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Since early 2024, a multistate outbreak of highly pathogenic avian influenza H5N1 has been affecting dairy cattle in the USA. The influenza viral RNA concentrations in milk make it an ideal matrix for surveillance purposes. However, viral RNA detection in multi-component fluids such as milk can be complex, and optimization of influenza detection methods is thus required. Raw bulk tank milk and mastitis milk samples were artificially contaminated with an avian influenza strain and subjected to five extraction methods. HCoV-229E and synthetic RNA were included as exogenous internal process controls. Given the high viral load usually observed in individual raw milk samples, four out of five tested methods would enable influenza detection in milk with normal texture, over a time window of at least 2 weeks post-onset of clinical signs. Nevertheless, sample dilution 1:3 in molecular transport medium prior to RNA extraction provided the best results for dilution of inhibitory substances and a good recovery rate of influenza RNA, that reached 12.5 ± 1.2% and 10.4 ± 3.8% in two independent experiments in bulk milk and 11.2 ± 3.6% and 10.0 ± 2.9% on two cohorts of mastitis milk samples. We have also shown compatibility of an influenza RT-qPCR system with synthetic RNA detection for simultaneous validation of the RNA extraction and RT-qPCR processes.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

The influenza A virus has been scarcely investigated in pigs in Africa, with rare detection prior to 2009. The spread of A(H1N1)pdm09 changed the epidemiology due to frequent human-to-swine transmission and the emergence of various new reassortants. This study therefore aimed at estimating the level of circulation and characterizing influenza A viruses at the interface between swine workers, who are crucial players in the inter-species transmission of influenza A viruses, and their animals in several farms in Nigeria, a hub for pig production in Africa. This cross-sectional study showed that 24.6% (58/236) of the pig serum samples collected in 2013-2014 had anti-influenza A antibodies in the absence of vaccination programs, but none of the pig swabs ( n = 1193) were positive according to RT-qPCR. Viral RNA was detected in 0.9% (2/229) of swine workers sampled at their place of work, and the strains were characterized as A(H1N1)pdm09 and seasonal A(H3N2). Our results highlight that more awareness of swine workers regarding the consequences of reverse zoonosis for animal and public health is warranted. Annual vaccination and the wearing of masks when experiencing influenza-like symptoms would help decrease influenza inter-species transmission, while surveillance should be adequately supported for early detection.

Background: Infections with SARS-CoV-2 have a pronounced impact on the gastrointestinal tract and its resident microbiome. Clear differences between severe cases of infection and healthy individuals have been reported, including the loss of commensal taxa. We aimed to understand if microbiome alterations including functional shifts are unique to severe cases or a common effect of COVID-19. We used high-resolution systematic multi-omic analyses to profile the gut microbiome in asymptomatic-to-moderate COVID-19 individuals compared to a control group., Results: We found a striking increase in the overall abundance and expression of both virulence factors and antimicrobial resistance genes in COVID-19. Importantly, these genes are encoded and expressed by commensal taxa from families such as Acidaminococcaceae and Erysipelatoclostridiaceae, which we found to be enriched in COVID-19-positive individuals. We also found an enrichment in the expression of a betaherpesvirus and rotavirus C genes in COVID-19-positive individuals compared to healthy controls., Conclusions: Our analyses identified an altered and increased infective competence of the gut microbiome in COVID-19 patients. Video Abstract., (© 2023. The Author(s).)

The COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been plaguing the world since late 2019/early 2020 and has changed the way we function as a society, halting both economic and social activities worldwide. Classrooms, offices, restaurants, public transport, and other enclosed spaces that typically gather large groups of people indoors, and are considered focal points for the spread of the virus. For society to be able to go "back to normal", it is crucial to keep these places open and functioning. An understanding of the transmission modes occurring in these contexts is essential to set up effective infection control strategies. This understanding was made using a systematic review, according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement (PRISMA) 2020 guidelines. We analyze the different parameters influencing airborne transmission indoors, the mathematical models proposed to understand it, and discuss how we can act on these parameters. Methods to judge infection risks through the analysis of the indoor air quality are described. Various mitigation measures are listed, and their efficiency, feasibility, and acceptability are ranked by a panel of experts in the field. Thus, effective ventilation procedures controlled by CO 2 -monitoring, continued mask wearing, and a strategic control of room occupancy, among other measures, are put forth to enable a safe return to these essential places.

Surveillance of hepatitis E virus (HEV) in risk groups is an important strategy to monitor its circulation pattern and to timely detect changes thereof. The aims of this cross-sectional study were to estimate the prevalence of HEV infections in pigs and humans from different regions of the country, to identify risk factors for increasing anti-HEV IgG prevalence and to characterize HEV strains. The presence of anti-HEV antibodies was assessed by commercial ELISA in serum samples from the general population, farm and slaughterhouse employees, as well as pigs sampled in the three regions of Cuba from February to September 2016. Overall, individuals with occupational exposure to swine or swine products (70/248, 28.2%) were 4 times more likely to be seropositive compared to the general population (25/285, 8.7%; OR: 4.18; p < .001). Within the risk group, risk factors included age, number of years working in a professional activity with direct exposure to swine, geographic region and distance between residence and closest professional swine setting, while wearing gloves had a protective effect. Prevalence of total anti-HEV antibodies in swine was 88.2% (165/187) and HEV RNA was detected by real-time RT-PCR in 9.2% (16/173) swine stools. All HEV strains sequenced clustered within genotype 3. Some strains clearly belonged to subtype 3a, while another group of strains was related with subtypes 3b and 3 k but partial HEV sequences did not allow unequivocal subtype assignment. These findings suggest that the high HEV exposure in Cuban individuals with swine-related occupations could be due to enzootic HEV in certain regions, direct contact with infectious animals or their products as well as environmental contamination., (© 2022 Wiley-VCH GmbH.)

Introduction: Children requiring multiple blood transfusions are at high risk of transfusion-transmissible infections (TTIs). Lao People's Democratic Republic is a low-resource setting where donor blood screening faces challenges. This study aimed to determine the burden of TTIs in children in Vientiane Capital., Methods: 300 children with transfusion history and 300 controls were recruited. In addition, 49 newly diagnosed transfusion recipients were followed for up to 12 months. Serum was tested for hepatitis B surface antigen and IgG antibodies against parvovirus B19, hepatitis B, C and E viruses., Results: The patients had a similar prevalence of anti-hepatitis B core antibodies (56; 18.7%) and hepatitis B surface antigen (8; 2.7%) as the controls (58; 19.3% and 9; 3.0%, respectively). However, there was a higher prevalence of an antibody profile suggestive of hepatitis B vaccination (anti-hepatitis B surface antibody positive/anti-hepatitis B core antibody negative) in the transfused group (140/299; 46.8%) than in controls (77/300; 25.7%, p<0.01). All other markers were similar in the patients and controls or higher in the controls: anti-hepatitis C virus (2.7% and 3.3%, p=0.6), anti-hepatitis E virus (7.5% and 12.7%, p=0.006) and anti-parvovirus B19 (2.4% and 8.5%, p=0.001). The longitudinal cohort did not show an increase in any marker over time., Conclusion: Our results suggest no significant role of TTIs in Lao children. The higher prevalence of the hepatitis B vaccination profile in transfusion recipients showed that recommendations to vaccinate before commencing transfusions is at least partially implemented, although there is room for improvement., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Influenza D virus (IDV) is an emerging influenza virus that was isolated for the first time in 2011 in the USA from swine with respiratory illness. Since then, IDV has been detected worldwide in different animal species, and it was also reported in humans. Molecular epidemiological studies revealed the circulation of two major clades, named D/OK and D/660. Additional divergent clades have been described but have been limited to specific geographic areas (i.e. Japan and California). In Europe, IDV was detected for the first time in France in 2012 and subsequently also in Italy, Luxembourg, Ireland, the UK, Switzerland, and Denmark. To understand the time of introduction and the evolutionary dynamics of IDV on the continent, molecular screening of bovine and swine clinical samples was carried out in different European countries, and phylogenetic analyses were performed on all available and newly generated sequences. Until recently, D/OK was the only clade detected in this area. Starting from 2019, an increase in D/660 clade detections was observed, accompanied by an increase in the overall viral genetic diversity and genetic reassortments. The time to the most recent common ancestor (tMRCA) of all existing IDV sequences was estimated as 1995-16 years before its discovery, indicating that the virus could have started its global spread in this time frame. Despite the D/OK and D/660 clades having a similar mean tMRCA (2007), the mean tMRCA for European D/OK sequences was estimated as January 2013 compared to July 2014 for European D/660 sequences. This indicated that the two clades were likely introduced on the European continent at different time points, as confirmed by virological screening findings. The mean nucleotide substitution rate of the hemagglutinin-esterase-fusion (HEF) glycoprotein segment was estimated as 1.403 × 10 -3  substitutions/site/year, which is significantly higher than the one of the HEF of human influenza C virus ( P  < 0.0001). IDV genetic drift, the introduction of new clades on the continent, and multiple reassortment patterns shape the increasing viral diversity observed in the last years. Its elevated substitution rate, diffusion in various animal species, and the growing evidence pointing towards zoonotic potential justify continuous surveillance of this emerging influenza virus., (© The Author(s) 2022. Published by Oxford University Press.)

SARS-CoV-2 variants raise concern because of their high transmissibility and their ability to evade neutralizing antibodies elicited by prior infection or by vaccination. Here, we compared the neutralizing abilities of sera from 70 unvaccinated COVID-19 patients infected before the emergence of variants of concern (VOCs) and of 16 vaccine breakthrough infection (BTI) cases infected with Gamma or Delta against the ancestral B.1 strain, the Gamma, Delta and Omicron BA.1 VOCs using live virus. We further determined antibody levels against the Nucleocapsid (N) and full Spike proteins, the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the Spike protein. Convalescent sera featured considerable variability in the neutralization of B.1 and in the cross-neutralization of different strains. Their neutralizing capacity moderately correlated with antibody levels against the Spike protein and the RBD. All but one convalescent serum failed to neutralize Omicron BA.1. Overall, convalescent sera from patients with moderate disease had higher antibody levels and displayed a higher neutralizing ability against all strains than patients with mild or severe forms of the disease. The sera from BTI cases fell into one of two categories: half the sera had a high neutralizing activity against the ancestral B.1 strain as well as against the infecting strain, while the other half had no or a very low neutralizing activity against all strains. Although antibody levels against the spike protein and the RBD were lower in BTI sera than in unvaccinated convalescent sera, most neutralizing sera also retained partial neutralizing activity against Omicron BA.1, suggestive of a better cross-neutralization and higher affinity of vaccine-elicited antibodies over virus-induced antibodies. Accordingly, the IC50: antibody level ratios were comparable for BTI and convalescent sera, but remained lower in the neutralizing convalescent sera from patients with moderate disease than in BTI sera. The neutralizing activity of BTI sera was strongly correlated with antibodies against the Spike protein and the RBD. Together, these findings highlight qualitative differences in antibody responses elicited by infection in vaccinated and unvaccinated individuals. They further indicate that breakthrough infection with a pre-Omicron variant boosts immunity and induces cross-neutralizing antibodies against different strains, including Omicron BA.1.

Background: Endemic circulation of human-specific hepatitis E virus (HEV) genotypes 1 and 2 may occult the importance of sporadic zoonotic HEV transmissions in Africa. Increasing numbers of studies reporting anti-HEV antibodies in cattle and the discovery of infectious HEV in cow milk has raised public health concern, but cattle exposure has seldom been investigated in Africa., Objectives: This study aimed at investigating the role of cows in the epidemiology of HEV in Burkina Faso and farmers habits in terms of dairy product consumption as a prerequisite to estimate the risk of transmission to humans., Methods: Sera from 475 cattle and 192 pigs were screened for the presence of anti-HEV antibodies while HEV RNA in swine stools was detected by reverse transcription polymerase chain reaction. Data on mixed farming, dairy product consumption and selling habits were gathered through questionnaires., Results: The overall seroprevalence in cattle was 5.1% and herd seroprevalence reached 32.4% (11/34). Herd seropositivity was not associated with husbandry practice or presence of rabbits on the farms. However, herd seropositivity was associated with on-site presence of pigs, 80.7% of which had anti-HEV antibodies. The majority of farmers reported to preferentially consume raw milk based dairy products., Conclusions: Concomitant presence of pigs on cattle farms constitutes a risk factor for HEV exposure of cattle. However, the risk of HEV infections associated with raw cow dairy product consumption is currently considered as low., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Korean Society of Veterinary Science.)

We detected Usutu virus in a dead Eurasian blackbird (Turdus merula) in Luxembourg in September 2020. The strain clustered within the Africa 3.1 lineage identified in Western Europe since 2016. Our results suggest maintenance of the virus in Europe despite little reporting during 2019-2020, rather than a new introduction.

While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral cellular or soluble immune features in a longitudinal cohort of 63 mild and 15 hospitalized patients versus 14 asymptomatic and 26 household controls. We observe a transient increase of IP10/CXCL10 and interferon-β levels, coordinated responses of dominant SARS-CoV-2-specific CD4 and fewer CD8 T cells, and various antigen-presenting and antibody-secreting cells in mild patients within 3 days of PCR diagnosis. The frequency of key innate immune cells and their functional marker expression are impaired in hospitalized patients at day 1 of inclusion. T cell and dendritic cell responses at day 1 are highly predictive for SARS-CoV-2-specific antibody responses after 3 weeks in mild but not hospitalized patients. Our systematic analysis reveals a combinatorial picture and trajectory of various arms of the highly coordinated early-stage immune responses in mild COVID-19 patients., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The influenza D virus (IDV) was first identified and characterized in 2011. Considering the virus' zoonotic potential, its genome nature (segmented RNA virus), its worldwide circulation in livestock and its role in bovine respiratory disease, an increased interest is given to IDV. However, few data are available on drivers of emergence of IDV. We first listed fifty possible drivers of emergence of IDV in ruminants and swine. As recently carried out for COVID-19 in pets (Transboundary and Emerging Diseases, 2020), a scoring system was developed per driver and scientific experts (N = 28) were elicited to (a) allocate a score to each driver, (b) weight the drivers' scores within each domain and (c) weight the different domains among themselves. An overall weighted score was calculated per driver, and drivers were ranked in decreasing order. Drivers with comparable likelihoods to play a role in the emergence of IDV in ruminants and swine in Europe were grouped using a regression tree analysis. Finally, the robustness of the expert elicitation was verified. Eight drivers were ranked with the highest probability to play a key role in the emergence of IDV: current species specificity of the causing agent of the disease; influence of (il)legal movements of live animals (ruminants, swine) from neighbouring/European Union member states and from third countries for the disease to (re-)emerge in a given country; detection of emergence; current knowledge of the pathogen; vaccine availability; animal density; and transport vehicles of live animals. As there is still limited scientific knowledge on the topic, expert elicitation of knowledge and multi-criteria decision analysis, in addition to clustering and sensitivity analyses, are very important to prioritize future studies, starting from the top eight drivers. The present methodology could be applied to other emerging animal diseases., (© 2020 Wiley-VCH GmbH.)

Respiratory infections are one of the most frequent reasons for medical consultations in children. In low resource settings such as in Lao People's Democratic Republic, knowledge gaps and the dearth of laboratory capacity to support differential diagnosis may contribute to antibiotic overuse. We studied the etiology, temporal trends, and genetic diversity of viral respiratory infections in children to provide evidence for prevention and treatment guidelines. From September 2014 to October 2015, throat swabs and nasopharyngeal aspirates from 445 children under 10 years old with symptoms of acute respiratory infection were collected at the Children Hospital in Vientiane. Rapid antigen tests were performed for influenza A and B and respiratory syncytial virus. Real-time reverse-transcription polymerase chain reactions (RT-PCRs) were performed to detect 16 viruses. Influenza infections were detected with a higher sensitivity using PCR than with the rapid antigen test. By RT-PCR screening, at least one pathogen could be identified for 71.7% of cases. Human rhinoviruses were most frequently detected (29.9%), followed by influenza A and B viruses combined (15.9%). We identify and discuss the seasonality of some of the infections. Altogether these data provide a detailed characterization of respiratory pathogens in Lao children and we provide recommendations for vaccination and further studies., (© 2021 The Authors. Journal of Medical Virology Published by Wiley Periodicals LLC.)

Foot and mouth disease (FMD) virus remains enzootic in Lao People's Democratic Republic (Lao PDR) due to insufficient control measures, including low vaccination coverage. We assessed virus epidemiology and evaluated knowledge, attitude and practice of Lao farmers and animal health stakeholders towards FMD to support politics in devising evidence-based control measures. Sera were collected from 972 domestic ruminants in three provinces of Lao PDR: Vientiane Capital, Vientiane and Xiengkhouang provinces. Seroprevalence of antibodies directed against non-structural proteins of FMD virus was assessed using a commercial ELISA. Positive sera were further characterized by detecting antibodies directed against the structural proteins of FMD serotypes O, A and Asia 1. Information about farm management, biosecurity practices, livestock trade, and past FMD outbreaks was obtained. Overall 35% (340/972) of the ruminants had antibodies against FMD virus with a similar seroprevalence in each province. Seroprevalence depended significantly on the ruminant species (p < .001): 61% of buffaloes, but only 41% of cattle and 15% of goats were seropositive. While antibodies against FMD serotype Asia 1 were absent, 87% (297/340) of the seropositive animals had antibodies against FMD serotype O and 32% (110/340) against FMD serotype A. Many seropositive animals (31%) had antibodies against both serotypes O and A. The majority of the farmers could name the symptoms of FMD and the susceptible animal species. Although many had likely observed FMD outbreaks in their herd and were aware of FMD vaccines, only few vaccinated their animals. This study confirms that FMD remains enzootic in at least three provinces of Lao PDR where vaccination coverage is low. It also shows the relevance of nationwide active and passive disease surveillance, as well as of vaccination campaigns using bivalent FMD vaccines and targeting all susceptible animal species., (© 2020 Wiley-VCH GmbH.)

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field., (© 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Asymptomatic individuals, called "silent spreaders" spread SARS-CoV-2 efficiently and have complicated control of the ongoing COVID-19 pandemic. As seen in previous influenza pandemics, socioeconomic and life-trajectory factors are important in disease progression and outcome. The demographics of the asymptomatic SARS-CoV-2 carriers are unknown. We used the CON-VINCE cohort of healthy, asymptomatic, and oligosymptomatic individuals that is statistically representative of the overall population of Luxembourg for age, gender, and residency to characterise this population. Gender (male), not smoking, and exposure to early-life or adult traumatic experiences increased the risk of IgA seropositivity, and the risk associated with early-life exposure was a dose-dependent metric, while some other known comorbidities of active COVID-19 do not impact it. As prior exposure to adversity is associated with negative psychobiological reactions to external stressors, we recorded psychological wellbeing during the study period. Exposure to traumatic events or concurrent autoimmune or rheumatic disease were associated with a worse evolution of anxiety and depressive symptoms throughout the lockdown period. The unique demographic profile of the "silent spreaders" highlights the role that the early-life period plays in determining our lifelong health trajectory and provides evidence that the developmental origins of health and disease is applicable to infectious diseases.

Background and Aims: Respiratory syncytial virus (RSV) is one of the main viral pathogens causing acute respiratory infections in children under 5 years of age but has seldom been studied in Central African Republic (CAF). Taking advantage of the national influenza surveillance network in CAF, this study aimed at providing the first insights into RSV prevalence and seasonality over 4 years of surveillance and the clinical manifestations of RSV in this population in CAF., Methods: A total of 3903 children under 5 years matching the influenza-like illness (ILI, 68.5%) or severe acute respiratory infection (SARI, 31.5%) case definitions were recruited from January 2015 to December 2018. The presence of RSV viral RNA in nasopharyngeal samples was assessed by RT-PCR, followed by RSV-A and RSV-B typing and Sanger sequencing on a subset of samples. Phylogenetic analyses were carried on partial G protein sequences. Associations between RSV and demographic or clinical manifestations were investigated by statistical analyses., Results: RSV prevalence was significantly higher in infants <6 months (13.4%), in hospitalized children (13.3% vs 5.5%) and in male patients (9.5% vs 6.4%). An overall prevalence of RSV of 8.0% in the period of 2015 to 2018 was shown, with significant annual (6.4%-10.6%) and seasonal (12.7% in rainy season vs 3.0% in dry season) fluctuations. While RSV seasons in 2015, 2016, and 2018 were relatively similar, 2017 showed deviations from the overall patterns with significantly higher RSV circulation and an outbreak peak 3 to 5 months earlier. Concomitant circulation of RSV-A and RSV-B with an alternating predominance of RSV-A and RSV-B strains and temporal RSV-A genotype replacement from NA1 to ON1 was observed., Conclusion: This study represents the first in-depth epidemiological analysis of RSV in CAF and provides first insights into RSV genetic diversity and seasonality in the country., Competing Interests: The authors declare that they have no conflict of interests., (© 2021 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Background: To accompany the lifting of COVID-19 lockdown measures, Luxembourg implemented a mass screening (MS) programme. The first phase coincided with an early summer epidemic wave in 2020., Methods: rRT-PCR-based screening for SARS-CoV-2 was performed by pooling of samples. The infrastructure allowed the testing of the entire resident and cross-border worker populations. The strategy relied on social connectivity within different activity sectors. Invitation frequencies were tactically increased in sectors and regions with higher prevalence. The results were analysed alongside contact tracing data., Findings: The voluntary programme covered 49% of the resident and 22% of the cross-border worker populations. It identified 850 index cases with an additional 249 cases from contact tracing. Over-representation was observed in the services, hospitality and construction sectors alongside regional differences. Asymptomatic cases had a significant but lower secondary attack rate when compared to symptomatic individuals. Based on simulations using an agent-based SEIR model, the total number of expected cases would have been 42·9% (90% CI [-0·3, 96·7]) higher without MS. Mandatory participation would have resulted in a further difference of 39·7% [19·6, 59·2]., Interpretation: Strategic and tactical MS allows the suppression of epidemic dynamics. Asymptomatic carriers represent a significant risk for transmission. Containment of future outbreaks will depend on early testing in sectors and regions. Higher participation rates must be assured through targeted incentivisation and recurrent invitation., Funding: This project was funded by the Luxembourg Ministries of Higher Education and Research, and Health., Competing Interests: All authors report grants from Luxembourg Ministry of Higher Education and Research, and Ministry of Health during the conduct of the study. Dr. Rodrigues reports working for the Ministry of Higher Education and Research as a public servant, during the conduct of the study. Dr. Snoeck reports that Fast Track Diagnostics provided a few SARS-CoV-2 rRT-PCR kits (RUO) free of charge at the time of evaluation of different commercial assays to be procured by the Luxembourg Government in order to do the mass screening intervention in the country., (© 2021 The Author(s).)

Influenza D virus (IDV) is a novel orthomyxovirus that was first isolated in 2011 in the United States from a swine exhibiting influenza-like disease. To date, its detection is extended to all continents and in a broad host range: IDV is circulating in cattle, swine, feral swine, camelids, small ruminants and horses. Evidence also suggests a possible species jump to humans, underlining the issue of zoonotic potential. In Europe, serological investigations in cattle have partially allowed the understanding of the virus diffusion in different countries such as Italy, France, Luxembourg and Ireland. The infection is widespread in cattle but limited in other investigated species, consolidating the assumption of cattle as IDV primary host. We hypothesize that commercial livestock trade could play a role in the observed differences in IDV seroprevalence among these areas. Indeed, the overall level of exposure in cattle and swine in destination countries (e.g. Italy) is higher than in origin countries (e.g. France), leading to the hypothesis of a viral shedding following the transportation of young cattle abroad and thus contributing to larger diffusion at countries of destination. IDV large geographic circulation in cattle from Northern to more Southern European countries also supports the hypothesis of a viral spread through livestock trade. This review summarizes available data on IDV seroprevalence in Europe collected so far and integrates unpublished data from IDV European surveillance framework of the last decade. In addition, the possible role of livestock trade and biosecurity measures in this pathogen's spread is discussed., (© 2020 Wiley-VCH GmbH.)

Despite improvement of human and avian influenza surveillance, swine influenza surveillance in sub-Saharan Africa is scarce and pandemic preparedness is still deemed inadequate, including in Burkina Faso. This cross-sectional study therefore aimed to investigate the (past) exposure of pigs to influenza A viruses. Practices of people with occupational contacts with pigs and their knowledge on influenza A were investigated in order to formulate future prevention guidelines. In 2016-2017, pig nasopharyngeal swabs and sera were collected and screened for the presence of influenza virus by RT-PCR or of anti-influenza antibodies by competitive ELISA. Seropositive samples were further characterized in virus microneutralization assays against human and swine H1N1 virus strains. Nasopharyngeal swabs were obtained from people with occupational contact with pigs and screened similarly. Demographic data as well as practices related to their profession were recorded. No influenza A virus was detected in nasopharyngeal swabs in humans (n = 358) or in pigs (n = 600). Seroprevalence in pigs reached 6.8 % (41/600) and seropositive animals were found in 50.0 % of extensive settings (10/20) and 19.0 % of (semi-)intensive farms (4/21). All positive sera reacted against the pandemic H1N1/2009 strain, while seropositivity against two Eurasian avian-like and one American swine H1N1 strains and individual titers were lower. These results suggested exposure to pandemic H1N1/2009 virus and cross-reactivity to other H1N1 strains. Farmers with higher frequency of contact to pigs, absence of protective equipment and lack of knowledge on zoonoses are likely key players in driving human-to-swine virus transmission., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Wild birds are natural hosts of multiple microbial agents, including a wide diversity of coronaviruses. Here we describe a pan-Coronavirus detection RT-PCR method to identify those viruses regardless of the coronavirus genus or nature of the specimen. We also describe a protocol using high-throughput sequencing technologies to obtain their entire genome, which overcomes the inherent difficulties of wild bird coronavirus sequencing, that is, their genetic diversity and the lack of virus isolation methods.

In backyard farms of Lao People's Democratic Republic, mixed-species rearing of poultry is a breeding-ground for cross-species transmission. Here, the epidemiology of viruses circulating among backyard poultry in Vientiane Province was assessed to guide future control strategies. Oral/tracheal and cloacal swabs, collected from 605 poultry (308 ducks, 297 chickens) between 2011 and 2015, were screened by PCR for Newcastle disease virus (NDV), coronavirus (CoV) and chicken anaemia virus (CAV). Chicken sera were screened for anti-NDV antibodies by ELISA. Statistical and phylogenetic analyses revealed transmission patterns and relationships. Closely related strains co-circulated in chickens and ducks. While CoV RNA was detected in oral/tracheal swabs of 9.3% of the chickens and 2.4% of the ducks, rates were higher in faecal swabs of both species (27.3% and 48.2%). RNA of infectious bronchitis virus (IBV) and duck CoV was found in faecal swabs of chickens (19.7% and 7.1%) and ducks (4.1% and 44.1%). Moreover, DNA of the generally chicken-specific CAV was detected in oral/tracheal swabs of chickens (18.1%) and, sporadically, of ducks (2.4%). Despite serological evidence of NDV circulation or vaccination (86.9%), NDV RNA was not detected. We found a high prevalence and indication for cross-species transmission of different CoV strains in backyard poultry. Interestingly, ducks served as biological, or at least mechanical, carriers of viral strains closely related not only to IBV, but also to CAV. Bird containment and poultry species separation could be first steps to avoid cross-species transmission and emergence of novel strains with broad host range and enhanced pathogenicity. RESEARCH HIGHLIGHTS High rates of avian viruses were detected by PCR in backyard poultry from Lao PDR. Diverse coronavirus and chicken anemia virus strains co-circulated. Phylogenetic analyses suggested virus transmission between chickens and ducks. Serological evidence of Newcastle disease was found, but viral RNA was not detected.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Epidemiological characteristics of hepatitis E Virus (HEV) differ due to the vast spatial heterogeneity in sanitation status, dietary habits and extent of exposure to animals. Although HEV infections are mostly asymptomatic, prognosis is poor for patients with compromised immune systems or pre-existing liver disease and during pregnancy. In developing countries, low hygienic standards and close human-animal interactions at farms and slaughterhouses likely favour zoonotic transmission of the virus. In this cross-sectional study from Lao People's Democratic Republic, anti-HEV IgG was detected by ELISA in 54.0% (136/252) of slaughter pigs, in 41.0% (57/139) of professionals exposed to pigs and in 18.1% of the nonrisk controls (38/210). We show that people occupationally exposed to pigs are at higher risk of infection (p < 0.001). In particular, contact to young piglets as compared to contact to the older slaughter pigs was a major risk factor (p = 0.011). Besides, consumption of bottled water significantly reduced the risk of infection (p = 0.018). In conclusion, we show that in Lao PDR, the high endemicity of HEV, the inadequate implementation of hygiene measures and the limited access to safe water jeopardize the health of professionals exposed to pigs., (© 2018 Blackwell Verlag GmbH.)

In Lao People's Democratic Republic (PDR), acute respiratory infections overburden the health care system, but viral etiology, genetic diversity, and seasonality, especially in light of the introduction of influenza vaccination in the country, are poorly understood. From August 2010 to April 2011, 309 outpatients were recruited at the Luang Prabang Provincial Hospital covering highland Lao communities. Nasopharyngeal swabs were screened for the presence of 13 respiratory viruses. At least one virus was detected in 69.6% and dual/triple viral infections in 12.9%/1.9% of the patients. Influenza A and B viruses combined were the most frequently detected pathogens, followed by human adenovirus and respiratory syncytial virus (RSV). The other viruses were detected in less than 10% of the patients. Phylogenetic analyses on a representative set of RSV strains revealed that, while otherwise very rare, the RSV-B CB1/THB genotype cocirculated with other common genotypes. A single wave of influenza virus and RSV activity was observed during the rainy season, providing further support to influenza vaccination before the onset of the rains. This study provides recommendations for influenza vaccination that still needs optimization and highlights the need for revised guidelines for treatment and prevention of respiratory infections in Lao PDR, as well as for increased surveillance efforts., (© 2018 Wiley Periodicals, Inc.)

Although pigs are the main reservoir, ruminants have also been shown to be susceptible to hepatitis E virus (HEV). We investigated zoonotic transmission of HEV in rural settings of Lao People's Democratic Republic (Lao PDR) where humans are in close contacts with ruminants and where pigs are rare. Villagers with (n = 171, risk group) and without (n = 155, control group) cattle were recruited in seven villages in Vientiane Capital. Owners of pigs were excluded. Blood, as well as information on socio-demographics, animal contact, dietary habits and awareness of zoonoses were collected to assess risk factors. Blood and rectal swabs were collected from cattle (n = 173) and other ruminants (27 goat, 5 buffaloes) to measure anti-HEV antibody and virus prevalence. A similar anti-HEV antibody seroprevalence was found in cattle (6.8%) and other ruminants (8%). HEV RNA was detected in none of the animal rectal swabs and human sera. Anti-HEV IgG seroprevalence was higher in cattle farmers than in the control group (59.1% vs. 43.9%, p = 0.008) and increased significantly with age. Other risk factors included male gender, close contact with cattle and consumption of undercooked meat. We find that HEV is highly endemic in rural Laos and provide first evidence that HEV circulates in free-roaming ruminants with open access to village water sources. Despite some awareness about hygiene, villagers are likely constantly exposed to zoonotic diseases by dietary and lifestyle habits. Cattle farmers had a higher risk of HEV infection than other villagers. Our study highlights the need to raise the awareness of the rural population about water- and food-borne pathogens, and about the role of cattle as a possible source of infection. The knowledge gained on local risk factors and husbandry conditions should guide future awareness raising campaigns and promote appropriate hygienic measures including handwashing and the consumption of safe food and water., (© 2018 The Authors Zoonoses and Public Health Published by Blackwell Verlag GmbH.)

Background: The importance of influenza viruses in respiratory infections in sub-Saharan Africa has been historically overlooked, including in Burkina Faso., Objectives: This study therefore aimed at evaluating the prevalence and seasonal occurrence of influenza viruses in children under 5 years old, at risk of influenza-related complications, presenting with influenza-like illness (ILI) or severe acute respiratory infection (SARI). The study also aimed at identifying the periods with increased influenza transmission for vaccination recommendations in Burkina Faso., Methods: From January 2014 to December 2015, ILI and SARI (2015 only) patients were recruited in six healthcare centers in Burkina Faso. Influenza A and B molecular detection and subtyping were performed. Clade clustering of a subset of A(H1N1)pdm09 and A(H3N2) strains was deduced by performing phylogenetic analyses on hemagglutinin gene sequences. Weekly surveillance data from FluNet (2011-2013; 2016) and this study (2014-2015) were used to identify periods of increased influenza activity., Results: Influenza A and B viruses were detected in 15.1% (112 of 743) of ILI and 6.6% (12 of 181) of SARI patients. Overall, influenza A viruses were largely predominant (81 of 124, 65.3%), with 69.1% of A(H3N2) and 30.9% of A(H1N1)pdm09 strains. Four waves of increased transmission were identified in 2014-2015, each dominated by different influenza subtypes and clades. Between 2011 and 2016, periods of increased influenza activity varied in their frequency, duration, and timing., Conclusion: Influenza A and B viruses were detected in a substantial number of ILI and SARI cases in Burkina Faso. Vaccination in September-October would likely protect the highest number of patients., (© 2018 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Several infectious disease outbreaks with high mortality in humans have been attributed to viruses that are thought to have evolved from bat viruses. In this study from Luxembourg, the genetic diversity and epidemiology of paramyxoviruses and coronaviruses shed by the bat species Rhinolophus ferrumequinum and Myotis emarginatus were evaluated. Feces collection ( n = 624) was performed longitudinally in a mixed-species colony in 2015 and 2016. In addition, feces ( n = 254) were collected cross-sectionally from six Myotis emarginatus colonies in 2016. By use of degenerate primers in a nested format, overall prevalences of 1.1% (10/878) and 4.9% (43/878) were determined for paramyxoviruses and coronaviruses. Sequences of the partial RNA-dependent RNA polymerase and spike glycoprotein genes of coronaviruses, as well as sequences of the partial L gene of paramyxoviruses, were obtained. Novel paramyxovirus and Alphacoronavirus strains were identified in different Myotis emarginatus colonies, and severe acute respiratory syndrome (SARS)-related Betacoronavirus strains were shed by Rhinolophus ferrumequinum Logistic regression revealed that the level of Alphacoronavirus shedding was highest in July (odds ratio, 2.8; P < 0.01), probably due to periparturient stress. Phylogenetic analyses point to close virus-host coevolution, and the high genetic similarity of the study strains suggests that the Myotis emarginatus colonies in Luxembourg are socially connected. Most interestingly, we show that bats also host Betacoronavirus 1 strains. The high similarity of the spike gene sequences of these viruses with mammalian Betacoronavirus 1 strains may be of concern. Both the SARS-related and Betacoronavirus 1 strains detected in bats in Luxembourg may cross the species barrier after a host adaptation process. IMPORTANCE Bats are a natural reservoir of a number of zoonotic pathogens. Several severe outbreaks in humans (e.g., a Nipah virus outbreak in Malaysia in 1998, and the almost global spread of severe acute respiratory syndrome in 2003) have been caused by bat-borne viruses that were transmitted to humans mostly after virus adaptation (e.g., in intermediate animal hosts). Despite the indigenousness of bat species that host viruses with suspected zoonotic potential and despite the zoonotic transmission of European bat 1 lyssavirus in Luxembourg, knowledge about the diversity and epidemiology of bat viruses remains limited in this country. Moreover, in contrast to other European countries, bat viruses are currently not included in the national surveillance activities of this land-locked country. We suggest that this gap in disease surveillance should be addressed, since we show here that synanthropic bats host viruses that may be able to cross the species barrier., (Copyright © 2017 American Society for Microbiology.)

Background: Avian rotaviruses (RVs) cause gastrointestinal diseases of birds worldwide. However, prevalence, diversity, epidemiology and phylogeny of RVs remain largely under-investigated in Africa., Methods: Fecal samples from 349 birds (158 symptomatic, 107 asymptomatic and 84 birds without recorded health status) were screened by reverse transcription PCR to detect RV groups A and D (RVA and RVD). Partial gene sequences of VP4, VP6, VP7 and NSP4 for RVA, and of VP6 and VP7 for RVD were obtained and analyzed to infer phylogenetic relationship. Fisher's exact test and logistic regression were applied to identify factors potentially influencing virus shedding in chickens., Results: A high prevalence of RVA (36.1%; 126/349) and RVD (31.8%; 111/349) shedding was revealed in birds. In chickens, RV shedding was age-dependent and highest RVD shedding rates were found in commercial farms. No negative health effect could be shown, and RVA and RVD shedding was significantly more likely in asymptomatic chickens: RVA/RVD were detected in 51.9/48.1% of the asymptomatic chickens, compared to 18.9/29.7% of the symptomatic chickens (p < 0.001/p = 0.01). First RVA sequences were obtained from mallard ducks (Anas platyrhynchos) and guinea fowls (Numida meleagris). Phylogenetic analyses illustrated the high genetic diversity of RVA and RVD in Nigerian birds and suggested cross-species transmission of RVA, especially at live bird markets. Indeed, RVA strains highly similar to a recently published fox rotavirus (RVA/Fox-tc/ITA/288356/2011/G18P[17]) and distantly related to other avian RVs were detected in different bird species, including pigeons, ducks, guinea fowls, quails and chickens., Conclusion: This study provides new insights into epidemiology, diversity and classification of avian RVA and RVD in Nigeria. We show that cross-species transmission of host permissive RV strains occurs when different bird species are mixed.

Besides birds, pigs are another important reservoir of influenza A viruses that can be transmitted to human, as highlighted by the emergence and spread of the pandemic (H1N1) virus (pdm/09) in 2009. Surveillance in pigs is therefore necessary for public health and influenza pandemic preparedness. Nevertheless, there is a serious lack of data on influenza in Africa, especially in swine. We therefore collected serum samples from pigs in Nigeria (2009, 2012) and Cameroon (2011) in which the presence of anti-influenza A neutralizing antibodies was investigated. Our serological survey suggests that, before the 2009 pandemic, only rare swine and human H3N2 or human H1N1 infections occurred in Nigeria in swine. However, in 2011-2012, 27.4% of pigs in Nigeria and 5.6% in Cameroon had antibodies against H1N1 viruses. Higher antibody titres against pdm/09 suggested that pigs were exposed to this or a similar virus, either by multiple introductions or sustained circulation, and that reactivity against American and European swine H1N1 viruses resulted from cross-reaction., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In West and Central Africa, virulent Newcastle disease virus (NDV) strains of the recently identified genotypes XIV, XVII, and XVIII are enzootic in poultry, representing a considerable threat to the sector. The increasing number of reports of virulent strains in wild birds at least in other parts of the world raised the question of a potential role of wild birds in the spread of virulent NDV in sub-Saharan Africa as well. We investigated 1,723 asymptomatic birds sampled at live-bird markets and sites important for wild-bird conservation in Nigeria and 19 sick or dead wild birds in Côte d'Ivoire for NDV class I and II. Typical avirulent wild-type genotype I strains were found in wild waterfowl in wetlands in northeastern Nigeria. They were unrelated to vaccine strains, and the involvement of inter- or intracontinental migratory birds in their circulation in the region is suggested. Phylogenetic analyses also revealed that genotype VI strains found in pigeons, including some putative new subgenotype VIh and VIi strains, were introduced on multiple separate occasions in Nigeria. A single virulent genotype XVIII strain was found in a dead wild bird in Côte d'Ivoire, probably as a result of spillover from sick poultry. In conclusion, screening of wild birds and pigeons for NDV revealed the presence a variety of virulent and avirulent strains in West Africa but did not provide strong evidence that wild birds play an important role in the spread of virulent strains in the region.

Despite rampant Newcastle disease virus (NDV) outbreaks in Africa for decades, the information about the genetic characteristics of the virulent strains circulating in West and Central Africa is still scarce. In this study, 96 complete NDV fusion gene sequences were obtained from poultry sampled in Cameroon, Central African Republic, Côte d'Ivoire, and Nigeria between 2006 and 2011. Based on rational criteria recently proposed for the classification of NDV strains into classes, genotypes, and subgenotypes, we revisited the classification of virulent strains, in particular those from West and Central Africa, leading to their grouping into genotype XIV and newly defined genotypes XVII and XVIII, each with two subgenotypes. Phylogenetic analyses revealed that several (sub)genotypes are found in almost every country. In Cameroon, most strains were related to vaccine strains, but a single genotype XVII strain was also found. Only three highly similar genotype XVII strains were detected in Central African Republic. Subgenotypes XVIIa, XVIIIa, and XVIIIb cocirculated in Côte d'Ivoire, while subgenotypes XIVa, XIVb, XVIIa, XVIIb, and XVIIIb were found in Nigeria. While these genotypes are so far geographically restricted, local and international trade of domestic and exotic birds may lead to their spread beyond West and Central Africa. A high genetic diversity, mutations in important neutralizing epitopes paired with suboptimal vaccination, various levels of clinical responses of poultry and wild birds to virulent strains, strains with new cleavage sites, and other genetic modifications found in these genotypes tend to undermine and complicate NDV management in Africa.

Newcastle disease virus (NDV) is one of the most important viral diseases of birds. Wild birds constitute a natural reservoir of low-virulence viruses, while poultry are the main reservoir of virulent strains. Exchange of virus between these reservoirs represents a risk for both bird populations. Samples from wild and domestic birds collected between 2006 and 2010 in Luxembourg were analyzed for NDV. Three similar avirulent genotype I strains were found in ducks during consecutive years, suggesting that the virus may have survived and spread locally. However, separate introductions cannot be excluded, because no recent complete F gene sequences of genotype I from other European countries are available. Detection of vaccine-like strains in wild waterbirds suggested the spread of vaccine strains, despite the nonvaccination policy in Luxembourg. Among domestic birds, only one chicken was positive for a genotype II strain differing from the LaSota vaccine and exhibiting a so-far-unrecognized fusion protein cleavage site of predicted low virulence. Three genotype VI strains from pigeons were the only virulent strains found. The circulation of NDV in wild and free-ranging domestic birds warrants continuous surveillance because of increased concern that low-virulence wild-bird viruses could become more virulent in domestic populations.

Background: Although chicken anemia virus (CAV) has been detected on all continents, little is known about this virus in sub-Saharan Africa. This study aimed to detect and characterize CAV for the first time in Central African Republic and in Cameroon., Results: An overall flock seroprevalence of 36.7% was found in Central African Republic during the 2008-2010 period. Virus prevalences were 34.2% (2008), 14.3% (2009) and 10.4% (2010) in Central African Republic and 39% (2007) and 34.9% (2009) in Cameroon. CAV DNA was found in cloacal swabs of 76.9% of seropositive chickens, suggesting that these animals excreted the virus despite antibodies. On the basis of VP1 sequences, most of the strains in Central African Republic and Cameroon belonged to 9 distinct phylogenetic clusters at the nucleotide level and were not intermixed with strains from other continent. Several cases of mixed infections in flocks and individual chickens were identified., Conclusions: Our results suggest multiple introductions of CAV in each country that later spread and diverged locally. Mixed genotype infections together with the observation of CAV DNA in cloacal samples despite antibodies suggest a suboptimal protection by antibodies or virus persistence.

To investigate the presence and persistence of avian influenza virus in African birds, we monitored avian influenza in wild and domestic birds in two different regions in Nigeria. We found low-pathogenic avian influenza (LPAI) H5N2 viruses in three spur-winged geese (Plectropterus gambensis) in the Hadejia-Nguru wetlands. Phylogenetic analyses revealed that all of the genes, except the non-structural (NS) genes, of the LPAI H5N2 viruses were more closely related to genes recently found in wild and domestic birds in Europe. The NS genes formed a sister group to South African and Zambian NS genes. This suggested that the Nigerian LPAI H5N2 viruses found in wild birds were reassortants exhibiting an NS gene that circulated for at least 7 years in African birds and is part of the African influenza gene pool, and genes that were more recently introduced into Africa from Eurasia, most probably by intercontinental migratory birds. Interestingly the haemagglutinin and neuraminidase genes formed a sister branch to highly pathogenic avian influenza (HPAI) H5N2 strains found in the same wild bird species in the same wetland only 1 year earlier. However, they were not the closest known relatives of each other, suggesting that their presence in the wetland resulted from two separate introductions. The presence of LPAI H5N2 in wild birds in the Hadejia-Nguru wetlands, where wild birds and poultry occasionally mix, provides ample opportunity for infection across species boundaries, with the potential risk of generating HPAI viruses after extensive circulation in poultry.

Between 2002 and 2007, more than 1000 chickens from commercial farms, live bird markets and backyard farms in Nigeria and Niger were tested for the presence of the infectious bronchitis virus (IBV) genome. Phylogenetic analysis of full-length sequences of the spike 1 (S1) gene revealed a new genotype of IBV that we refer to as 'IBADAN'. The minimum genetic distance to the closest 'non-IBADAN' strains (UK/7/93 at the nucleotide level; H120 and M41 at the amino acid level) reached 24 and 32 % at the nucleotide and amino acid levels, respectively. The full genome of the IBADAN reference strain (NGA/A116E7/2006) had a genetic distance of 9.7-16.4 % at the nucleotide level with all available fully sequenced strains. As IBV S1 plays a major role in antigenicity, the antigenic relatedness of NGA/A116E7/2006 was compared with strains of other serotypes. NGA/A116E7/2006 did not cross-react with antisera against IT02, M41, D274, Connecticut or 793/B strains in virus neutralization assays. NGA/A116E7/2006 cross-reacted with the QX-like strain ITA/90254/2005 but only to a low level (antigenic relatedness of 33 %), suggesting that IBADAN also represents a new serotype. A comparison of S1 sequences identified several amino acids that may play a role in IBV antigenicity. Despite the absence of obvious clinical signs in poultry infected by IBADAN strains, it is important to test the cross-protection of current vaccine strains.

Forty-four Newcastle disease virus (NDV) strains, obtained between 2002 and 2007 from different poultry species in Nigeria, Niger, Burkina Faso and Cameroon, were phylogenetically analysed based on partial F sequences. Lineage 2 viruses were genetically identical or similar to the locally used LaSota vaccine strain and were mostly detected in commercial farms. Lineage 1, 3 and 4 strains were only sporadically found, and their origin was less clear. Twenty-one strains from backyard farms and live bird markets formed three new clusters within lineage 5, tentatively named 5f, 5g and 5h. All of these strains were predicted to be virulent based on their F protein cleavage site sequence. Minimal genetic distances between new and previously established sublineages ranged from 9.4 to 15.9%, and minimal distances between the new sublineages were 11.5 to 17.3%. Their high genetic diversity and their presence in three different Sub-Saharan countries suggest that these new sublineages represent the NDV variants indigenous to West Africa.