Highly Pathogenic Avian Influenza H5N1 Research Articles

Estimating the introduction time of highly pathogenic avian influenza into poultry flocks

The estimation of farm-specific time windows for the introduction of highly-pathogenic avian influenza (HPAI) virus can be used to increase the efficiency of disease control measures such as contact tracing and may help to identify risk factors for virus introduction. The aims of this research are to (1) develop and test an accurate approach for estimating farm-specific virus introduction windows and (2) evaluate this approach by applying it to 11 outbreaks of HPAI (H5N8) on Dutch commercial poultry farms during the years 2014 and 2016. We used a stochastic simulation model with susceptible, infectious and recovered/removed disease stages to generate distributions for the period from virus introduction to detection. The model was parameterized using data from the literature, except for the within-flock transmission rate, which was estimated from disease-induced mortality data using two newly developed methods that describe HPAI outbreaks using either a deterministic model (A) or a stochastic approach (B). Model testing using simulated outbreaks showed that both method A and B performed well. Application to field data showed that method A could be successfully applied to 8 out of 11 HPAI H5N8 outbreaks and is the most generally applicable one, when data on disease-induced mortality is scarce.

Investigation of Avian Influenza Viruses (H9N2-H5nx) in Pigeons during Highly Pathogenic Avian Influenza Outbreaks in Iran, in 2016.

Avian influenza (AI) virus (H9N2 and H5 subtypes) infections in birds cause major concerns around the world. The majority of the avian species, such as domestic, pet, and wild birds, are natural and experimental hosts of avian influenza viruses. There are global concerns about members of the Columbidae family, namely pigeons or doves, for their role as the potential interspecies bridge in influenza A viruses ecology. The acquired scientific data in this regard is still not clear since there are doubts about whether or not they transmit viruses between susceptible populations, and spread viruses among farms during outbreaks. To monitor H5 and H9 influenza virus infection status in the rural, backyard, and domestic birds, an annual active surveillance program was performed from September to October 2016. In December 2016, an outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N8 was detected in a layer farm in Tehran province, Iran. The present research was conducted to study H9N2 or H5 infections in pigeons within HPAI H5N8 2016 outbreaks and annual national AI surveillance in Iran. For this purpose, cloacal swabs and tissue samples (trachea, lung, brain, liver, heart, pancreas, and cecal tonsil) were collected and examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) method and virus isolation. Results of the tests performed on the swab and tissue samples were negative for H5 nor H9N2 viruses. The samples in real-time RT-PCR that after three passages still showed negative results in HA and molecular tests were considered negative. Moreover, the Newcastle disease virus was isolated in most of the samples taken from dead pigeons, after inoculation in embryonated chicken eggs.

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014-2018: Clinical signs and mortality.

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg‐producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017–2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.

Highly pathogenic H5N6 avian influenza virus subtype clade 2.3.4.4 indigenous in South Korea

The outbreaks of the highly pathogenic avian influenza (HPAI) in 2016–2017 and 2017–2018, caused by novel reassortant clade 2.3.4.4 H5N6 viruses, resulted in the loss of one billion birds in South Korea. Here, we characterized the H5N6 viruses isolated from wild birds in South Korea from December 2017 to August 2019 by next-generation sequencing. The results indicated that clade 2.3.4.4 H5N6 viruses isolated in 2017 and 2019 shared almost identical nucleotide sequences with the HPAI H5N6 viruses from 2016 in South Korea. This repeated detection of evolutionarily identical H5N6 viruses in same region for more than three years may suggest indigenization of the HPAI H5N6 virus in South Korea. Phylogenetic analysis demonstrated that the clade 2.3.4.4 H5N6 viruses isolated in 2017 and 2019 were evolutionarily distinct from those isolated in 2018. Molecular analysis revealed that the H5N6 viruses isolated in 2017 and 2019 had features associated with an increased risk of human infection (e.g. a deletion at position 133 of HA and glutamic acid residue at position 92 of NS1). Overall, these genomic features of HPAI H5N6 viruses highlight the need for continuous monitoring of avian influenza viruses in wild migratory birds as well as in domestic birds.

Immunization with the H5N1 Recombinant Vaccine Candidate Induces High Protection in Chickens against Vietnamese Highly Pathogenic Avian Influenza Virus Strains.

Vietnam is one of the countries most affected worldwide by the highly pathogenic avian influenza (HPAI) virus, which caused enormous economic loss and posed threats to public health. Over nearly two decades, with the antigenic changes in the diversified H5Ny viruses, the limited protective efficacy of the available vaccines was encountered. Therefore, it is necessary to approach a technology platform for the country to accelerate vaccine production that enables quick response to new influenza subtypes. This study utilized a powerful reverse genetics technique to successfully generate a recombinant H5N1 vaccine strain (designated as IBT-RG02) containing two surface proteins (haemagglutinin (HA) and neuraminidase (NA)) from the HPAI H5N1 (A/duck/Vietnam/HT2/2014(H5N1)) of the dominant clade 2.3.2.1c in Vietnam during 2012–2014. Importantly, the IBT-RG02 vaccine candidate has elicited high antibody titres in chickens (geometric mean titre (GMT) of 6.42 and 6.92, log2 on day 14 and day 28 p.i., respectively). To test the efficacy, immunized chickens were challenged with the circulating virulent strains. As results, there was a high protection rate of 91.6% chickens against the virulent A/DK/VN/Bacninh/NCVD-17A384/2017 of the same clade and a cross-protection of 83.3% against A/duck/TG/NAVET(3)/2013 virus of clade 1.1. Our promising results showed that we can independently master the reverse genetics technology for generation of highly immunogenic vaccine candidates, and henceforth, it is a timely manner to reformulate avian influenza virus vaccines against variable H5 clade HPAI viruses in Vietnam.

Study on the Antiviral Activities and Hemagglutinin-Based Molecular Mechanism of Novel Chlorogenin 3-O-β-Chacotrioside Derivatives Against H5N1 Subtype Viruses.

The objective of this study was to investigate the inhibitory effect of chlorogenin 3-O-β-chacotrioside derivatives against H5N1 subtype of the highly pathogenic avian influenza (HPAI) viruses and its molecular mechanism. A series of novel small molecule pentacyclic triterpene derivatives were designed and synthesized and their antiviral activities on HPAI H5N1 viruses were detected. The results displayed that the derivatives UA-Nu-ph-5, XC-27-1 and XC-27-2 strongly inhibited wild-type A/Duck/Guangdong/212/2004 H5N1 viruses with the IC50 values of 15.59 ± 2.4 μM, 16.83 ± 1.45 μM, and 12.45 ± 2.27 μM, respectively, and had the selectivity index (SI) > 3, which was consistent with the efficacy against A/Thailand/kan353/2004 pseudo-typed viruses. Four dealt patterns were compared via PRNT. The prevention dealt pattern showed the strongest inhibitory effects than other patterns, suggesting that these derivatives act on the entry process at the early stages of H5N1 viral infection, providing protection for cells against infection. Further studies through hemagglutinin inhibition (HI) and neuraminidase inhibitory (NAI) assay confirmed that these derivatives inhibited H5N1 virus replication by interfering with the viral hemagglutinin function. The derivatives could recognize specifically HA protein with binding affinity constant KD values of 2.57 × 10−4 M and 3.67 × 10−4 M. In addition, through site-directed mutagenesis combined with a pseudovirion system, we identified that the high-affinity docking sites underlying interaction were closely associated with amino acid residues I391 and T395 of HA. However, the potential binding sites of the derivatives with HA did not locate at HA1 sialic acids receptor binding domain (RBD). Taken together, these study data manifested that chlorogenin 3-O-β-chacotrioside derivatives generated antiviral effect against HPAI H5N1 viruses by targeting the hemagglutinin fusion machinery.

Outbreak of highly pathogenic avian influenza subtype H5N8 in two multi-age chicken farms in Jos, Plateau State, Nigeria

An outbreak of highly pathogenic avian influenza (HPAI) subtype H5N8 in two multi-age chicken flocks in Jos, Plateau State, Nigeria, is described. Diagnosis was made using tissue samples from the affected farms that were submitted to the Regional Lab for Animal Influenza and other Transboundary Animal Diseases, National Veterinary Research Institute, Vom. Pullets with broilers of 9 and 4 weeks of age respectively, from farm A (fA) and broilers, 4 weeks of age from farm B (fB) were submitted from flocks experiencing a drastic increase in mortality. The February 2019 outbreak resulted in10 – 60 % and 60 % mortality on fA and fB respectively. Clinical signs included anorexia, depression, hock sitting and sudden onset of increasing mortality. Gross lesions included edema of the head with cyanosis of comb and wattle; swollen abdomen due to ascites; generalized congestion of visceral organs with frank blood in the abdomen; congested mesenteric vessels with haemorrhages in the mucosa of small and large intestines; congested and frothy lungs with severe hemorrhagic tracheitis among others. In view of the clinical and gross pathology, HPAI was considered as the tentative diagnosis. Diagnosis of HPAI subtype H5N8 was confirmed by reverse transcriptase polymerase chain reaction and viral isolation in embryonated chicken eggs. The current outbreak has shown the presence of HPAI H5N8 in the country different from the H5N1 subtype that was introduced since 2006 in Nigeria. The co-circulation of these two subtypes if the current wave of infection is left unchecked will have adverse effects on poultry production, international trade and human health. Hence, the need for the re-evaluation and strengthening of the Government control policy to save the poultry industry from perennial economic loses.Keywords: Broilers, Highly pathogenic avian influenza (HPAI H5N8), Jos, Nigeria, Outbreaks, Pullets

Innate immunemodulator containing adjuvant formulated HA based vaccine protects mice from lethal infection of highly pathogenic avian influenza H5N1 virus

The highly pathogenic avian influenza (HPAI) H5N1 viruses and their spillover into the human population pose substantial economic and public health threats. Although antiviral drugs have some effect in treating influenza infection, vaccination is still the most effective intervention to prevent possible pandemic outbreaks. We have developed a novel H5 influenza vaccine to improve the world’s pandemic preparedness. We produced a hemagglutinin (HA) of HPAI H5N1 virus A/Alberta/01/2014 (AB14) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj) and their efficacy as vaccine candidates was evaluated in mice. Intramuscular delivery of two doses of TriAdj formulated mammalian expressed HA (m-HA/TriAdj) was shown to provide full protection against a lethal challenge of AB14 in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant, and m-HA without adjuvant resulted in no protection in immunized mice. Furthermore, m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody titres. All the mice in the m-HA/TriAdj group survived a lethal AB14 H5N1 challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. Our results suggest that m-HA formulated with TriAdj has potential to protect against pandemic H5N1 in the event of its cross over to the human host.

Agricultural and geographic factors shaped the North American 2015 highly pathogenic avian influenza H5N2 outbreak.

The 2014–2015 highly pathogenic avian influenza (HPAI) H5NX outbreak represents the largest and most expensive HPAI outbreak in the United States to date. Despite extensive traditional and molecular epidemiological studies, factors associated with the spread of HPAI among midwestern poultry premises remain unclear. To better understand the dynamics of this outbreak, 182 full genome HPAI H5N2 sequences isolated from commercial layer chicken and turkey production premises were analyzed using evolutionary models able to accommodate epidemiological and geographic information. Epidemiological compartmental models embedded in a phylogenetic framework provided evidence that poultry type acted as a barrier to the transmission of virus among midwestern poultry farms. Furthermore, after initial introduction, the propagation of HPAI cases was self-sustainable within the commercial poultry industries. Discrete trait diffusion models indicated that within state viral transitions occurred more frequently than inter-state transitions. Distance and sample size were very strongly supported as associated with viral transition between county groups (Bayes Factor > 30.0). Together these findings indicate that the different types of midwestern poultry industries were not a single homogenous population, but rather, the outbreak was shaped by poultry industries and geographic factors.

Determinants of biosecurity practices in French duck farms after a H5N8 Highly Pathogenic Avian Influenza epidemic: The effect of farmer knowledge, attitudes and personality traits.

Biosecurity is crucial for infectious disease prevention, more importantly in the absence of vaccination. The need for improving the implementation of biosecurity practices was highlighted in French duck farms following the 2016-2017 H5N8 Highly Pathogenic Avian Influenza (HPAI) epidemic. Farmers have multiple reasons for not implementing biosecurity practices: external (time, money) and internal (socio-psychological). The purpose of this study was to determine how sets of socio-psychological factors (i.e. knowledge on biosecurity and avian influenza transmission, attitudes, personality traits, social background) affect the adoption of on-farm biosecurity practices. Biosecurity practices and socio-psychological determinants were assessed during 127 duck farm visits, in South West France, using both questionnaires and on-farm observations. Factorial analysis of mixed data (FAMD) and hierarchical clustering analysis (HCA) identified three groups of farmers with different socio-psychological profiles: the first group was characterized by minimal knowledge, negative attitudes towards biosecurity, little social pressure and a low level of conscientiousness. The second group was characterized by more extensive experience in poultry production, higher stress and social pressure. The third group was characterized by less experience in poultry production, better knowledge and positive attitudes towards biosecurity, increased self-confidence and orientation towards action. The first group had a significantly lower adoption of biosecurity measures than the two other groups. A better understanding of the factors involved in farmers' decision-making could improve the efficiency of interventions aiming at improving and maintaining the level of on-farm biosecurity in the duck industry.

H7N9 Influenza Virus Containing a Polybasic HA Cleavage Site Requires Minimal Host Adaptation to Obtain a Highly Pathogenic Disease Phenotype in Mice.

Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease; however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species.

Novel pathogenic characteristics of highly pathogenic avian influenza virus H7N9: viraemia and extrapulmonary infection

ABSTRACT The H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus’s pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient’s plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment.

Outbreaks of Clade 2.3.4.4 H5N8 highly pathogenic avian influenza in 2018 in the northern regions of South Africa were unrelated to those of 2017.

Asian-origin H5N8 highly pathogenic avian influenza (HPAI) viruses of the H5 Goose/Guangdong/96 lineage, clade 2.3.4.4 group B, reached South Africa by June 2017. By the end of that year, 5.4 million layers and broiler chickens died or were culled, with total losses in the poultry industry estimated at US$ 140 million, and thousands of exotic birds in zoological collections, endangered endemic species and backyard poultry and pet birds also perished. The 2017 H5N8 HPAI outbreaks were characterized by two distinct spatial clusters, each associated with specific reassortant viral genotypes. Genotypes 1, 2, 3 and 5 were restricted to the northern regions, spanning the provinces of Limpopo, Gauteng, North West, Mpumalanga, KwaZulu-Natal and Free State. The second, much larger cluster of outbreaks was in the south, in the Western and Eastern Cape provinces, wherein 2017 and 2018 outbreaks were caused solely by genotype 4. The last confirmed case of H5N8 HPAI in the northern region in 2017 was in early October, and the viruses seemed to disappear over the summer. However, starting in mid-February 2018, H5N8 HPAI outbreaks resurged in the north. Viruses from two of the eight outbreaks were sequenced, one from an outbreak in quails (Coturnix japonica) in the North West Province, and another from commercial pullets in the Gauteng province. Phylogenetic analysis identified the viruses as a distinct sixth genotype that was most likely a new introduction to South Africa in early 2018.

Development of an effective contemporary trivalent avian influenza vaccine against circulating H5N1, H5N8, and H9N2 in Egypt

Low pathogenicity avian influenza (LPAI) H9N2, highly pathogenic avian influenza (HPAI) H5N1, and H5N8 circulate in Egyptian poultry and cause veterinary and public health burdens. In response, AIV vaccines are commonly used. The main objective of this study was to develop a broad, cross-protective, trivalent vaccine based on circulating AIVs in Egypt. We generated highly replicating avirulent AIVs, H5N1, and H5N8, to be used in combination with H9N2 strain for the generation of an inactivated vaccine. Immunogenicity and protective efficacy of this vaccine were tested. Results showed that a single immunization dose enhanced humoral immune responses giving full protection against challenges with LPAI H9N2, HPAI H5N1, and H5N8 viruses. This efficacious vaccine will reduce the cost of vaccination for poultry growers and is expected to be effective in the field as it is based on contemporary viruses currently in circulation among Egyptian poultry.

Characterization of H7N9 avian influenza viruses isolated from duck meat products.

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi-basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ-HE29-22/2017 (HE29-22) and A/duck/Japan/AQ-HE29-52/2017 (HE29-52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ-HE28-3/2016 (HE28-3)] in mice and ferrets. In mice, HE29-52 was more pathogenic than HE29-22 and HE28-3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.

Transmission dynamics between infected waterfowl and terrestrial poultry: Differences between the transmission and tropism of H5N8 highly pathogenic avian influenza virus (clade 2.3.4.4a) among ducks, chickens and turkeys

H5N8 highly-pathogenic avian influenza viruses (HPAIVs, clade 2.3.4.4) have spread globally via migratory waterfowl. Pekin ducks infected with a UK virus (H5N8-2014) served as the donors of infection in three separate cohousing experiments to attempt onward transmission chains to sequentially introduced groups of contact ducks, chickens and turkeys. Efficient transmission occurred among ducks and turkeys up to the third contact stage, with all (100%) birds becoming infected. Introduction of an additional fourth contact group of ducks to the turkey transmission chain demonstrated retention of H5N8-2014's waterfowl-competent adaptation. However, onward transmission ceased in chickens at the second contact stage where only 13% became infected. Analysis of viral progeny at this contact stage revealed no emergent polymorphisms in the intra-species (duck) transmission chain, but both terrestrial species included changes in the polymerase and accessory genes. Typical HPAIV pathogenesis and mortality occurred in infected chickens and turkeys, contrasting with 5% mortality among ducks.

Identification the Presence of Clade 2.3.2.1c-Avian Influenza H5N1, a Highly Pathogenic Virus in Iraq, 2018

Abstract For the first time in Iraq, we identified in March, 2018 the presence of a highly virulent avian influenza virus (AIV), H5N1 (Clade 2.3.2.1c), causing highly pathogenic avian influenza (HPAI) in poultry farms, Iraq,. The identification of the virus was done using a rapid serological test, a real time-qPCR, and glycoprotein gene sequencing. Using sequencing and phylogenetic analyses, the clade 2.3.2.1c virus was recorded to be clustered, with high similarity to Asian and West African AIV, HPAI H5N1 from Ivory Coast identified in 2015. According to our knowledge, there was no previous detection of the clade 2.3.2.1c made in Iraq. Our results provide evidence that high risk of HPAI H5 outbreaks might be present in Iraq, and this needs to lead to high quality surveillance targeting of wild and domestic birds for early diagnosis of HPAI. The current work provides feasible and accurate approaches for understanding the evolution of HPAI H5 virus in different countries around the world.

Fireworks-like surveillance approach: The case of HPAI H5N1 in wild birds in Europe.

Highly pathogenic avian influenza (HPAI) risk management requires efficient surveillance of the infection in wild birds for early warning purposes. In this study, our aim was to describe the spread of continent-wide infection cases using a fireworks model and therefore improve current surveillance systems. The fireworks model is a metaphor illustrating the spread of HPAI as a point source epizootic. The approach is based on early detection of the outbreak seeds (sparks from the fireworks) and uses a predictive model of the probability of the occurrence of new cases following a seed introduction; this then determines the spatiotemporal perimeter for intense surveillance investigations. For a case study, we used surveillance data on HPAI H5N1 in wild birds across Europe between 2005 and 2010 to describe the outbreaks and determine the success of the case detection used to inform management of the disease. The fireworks description assumes simultaneous introductions of 'seeds' of cases, which then 'explode' in local foci but do not merge into a progressive disease wave. This model fits the data well. Using this predictive approach for HPAI cases in EU countries, we found that the investigation radius needed to achieve a detection level of 90% of new cases after an outbreak ranged from 10km to more than 300km, depending on the outbreak pattern. Based on these findings, the fireworks approach can be a valuable method for identifying the perimeters and risk areas to be targeted for enhanced surveillance. The rationale of the fireworks approach is quite generic and can easily be adapted to different situations and contexts.

Rapid evolution of Mexican H7N3 highly pathogenic avian influenza viruses in poultry.

Highly pathogenic avian influenza (HPAI) virus subtype H7N3 has been circulating in poultry in Mexico since 2012 and vaccination has been used to control the disease. In this study, eight Mexican H7N3 HPAI viruses from 2015–2017 were isolated and fully sequenced. No evidence of reassortment was detected with other avian influenza (AI) viruses, but phylogenetic analyses show divergence of all eight gene segments into three genetic clusters by 2015, with 94.94 to 98.78 percent nucleotide homology of the HA genes when compared to the index virus from 2012. The HA protein of viruses from each cluster showed a different number of basic amino acids (n = 5–7) in the cleavage site, and six different patterns at the predicted N-glycosylation sites. Comparison of the sequences of the Mexican lineage H7N3 HPAI viruses and American ancestral wild bird AI viruses to characterize the virus evolutionary dynamics showed that the nucleotide substitution rates in PB2, PB1, PA, HA, NP, and NS genes greatly increased once the virus was introduced into poultry. The global nonsynonymous and synonymous ratios imply strong purifying selection driving the evolution of the virus. Forty-nine positively selected sites out of 171 nonsynonymous mutations were identified in the Mexican H7N3 HPAI viruses, including 7 amino acid changes observed in higher proportion in North American poultry origin AI viruses isolates than in wild bird-origin viruses. Continuous monitoring and molecular characterization of the H7N3 HPAI virus is important for better understanding of the virus evolutionary dynamics and further improving control measures including vaccination.

Avian Influenza H5N8 Outbreak in African Penguins (Spheniscus demersus), Namibia, 2019

In January 2019, high mortalities were reported among African Penguins (Spheniscus demersus) in a breeding colony on Halifax Island, Namibia, Africa. Analysis of samples by reverse transcription quantitative PCR indicated the presence of highly pathogenic avian influenza (HPAI) subtype H5N8. Sequence analysis of the hemagglutinin and neuraminidase genes confirmed the presence of the virus in the birds and its high similarity to HPAI subtype H5N8 identified in South Africa in 2017. There have been no previous reports of HPAI H5N8 in Namibia.