Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

Protective efficacy of a plant-produced beta variant rSARS-CoV-2 VLP vaccine in golden Syrian hamsters.

In the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.

Spillover of an endemic avian Influenza H6N2 chicken lineage to ostriches and reassortment with clade 2.3.4.4b H5N1 high pathogenicity viruses in chickens.

Prior to 2017, chicken production in South Africa had only ever been affected by an endemic strain of H6N2 low pathogenic avian influenza (LPAI), but since 2017, an outbreak of Goose/Guangdong clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) introduced by wild birds, followed by clade 2.3.4.4b H5N1 HPAI (2021-present), affected the country. In the present study, the viruses from seven cases of H6N2 LPAI from commercial poultry between October 2019 and August 2020 were genome-sequenced along with an H5N2 HPAI virus, and phylogenetic analysis was performed. The H5N2 HPAI virus caused localized outbreaks in a small-scale chicken farm and a large commercial layer farm in the KwaZulu-Natal province between late October and early December 2022. The phylogenetic results confirmed the first incidence of the chicken-adapted H6N2 lineage in commercial ostriches in the Western Cape province, with a likely epidemiological origin in chickens from the KwaZulu Natal province. The results also showed that the H5N2 HPAI virus was a novel reassortant of PB2, PB1, PA, NP and NA genome segments derived from a parental H6N2 virus that circulated in region, whereas the HA, M and NS genome segments were derived from sub-genotype SA10 H5N1 HPAI parental virus that had circulated in the local wild bird reservoir since July 2021.

Vaccination of African penguins (Spheniscus demersus) against high-pathogenicity avian influenza.

BACKGROUND: High-pathogenicity avian influenza (HPAI) has become a conservation threat to wild birds. Therefore, suitable vaccine technology and practical application methods require investigation. METHODS: Twenty-four African penguins (Spheniscus demersus) were vaccinated with either a conventional inactivated clade 2.3.4.4b H5N8 HPAI whole virus or a tobacco leaf-produced H5 haemagglutinin-based virus-like particle (VLP). Six birds received a second dose of the inactivated vaccine. Antibody responses were assessed and compared by employing haemagglutination inhibition tests. RESULTS: A second dose of inactivated vaccine was required to induce antibody titres above the level required to suppress virus shedding, while a single dose of VLP vaccine produced these levels by day 14, and one bird still had antibodies on day 430. LIMITATIONS: Bacterial contamination of the VLP vaccine limited the monitoring period and sample size in that treatment group, and it was not possible to perform a challenge study with field virus. CONCLUSION: VLP vaccines offer a more practical option than inactivated whole viruses, especially in logistically challenging situations involving wild birds.

Molecular characterization of Newcastle disease virus obtained from Mawenzi live bird market in Morogoro, Tanzania in 2020-2021.

Newcastle disease (ND) is among the most important poultry diseases worldwide. It is the major threat to poultry production in Africa and causes major economic losses for both local and commercial chickens. To date, half of ND class II genotypes have been reported in Africa (I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII, and XXI). The information on the circulating NDV genotypes is still scarce despite the endemic nature of ND in most countries on the African continent.A total of 659 oro-cloacal swabs were collected from local chickens in Mawenzi live bird market located in Morogoro, Tanzania, between June 2020 and May 2021. Newcastle disease virus was detected by using reverse transcription real-time polymerase chain reaction (RT-qPCR) and conventional PCR followed by sequencing of PCR products. The prevalence of NDV in the surveilled live bird markets was 23.5%. Sequencing and phylogenetic analysis revealed the presence of sub-genotype VII.2. The detected sub-genotype VII.2 has phylogenetic links to Zambian NDV strains implying a Southeast dissemination of the virus, considering that it was first detected in Mozambique. This study underscores the need of active NDV surveillance to determine the distribution of this NDV genotype in the country and monitor its spread and contribution to the emergence of new ND viruses.

Efficacy of a plant-produced infectious bronchitis virus-like particle vaccine in specific pathogen-free chickens.

Infectious bronchitis (IB) Gammacoronavirus causes a highly contagious respiratory disease in chickens that is listed by the World Organisation for Animal Health (WOAH). Its high mutation ability has resulted in numerous variants against which the commercially available live or recombinant vaccines singly offer limited protection. Agrobacterium-mediated transient expression in Nicotiana benthamiana (tobacco) plants was used here to produce a virus-like particle (VLP) vaccine expressing a modified full-length IBV spike (S) protein of a QX-like IB variant. In a challenge study with the homologous live IB QX-like virus, VLP-vaccinated birds produced S protein-specific antibodies comparable to those produced by live-vaccinated birds seroconverting with mean geometric titers of 6.8 and 7.2 log2, respectively. The VLP-vaccinated birds had reduced oropharyngeal and cloacal viral shedding compared to an unvaccinated challenged control and were more protected against tracheal ciliostasis than the live-vaccinated birds. While the results appeared similar, plant-produced IB VLPs are safer, more affordable, easier to produce and update to antigenically match any emerging IB variant, making them a more suitable alternative to IBV control than live-attenuated vaccines.

Salmonella gallinarum strains from outbreaks of fowl typhoid fever in Southern Africa closely related to SG9R vaccines.

Introduction: Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SG) is associated with fowl typhoid fever, and the attenuated rough strain SG9R is widely used as a vaccine in many regions. Reversion to virulence of vaccine strains was suspected as the cause during recent fowl typhoid fever outbreaks in poultry in South Africa and Eswatini. Methods: To compare nine field isolates with global wild-type SG9 strains and the two commercial SG9R vaccines in use, Nobilis® SG9R and Cevac®-SG, we used whole-genome comparison with single-nucleotide polymorphism (SNP) detection. Results: SNP phylogenic analysis showed that all the southern African field isolates were more closely related to the vaccine strains than wild-type SG9 strains. Furthermore, SNPs in the pyruvate dehydrogenase (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes, which are known markers of attenuation, were found in four of the field isolates along with intact spv, SPI-1, and SPI-2 gene clusters, providing conclusive evidence that these four isolates were originally vaccine strains that reverted to virulence. Five other field isolates lacked the SG9R attenuation markers, but variant analysis identified an SNP in the yihX gene, insertions in the ybjX and hydH genes, and deletions in the ftsK and sadA genes that were shared between the field isolates and vaccine strains but absent in wild-type SG9, indicating that these field isolates were also likely revertant vaccines. Discussion: Overall, this study highlights different mechanisms of reversion of two commercial vaccines, where virulence caused by field isolates closely related to the Nobilis® SG9R vaccine was associated with the restoration of intact virulence gene clusters, and those derived from the Cevac®-SG vaccine were characterized by point mutations resulting in restored aceE and rfaJ genes. A possible new marker of attenuation was identified as a point mutation in the yihX gene, as well as four new candidate genes that could potentially be used to distinguish current vaccine strains from wild-type strains using PCR assays.

Optimization of infectious bronchitis virus-like particle expression in Nicotiana benthamiana as potential poultry vaccines.

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease in chickens, with a severe economic impact on poultry production globally. The rapid emergence of regional variants of this Gammacoronavirus warrants new vaccine approaches that are more humane and rapid to produce than the current embryonated chicken egg-based method used for IB variant vaccine propagation (chemically-inactivated whole viruses). The production of virus-like particles (VLPs) expressing the Spike (S) glycoprotein, the major antigen which induces neutralizing antibodies, has not been achieved in planta up until now. In this study, using the Agrobacterium-mediated Nicotiana benthamiana (tobacco plant) transient expression system, the highest levels of VLPs displaying a modified S protein of a QX-like IB variant were obtained when the native transmembrane (TM) domain and cytoplasmic tail were substituted with that of the Newcastle disease virus (NDV) fusion glycoprotein, co-infiltrated with the NDV Matrix protein. In comparison, the native IB modified S co-infiltrated with IB virus membrane, envelope and nucleocapsid proteins, or substituted with the TM and CT of an H6-subtype influenza A virus hemagglutinin glycoprotein yielded lower VLP expression levels. Strong immunogenicity was confirmed in specific pathogen free chickens immunized intramuscularly with VLPs adjuvanted with Emulsigen®-P, where birds that received doses of 5 µg or 20 µg (S protein content) seroconverted after two weeks with mean hemaggluttination inhibition titres of 9.1 and 10 log2, respectively. Plant-produced IB VLP variant vaccines are safer, more rapid and cost effective to produce than VLPs produced in insect cell expression systems or the traditional egg-produced inactivated whole virus oil emulsion vaccines currently in use, with great potential for improved IB disease control in future.

The Molecular Epidemiology of Clade 2.3.4.4B H5N1 High Pathogenicity Avian Influenza in Southern Africa, 2021-2022.

In southern Africa, clade 2.3.4.4B H5N1 high pathogenicity avian influenza (HPAI) was first detected in South African (SA) poultry in April 2021, followed by outbreaks in poultry or wild birds in Lesotho and Botswana. In this study, the complete or partial genomes of 117 viruses from the SA outbreaks in 2021-2022 were analyzed to decipher the sub-regional spread of the disease. Our analysis showed that seven H5N1 sub-genotypes were associated with the initial outbreaks, but by late 2022 only two sub-genotypes still circulated. Furthermore, SA poultry was not the source of Lesotho's outbreaks, and the latter was most likely an introduction from wild birds. Similarly, SA and Botswana's outbreaks in 2021 were unrelated, but viruses of Botswana's unique sub-genotype were introduced into SA later in 2022 causing an outbreak in ostriches. At least 83% of SA's commercial poultry cases in 2021-2022 were point introductions from wild birds. Like H5N8 HPAI in 2017-2018, a coastal seabird-restricted sub-lineage of H5N1 viruses emerged in the Western Cape province in 2021 and spread to Namibia, causing mortalities in Cape Cormorants. In SA ~24,000 of this endangered species died, and the loss of >300 endangered African penguins further threatens biodiversity.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits.

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.

Propagation of avian influenza virus in embryonated ostrich eggs.

Influenza A viruses (IAVs) are typically isolated and cultured by successive passages using 9- to 11-day-old embryonated chicken eggs (ECEs) and in 14-day old ECEs for virus mutational studies. Real-time reverse transcription-polymerase chain reaction tests (RT-PCRs) are commonly used for IAV diagnosis, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus. Efforts at isolating ostrich-origin IAVs from RT-PCR positive specimens using ECEs have often been unsuccessful, raising the possibility of a species bottleneck, whereby ostrich-adapted IAVs may not readily infect and replicate in ECEs, yet the capacity of an ostrich embryo to support the replication of influenza viruses has not been previously demonstrated. This study describes an optimised method for H5 and H7 subtype IAV isolation and propagation in 28-day old embryonated ostrich eggs (EOEs), the biological equivalent of 14-day old ECEs. The viability of EOEs transported from breeding sites could be maximised by pre-incubating the eggs for 12 to 14 days prior to long-distance transportation. This method applied to studies for ostrich-adapted virus isolation and in ovo studies will enable better understanding of the virus-host interaction in ostriches and the emergence of potentially zoonotic diseases.

Evolutionary dynamics of the clade 2.3.4.4B H5N8 high-pathogenicity avian influenza outbreaks in coastal seabirds and other species in southern Africa from 2017 to 2019.

From late 2017 to early 2018, clade 2.3.4.4B H5N8 highly pathogenic avian influenza (HPAI) viruses caused mass die-offs of thousands of coastal seabirds along the southern coastline of South Africa. Terns (Laridae) especially were affected, but high mortalities in critically endangered and threatened species like African Penguins (Spheniscus demersus) caused international concern and, exactly a year later, the disease recurred at a key African Penguin breeding site on Halifax Island, Namibia. Twenty-five clade 2.3.4.4B H5N8 HPAI viruses from coastal seabirds and a Jackal Buzzard (Buteo rufofuscus) were isolated and/or sequenced in this study. Phylogenetic analyses of the full viral genomes and time to the most recent common ancestor (tMRCA) analyses of the HA, NA, PB1 and PA genes determined that the South African coastal seabird viruses formed a monophyletic group nested within the South African genotype 4 viruses. This sub-lineage likely originated from a single introduction by terrestrial birds around October 2017. Only the HA and NA sequences were available for the Namibian penguin viruses, but the phylogenetic data confirmed that the South African coastal seabird viruses from 2017 to 2018 were the source and the most closely related South African virus was found in a gull. tMRCA analyses furthermore determined that the progenitors of the five genotypes implicated in the earlier 2017 South African outbreaks in wild birds and poultry were dated at between 2 and 4 months prior to the index cases. tMRCA and phylogenetic data also showed that the novel genotype 6 virus introduced to South Africa in 2018, and later also detected in Nigeria and Poland in 2019, most likely arose in late 2017 in West, Central or East Africa. We propose that it continued to circulate there, and that an unidentified reservoir was the source of both the South African outbreaks in early 2018 and in Nigeria in mid-2019.

Wild Bird Surveillance in the Gauteng Province of South Africa during the High-Risk Period for Highly Pathogenic Avian Influenza Virus Introduction.

Migratory birds carried clade 2.3.4.4B H5Nx highly pathogenic avian influenza (HPAI) viruses to South Africa in 2017, 2018 and 2021, where the Gauteng Province is a high-risk zone for virus introduction. Here, we combined environmental faecal sampling with sensitive rRT-PCR methods and direct Ion Torrent sequencing to survey wild populations between February and May 2022. An overall IAV incidence of 42.92% (100/231) in water bird faecal swab pools or swabs from moribund or dead European White Storks (Ciconia ciconia) was detected. In total, 7% of the IAV-positive pools tested H5-positive, with clade 2.3.4.4B H5N1 HPAI confirmed in the storks; 10% of the IAV-positive samples were identified as H9N2, and five complete H9N2 genomes were phylogenetically closely related to a local 2021 wild duck H9N2 virus, recent Eurasian LPAI viruses or those detected in commercial ostriches in the Western and Eastern Cape Provinces since 2018. H3N1, H4N2, H5N2 and H8Nx subtypes were also identified. Targeted surveillance of wild birds using environmental faecal sampling can thus be effectively applied under sub-Saharan African conditions, but region-specific studies should first be used to identify peak prevalence times which, in southern Africa, is linked to the peak rainfall period, when ducks are reproductively active.

Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens.

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log2, for at least four months before dropping to approximately 5-6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.

Effects of swab pool size and transport medium on the detection and isolation of avian influenza viruses in ostriches.

BACKGROUND: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor. RESULTS: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (106.7 - 103.0 vs. 106.6 - 103.1 EID50 for H5N8 HPAIV and 105.5 - 101.4 vs. 105.1 - 101.3 EID50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains. CONCLUSION: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics.

Experimental infection of ostriches with H7N1 low pathogenic and H5N8 clade 2.3.4.4B highly pathogenic influenza A viruses.

Infection dynamics data for influenza A virus in a species is important for understanding host-pathogen interactions and developing effective control strategies. Seven-week-old ostriches challenged with H7N1 low pathogenic viruses (LPAIV) or clade 2.3.4.4B H5N8 high pathogenic viruses (HPAIV) were co- housed with non-challenged contacts. Clinical signs, virus shed in the trachea, cloaca, and feather pulp, and antibody responses were quantified over 14 days. H7N1 LPAIV-infected ostriches remained generally healthy with some showing signs of mild conjunctivitis and rhinitis attributed to Mycoplasma co-infection. Mean tracheal virus shedding titres in contact birds peaked 3 days (106.2 EID50 equivalents / ml) and 9 days (105.28 EID50 equivalents / ml) after introduction, lasting for at least 13 days post infection. Cloacal shedding was substantially lower and ceased within 10 days of onset, and low virus levels were detected in wing feather pulp up until day 14. H5N8 HPAIV -infected ostriches showed various degrees of morbidity, with 2/3 mortalities in the in-contact group. Mean tracheal shedding in contact birds peaked 8 days after introduction (106.32 EID50 equivalents/ ml) and lasted beyond 14 days in survivors. Cloacal shedding and virus in feather pulp was generally higher and more consistently positive compared to H7N1 LPAIV, and was also detectable at least until 14 days post infection in survivors. Antibodies against H5N8 HPAIV and H7N1 LPAIV only appeared after day 7 post exposure, with higher titres induced by the HPAIV compared to the LPAIV, and neuraminidase treatment was essential to remove non-specific inhibitors from the H5N8-positive antisera.

The spatiotemporal epidemiology of high pathogenicity avian influenza outbreaks in key ostrich producing areas of South Africa.

High pathogenicity avian influenza (HPAI) has become a major focus point worldwide due to its zoonotic potential and economic effects resulting from trade restrictions and high mortality rates in poultry. Key ostrich producing provinces of South Africa have experienced three H5N2 HPAI outbreaks (2004, 2006 and 2011) and one H5N8 HPAI (2017) outbreak over the past two decades. The Klein Karoo region in the Western Cape Province, a province with a largely Mediterranean climate, is the predominant ostrich producing region in the country. Understanding the epidemiology of HPAI in ostrich producing areas is an essential first step in developing effective and efficient control measures. This study investigated the spatiotemporal patterns associated with the 2011 (H5N2) and 2017 (H5N8) HPAI outbreaks in the key ostrich producing areas of South Africa. Six hundred and nine and 340 active ostrich farms/holdings were subjected to surveillance during 2011 and 2017 respectively, with over 70 % of these farms located within five local municipalities of the study area. Forty-two and fifty-one farms were affected in the 2011 and 2017 outbreaks respectively. Both HPAI outbreaks occurred predominantly in areas of high ostrich farm density. However, the temporal occurrence, spatial and directional distributions of the outbreaks were different. The 2011 outbreak occurred earlier in the South African autumn months with a predominantly contiguous and stationary distribution, whilst the 2017 outbreak onset was during the winter with a more expansive multidirectional spatial distribution. Results suggest potential dissimilarities in the important risk factors for introduction and possible mode of spread. The 2011 outbreak pattern resembled an outbreak characterised by point introductions with the risk of introduction possibly being linked to high ostrich farm density and common management and husbandry practices in the ostrich industry. In contrast, the 2017 outbreak appeared to have a more propagating mode of transmission. The findings highlight epidemiological features of HPAI outbreak occurrence within ostrich populations that could be used to inform surveillance and control measures including targeted surveillance within high-risk spatial clusters. The study emphasizes the importance of both; implementation of a multi-pronged approach to HPAI control and the need for constant evaluation of the interaction of the host, environment and agent with each outbreak, in order to strengthen disease control.

Identification of bacteria in the tracheal swabs of farmed ostriches and their effect on the viability of influenza A virus.

Avian influenza surveillance is a requirement for commercial trade in ostrich products, but influenza A viruses (IAVs) have proven difficult to isolate from ostrich tracheal swabs that test positive using molecular methods. We hypothesized that microbes unique to the ostrich trachea propagate in the transport medium after sampling and affect viral viability. We cultured tracheal swabs from 50 ostriches on 4 farms in South Africa, and recovered and identified 13 bacterial, 1 yeast, and 2 fungal species. Dietzia sp. had not been identified previously in the oropharyngeal tract of a bird, to our knowledge. The bacteria were tested for antimicrobial susceptibility, and most aerobic species, except for Streptococcus sp. and Pseudomonas sp., were sensitive to enrofloxacin; all were susceptible to sulfonamide. Virus inhibition experiments determined that ostrich-source Streptococcus sp., Pantoea sp., and Citrobacter freundii produced extracellular metabolites that caused a substantial reduction in the IAV titers of 99.9%. Streptomyces, Corynebacterium, Staphylococcus, Arthrobacter gandavensis, Pseudomonas putida, and Acinetobacter spp. similarly reduced the viability of IAV from 77.6% to 24.1%. Dietzia appeared to have no effect, but Rothia dentocariosa, Rhodotorula spp., and Clostridium spp. slightly increased the viability of IAV by 25.9, 34.9, and 58.5%, respectively.

Emergence of highly pathogenic H5N2 and H7N1 influenza A viruses from low pathogenic precursors by serial passage in ovo.

Highly pathogenic (HPAI) strains emerge from their low pathogenic (LPAI) precursors and cause severe disease in poultry with enormous economic losses, and zoonotic potential. Understanding the mechanisms involved in HPAI emergence is thus an important goal for risk assessments. In this study ostrich-origin H5N2 and H7N1 LPAI progenitor viruses were serially passaged seventeen times in 14-day old embryonated chicken eggs and Ion Torrent ultra-deep sequencing was used to monitor the incremental changes in the consensus genome sequences. Both virus strains increased in virulence with successive passages, but the H7N1 virus attained a virulent phenotype sooner. Mutations V63M, E228V and D272G in the HA protein, Q357K in the nucleoprotein (NP) and H155P in the neuraminidase protein correlated with the increased pathogenicity of the H5N2 virus; whereas R584H and L589I substitutions in the polymerase B2 protein, A146T and Q220E in HA plus D231N in the matrix 1 protein correlated with increased pathogenicity of the H7N1 virus in embryos. Enzymatic cleavage of HA protein is the critical virulence determinant, and HA cleavage site motifs containing multibasic amino acids were detected at the sub-consensus level. The motifs PQERRR/GLF and PQRERR/GLF were first detected in passages 11 and 15 respectively of the H5N2 virus, and in the H7N1 virus the motifs PELPKGKK/GLF and PELPKRR/GLF were detected as early as passage 7. Most significantly, a 13 nucleotide insert of unknown origin was identified at passage 6 of the H5N2 virus, and at passage 17 a 42 nucleotide insert derived from the influenza NP gene was identified. This is the first report of non-homologous recombination at the HA cleavage site in an H5 subtype virus. This study provides insights into how HPAI viruses emerge from low pathogenic precursors and demonstrated the pathogenic potential of H5N2 and H7N1 strains that have not yet been implicated in HPAI outbreaks.