surveillance. Objectives: The primary objectives of this research included molecular characterization and phylogenetic analysis of the influenza A/H3N2 HA gene. Therefore, this research aimed to contribute valuable insights to influenza surveillance efforts on a global scale by identifying the variants associated with vaccine escape and antigenic shift. Methods: RNA extraction was performed on 300 nasopharyngeal swab samples from patients with influenza-like illness using a commercial kit. The presence of influenza A/H3N2 was determined using qRT-PCR. Subsequently, positive samples underwent HA gene amplification through RT-PCR using the Sanger method. Variability in HA immunological sites was compared with reference sequences, and genotype/subtype was determined. Alignment and phylogenetic analyses were conducted to construct a tree utilizing an optimal nucleotide substitution model Results: Among the 300 samples, 22 (7.33%) tested positive for influenza A/H3N2, with a male distribution of 13 cases. The mean age of the participants was 35 ± 2.40 years. The analysis of antigenic site mutations revealed significant variations (A, B, D, and E) in the HA gene, with major mutations at positions 140 and 186. Noteworthy mutations such as S159Y and the previously undocumented K83R substitution at antigenic site E. Phylogenetic analysis classified the A/H3N2 strain into clades 3C.2a and 3C.3. Conclusions: The identified mutations in the HA gene suggest potential changes in antigenicity, emphasizing their relevance to vaccine development and surveillance strategies. It is recommended that ongoing molecular investigations be conducted for effective influenza control; also, the ongoing need to monitor influenza viruses is suggested to be highlighted. This research provides valuable insights necessary for maintaining efficient influenza control measures globally.

The Influenza B Virus Victoria and Yamagata Lineages Display Distinct Cell Tropism and Infection-Induced Host Gene Expression in Human Nasal Epithelial Cell Cultures.

Understanding Influenza B virus infections is of critical importance in our efforts to control severe influenza and influenza-related diseases. Until 2020, two genetic lineages of influenza B virus-Yamagata and Victoria-circulated in the population. These lineages are antigenically distinct, but the differences in virus replication or the induction of host cell responses after infection have not been carefully studied. Recent IBV clinical isolates of both lineages were obtained from influenza surveillance efforts of the Johns Hopkins Center of Excellence in Influenza Research and Response and characterized in vitro. B/Victoria and B/Yamagata clinical isolates were recognized less efficiently by serum from influenza-vaccinated individuals in comparison to the vaccine strains. B/Victoria lineages formed smaller plaques on MDCK cells compared to B/Yamagata, but infectious virus production in primary human nasal epithelial cell (hNEC) cultures showed no differences. While ciliated epithelial cells were the dominant cell type infected by both lineages, B/Victoria lineages had a slight preference for MUC5AC-positive cells, and B/Yamagata lineages infected more basal cells. Finally, while both lineages induced a strong interferon response 48 h after infection of hNEC cultures, the B/Victoria lineages showed a much stronger induction of interferon-related signaling pathways compared to B/Yamagata. This demonstrates that the two influenza B virus lineages differ not only in their antigenic structure but also in their ability to induce host innate immune responses.

Influenza A and D Viruses in Non-Human Mammalian Hosts in Africa: A Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis to investigate the prevalence and current knowledge of influenza A virus (IAV) and influenza D virus (IDV) in non-human mammalian hosts in Africa. PubMed, Google Scholar, Wiley Online Library and World Organisation for Animal Health (OIE-WAHIS) were searched for studies on IAV and IDV from 2000 to 2020. Pooled prevalence and seroprevalences were estimated using the quality effects meta-analysis model. The estimated pooled prevalence and seroprevalence of IAV in pigs in Africa was 1.6% (95% CI: 0–5%) and 14.9% (95% CI: 5–28%), respectively. The seroprevalence of IDV was 87.2% (95% CI: 24–100%) in camels, 9.3% (95% CI: 0–24%) in cattle, 2.2% (95% CI: 0–4%) in small ruminants and 0.0% (95% CI: 0–2%) in pigs. In pigs, H1N1 and H1N1pdm09 IAVs were commonly detected. Notably, the highly pathogenic H5N1 virus was also detected in pigs. Other subtypes detected serologically and/or virologically included H3N8 and H7N7 in equids, H1N1, and H3N8 and H5N1 in dogs and cats. Furthermore, various wildlife animals were exposed to different IAV subtypes. For prudent mitigation of influenza epizootics and possible human infections, influenza surveillance efforts in Africa should not neglect non-human mammalian hosts. The impact of IAV and IDV in non-human mammalian hosts in Africa deserves further investigation.

Spatiotemporal Analysis of Influenza in China, 2005\u20132018

Influenza is a major cause of morbidity and mortality worldwide, as well as in China. Knowledge of the spatial and temporal characteristics of influenza is important in evaluating and developing disease control programs. This study aims to describe an accurate spatiotemporal pattern of influenza at the prefecture level and explore the risk factors associated with influenza incidence risk in mainland China from 2005 to 2018. The incidence data of influenza were obtained from the Chinese Notifiable Infectious Disease Reporting System (CNIDRS). The Besag York Mollié (BYM) model was extended to include temporal and space-time interaction terms. The parameters for this extended Bayesian spatiotemporal model were estimated through integrated nested Laplace approximations (INLA) using the package R-INLA in R. A total of 702,226 influenza cases were reported in mainland China in CNIDRS from 2005–2018. The yearly reported incidence rate of influenza increased 15.6 times over the study period, from 3.51 in 2005 to 55.09 in 2008 per 100,000 populations. The temporal term in the spatiotemporal model showed that much of the increase occurred during the last 3 years of the study period. The risk factor analysis showed that the decreased number of influenza vaccines for sale, the new update of the influenza surveillance protocol, the increase in the rate of influenza A (H1N1)pdm09 among all processed specimens from influenza-like illness (ILI) patients, and the increase in the latitude and longitude of geographic location were associated with an increase in the influenza incidence risk. After the adjusting for fixed covariate effects and time random effects, the map of the spatial structured term shows that high-risk areas clustered in the central part of China and the lowest-risk areas in the east and west. Large space-time variations in influenza have been found since 2009. In conclusion, an increasing trend of influenza was observed from 2005 to 2018. The insufficient flu vaccine supplements, the newly emerging influenza A (H1N1)pdm09 and expansion of influenza surveillance efforts might be the major causes of the dramatic changes in outbreak and spatio-temporal epidemic patterns. Clusters of prefectures with high relative risks of influenza were identified in the central part of China. Future research with more risk factors at both national and local levels is necessary to explain the changing spatiotemporal patterns of influenza in China.

Global flu view: a platform to connect crowdsourced disease surveillance around the world

Purpose: Participatory disease surveillance actively engages the public in reporting on symptoms of their health to provide community-level data that complements traditional healthcare-based surveillance. Participatory surveillance is timely, low-cost, can account for non-medically attended populations, and allows for direct engagement with local populations. Three examples of participatory surveillance systems – Flu Near You in North America, Influenzanet in Europe, and Flu Tracking in Australia and New Zealand – have recently collaborated to develop Global Flu View, a shared platform for aggregation and dissemination of crowdsourced data on influenza-like illness. Methods & Materials: The collaborating surveillance systems have developed a shared application programming interface (API) for data exchange among systems. The API specifications outline specific variables that are called into a shared, cloud-based database. Data is processed via an ETL layer resulting in aggregate data at the postcode level. Key variables include reporting week, postal and country codes, user birth month and birth year, gender, vaccination status, and a series of symptom variables that include fever, cough, sore throat, headache, fatigue, and other indications of influenza-like illness. Processed data will be available to view in both graph and map formats on the website www.globalfluview.org. Results: The Global Flu View platform connects community-level influenza surveillance efforts across 15 countries on 3 continents. Tens of thousands of weekly reports are made available for users to explore through graphs, maps, and various filtering tools. The Global Flu View database also serves as a repository of influenza surveillance data that can be shared with disease modelers, researchers, and epidemiologists around the world. Conclusion: Global Flu View can highlight the value of the participatory surveillance approach, serving as a model for other countries. The platform is poised to incorporate data from additional countries with similar tools and serves as a case study for collaborative, multi-country data sharing for global health surveillance. As of July 2018 the platform is in beta-testing mode and will be made publicly available by late August 2018.

Viral factors in influenza pandemic risk assessment.

The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk.DOI: http://dx.doi.org/10.7554/eLife.18491.001

PB2-E627K and PA-T97I substitutions enhance polymerase activity and confer a virulent phenotype to an H6N1 avian influenza virus in mice

H6N1 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H6N1 virus infection in 2013. Here, we set out to elucidate viral determinants critical to the pathogenesis of this virus using a mouse model. We found that the recombinant H6N1 viruses possessing both the PA-T97I and PB2-E627K substitutions displayed the greatest enhancement of replication in vitro and in vivo. Polymerase complexes possessing either PB2-E627K, PA-T97I, and PB2-E627K/PA-T97I displayed higher virus polymerase activity when compared to the wild-type virus, which may account for the increased replication kinetics and enhanced virulence of variant viruses. Our results demonstrate that PB2-E627K and PA-T97I enhance the ability of H6N1 virus to replicate and cause disease in mammals. Influenza surveillance efforts should include scrutiny of these regions of PB2 and PA because of their impact on the increased virulence of H6N1 AIVs in mice.

Sequential Reassortments Underlie Diverse Influenza H7N9 Genotypes in China

Initial genetic characterizations have suggested that the influenza A (H7N9) viruses responsible for the current outbreak in China are novel reassortants. However, little is known about the pathways of their evolution and, in particular, the generation of diverse viral genotypes. Here we report an in-depth evolutionary analysis of whole-genome sequence data of 45 H7N9 and 42 H9N2 viruses isolated from humans, poultry, and wild birds during recent influenza surveillance efforts in China. Our analysis shows that the H7N9 viruses were generated by at least two steps of sequential reassortments involving distinct H9N2 donor viruses in different hosts. The first reassortment likely occurred in wild birds and the second in domestic birds in east China in early 2012. Our study identifies the pathways for the generation of diverse H7N9 genotypes in China and highlights the importance of monitoring multiple sources for effective surveillance of potential influenza outbreaks.

Where Do Avian Influenza Viruses Meet in the Americas?

Avian influenza virus (AIV) surveillance has been scarce in most countries of Latin America and the Caribbean. Historically, avian influenza surveillance efforts in Central and South America have been localized in places where outbreaks in poultry have occurred. Since the emergence of the H5N1 subtype in Asia, active surveillance in wild birds has increased in a number of Latin American countries, including Barbados, Guatemala, Argentina, Brazil, Mexico, and Peru. A broad diversity of virus subtypes has been detected; however, nucleotide sequence data are still limited in comparison to other regions of the world. Here we review the current knowledge of AIV in Latin America, including phylogenetic relationships among publicly available viral genomes. Overall AIV reports are sparse across the region and the cocirculation of two distinct genetic lineages is puzzling. Phylogenetic analysis reflects bias in time and location where sampling has been conducted. Increased surveillance is needed to address the major determinants for AIV ecology, evolution, and transmission in the region.

Lessons from Emergence of A/Goose/Guangdong/1996‐Like H5N1 Highly Pathogenic Avian Influenza Viruses and Recent Influenza Surveillance Efforts in Southern China

Southern China is proposed as an influenza epicentre. At least two of the three pandemics in the last century, including 1957 and 1968 influenza pandemics, originated from this area. In 1996, A/goose/Guangdong/1/1996 (H5N1), the precursor of currently circulating highly pathogenic H5N1 avian influenza viruses (HPAIVs) was identified in farmed geese in southern China. These H5N1 HPAIVs have been spread across Asia, Europe and Africa and poses a continuous threat to both animal and human health. However, how and where this H5N1 HPAIV emerged are not fully understood. In the past decade, many influenza surveillance efforts have been carried out in southern China, and our understanding of the genetic diversity of non-human influenza A viruses in this area has been much better than ever. Here, the historical and first-hand experimental data on A/goose/Guangdong/1/1996(H5N1)-like HPAIVs are reviewed within the context of the findings from recent surveillance efforts on H5N1 HPAIVs and other non-human influenza A viruses. Such a retrospective recapitulation suggests that long-term and systematic surveillance programmes should continue to be implemented in southern China that the wet markets on the animal-human interface shall be the priority area and that the surveillance on the animal species bridging the interface between wildlife and domestic animal populations and the interface between the aquatics and territories shall be the strengthened.

Isolation and characterization of an H9N2 influenza virus isolated in Argentina

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) – herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.

Monitoring seasonal influenza A evolution: Rapid 2009 pandemic H1N1 surveillance with an reverse transcription-polymerase chain reaction/electro-spray ionization mass spectrometry assay

BackgroundThe emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts. ObjectivesTo determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity. Study designTwo sets of pandemic H1N1 samples, 190 collected between April and June of 2009, and 69 collected between October 2009 and January 2010, were processed by the RT-PCR/ESI-MS influenza typing assay, and the spectral results were compared to reference laboratory results and historical sequencing data from the Nucleotide Database of the National Center for Biotechnology Information (NCBI). ResultsStrain typing concordance with reference standard testing was 100% in both sample sets, and the assay demonstrated a significant increase in influenza genetic diversity, from 10.5% non-wildtype genotypes in early samples to 69.9% in late samples (P<0.001). An NCBI search demonstrated a similar increase, from 13.4% to 45.2% (P<0.001). ConclusionsThis comparison of early versus late influenza samples analyzed by RT-PCR/ESI-MS demonstrates the influenza typing assay's ability as a universal influenza detection platform to provide high-fidelity pH1N1 strain identification over time, despite increasing genetic diversity in the circulating virus. The genotyping data can also be leveraged for high-throughput influenza surveillance.

Phylogenetic and molecular characterization of equine H3N8 influenza viruses from Greece (2003 and 2007): evidence for reassortment between evolutionary lineages.

BackgroundFor first time in Greece equine influenza virus infection was confirmed, by isolation and molecular analysis, as the cause of clinical respiratory disease among unvaccinated horses during 2003 and 2007 outbreaks.MethodsEquine influenza virus (EIV) H3N8 was isolated in MDCK cells from 30 nasal swabs from horses with acute respiratory disease, which were tested positive by Directigen Flu A. Isolation was confirmed by haemagglutination assay and RT-PCR assay of the M, HA and NA gene.ResultsHA sequences of the Greek isolates appeared to be more closely related to viruses isolated in early 1990s in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. On the other hand, analysis of deduced NA amino acid sequences were more closely related to viruses isolated in outbreaks in Europe and Asia during 2003-2007. Phylogenetic analysis characterized the Greek isolates as a member of the Eurasian lineage by the haemagglutinin (HA) protein alignment, but appeared to be a member of the Florida sublineage clade 2 by the neuraminidase (NA) protein sequence suggesting that reassortment might be a possible explanation.ConclusionOur findings suggest that the Greek strains represent an example of "frozen evolution" and probably reassortment between genetically distinct co-circulated strains. Therefore expanding current equine influenza surveillance efforts is a necessity.

Two Distinct Surveillance Methods to Track Hospitalized Influenza Patients in New York State During the 2009–2010 Influenza Season

To better understand the severity of 2009 H1N1 influenza disease, enhanced surveillance of patients hospitalized with influenza was conducted during the 2009-2010 influenza season in New York State through existing Emerging Infections Program surveillance and a newly established sentinel hospital surveillance program. The 2 surveillance systems were compared to determine consistency across surveillance modalities and reveal the strengths and weaknesses of each to accomplish comprehensive influenza surveillance. Similar variables from the aggregate data collected from each system were compared and differences were analyzed in detail. New York State. Hospitalized adult and pediatric patients detected through 2 influenza surveillance programs. Significant differences in age distribution, timing of illness onset, illness complications, underlying medical conditions, critical care admissions, use of mechanical ventilation, and illness outcomes. Both surveillance systems saw the highest numbers of confirmed influenza infection among patients hospitalized in early fall 2009, with sharp declines thereafter. Sentinel hospital surveillance continued to detect hospitalizations for influenza-like illness that were not due to 2009 H1N1 influenza well into March 2010. Compared to influenza surveillance conducted through the Emerging Infections Program, the sentinel hospital influenza surveillance program tended to detect a sicker population of children and adults, including a higher rate of critical illness and mechanical ventilation, and among adults, higher rates of some underlying medical conditions. There were no differences in disease outcomes detected between the 2 systems. Although the 2 surveillance systems were complementary, inherent methodologic variations revealed important differences at season conclusion. The lessons learned should be used to determine the best way to allocate resources to meet the needs of future state and national influenza surveillance efforts.

Avian biology, the human influence on global avian influenza transmission, and performing surveillance in wild birds

This paper takes a closer look at three interrelated areas of study: avian host biology, the role of human activities in virus transmission, and the surveillance activities centered on avian influenza in wild birds. There are few ecosystems in which birds are not found. Correspondingly, avian influenza viruses are equally global in distribution, relying on competent avian hosts. The immune systems, annual cycles, feeding behaviors, and migration patterns of these hosts influence the ecology of the disease. Decreased biodiversity has also been linked to heightened disease transmission in several disease systems, and it is evident that active destruction and modification of wetland environments for human use is impacting avian populations drastically. Legal and illegal trade in wild birds present a significant risk for introduction and maintenance of exotic diseases. After the emergence of HPAI H5N1 in Hong Kong in 1996 and the ensuing geographic spread of outbreaks after 2003, both infected countries and those at risk of introduction began intensifying avian influenza surveillance efforts. Several techniques for sampling wild birds for influenza viruses have been applied. Benefits, problems, and biases exist for each method. The wild bird avian influenza surveillance programs taking place across the continents are now scaling back due to the rise of other spending priorities; hopefully the lessons learned from this work will be preserved and will inform future research and disease outbreak response priorities.

Effect of Preservative on Recoverable RT-PCR Amplicon Length from Influenza A Virus in Bird Feces

Surveillance for avian influenza A viruses in wild bird populations is often limited by requirements for a cold chain from time of specimen collection, by availability of ultra-low temperature specimen storage within a few hours or days, and by laborious classical virologic procedures. Successful storage of specimens in preservatives at ambient temperature and subsequent detection of RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) would assist in helping influenza surveillance efforts become more widespread in remote areas, as well as more timely and inexpensive. Here, we describe bird feces spiked with influenza A virus preserved with guanidine and commercial buffers or alcohols at ambient temperature and analyzed by RT-PCR protocols. Virus-specific RT-PCR products of, at most, 206 bp were recovered for samples preserved with alcohols and up to 521 bp for samples preserved with guanidine or commercial buffers. These results suggest that this approach is feasible in the field and that preserved specimens might be better assayed molecularly when preserved in guanidine or commercial buffers.

Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.

Characterisation of three equine influenza A H3N8 viruses from Germany (2000 and 2002): Evidence for frozen evolution

Reported here are the results of antigenic and genetic characterisation of equine influenza strains causing local outbreaks reported to the Equine Diagnostic Centre in Berlin, Germany. In 2000, equine influenza virus was detected in a nasal swab from a non-vaccinated horse using a rapid diagnostic kit, but was not successfully isolated. Partial direct sequencing of the haemagglutinin (HA1) gene, indicated that the virus was a European lineage H3N8 subtype strain representative of strains isolated in several European countries during 2000. In 2002, two equine influenza viruses were isolated from nasal swabs both taken from unvaccinated horses with acute respiratory symptoms housed at the same stables. Antigenic characterisation using a panel of ferret antisera suggested that these isolates also belonged to the European lineage of H3N8 viruses. Analysis of deduced HA1 amino acid sequences confirmed that the HA1 of both isolates were identical and belonged to the European lineage. However, from phylogenetic analysis, both strains appeared to be more closely related to viruses isolated between 1989 and 1995 than to viruses isolated more recently in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. The importance of expanding current equine influenza surveillance efforts is emphasised.