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.

An African horse sickness (AHS) outbreak occurred in South Africa's AHS controlled area in autumn 2016. A freedom from disease survey was performed to establish the likelihood of ongoing circulation of the associated virus during the same period the following year. A single‐stage surveillance strategy was employed with a population‐level design prevalence of 1% to establish a survey population sensitivity of 95% (probability that one or more positive horses would be detected if AHS was present at a prevalence greater than or equal to the design prevalence). In March 2017, a total of 262 randomly selected horses from 51 herds were sampled from the 2016 outbreak containment zone. Three within‐herd and herd‐level design prevalence scenarios were used in evaluating the post‐survey probability of freedom. Depending on the underlying design prevalence scenarios, effectively ranging between 0.8% and 6.4%, and the use of informed or uninformed priors, the probability of freedom derived from this surveillance ranged between 73.1% and 99.9% (uninformed prior) and between 96.6% and 100% (informed prior). Based on the results, the authors conclude that it is unlikely that the 2016 AHS virus was still circulating in the autumn of 2017 in the 2016 outbreak containment zone. The ability to perform freedom from disease surveys, and also to include risk‐based methods, in the AHS controlled area of South Africa is influenced by the changing underlying population at risk and the high level of vaccination coverage in the horse population. Ongoing census post‐outbreak must be undertaken to maintain a valid sampling frame for future surveillance activity. The seasonality of AHS, the restricted AHS vaccination period and the inability to easily differentiate infected from vaccinated animals by laboratory testing impact the ability to perform a freedom from disease survey for AHS in the 12 months following an outbreak in the controlled area.

Background: In June 2017, an outbreak of the highly pathogenic avian influenza A(H5N8) was detected in commercial poultry farms in South Africa, which rapidly spread to all nine South African provinces. Objectives:We conducted active surveillance for the transmission of influenza A(H5N8) to humans working with infected birds during the South African outbreak.Methods: Influenza A(H5N8)-positive veterinary specimens were used to evaluate the ability of real-time PCR-based assays to detect contemporary avian influenza A(H5N8) strains. Whole genome sequences were generated from these specimens by next-generation sequencing for phylogenetic characterization and screening for mammalian-adaptive mutations.Results: Human respiratory samples from 74 individuals meeting our case definition, all tested negative for avian influenza A(H5) by real-time PCR, but 2 (3%) were positive for human influenza A(H3N2). 54% (40/74) reported wearing personal protective equipment including overalls, boots, gloves, masks, and goggles. 94% (59/63) of veterinary specimens positive for H5N8 were detected on an influenza A(H5) assay for human diagnostics. A commercial H5N8 assay detected H5 in only 6% (3/48) and N8 in 92% (44/48). Thirteen (13/25; 52%) A(H5N8) genomes generated from veterinary specimens clustered in a single monophyletic clade. These sequences contained the NS (P42S) and PB2 (L89V) mutations noted as markers of mammalian adaptation. Conclusions:Diagnostic assays were able to detect and characterize influenza A(H5N8) viruses, but poor performance is reported for a commercial assay. Absence of influenza A(H5N8) in humans with occupational exposure and no clear impression | 267 VALLEY-OMAR Et AL.

Rift Valley fever (RVF) is a zoonotic, viral, mosquito-borne disease that causes considerable morbidity and mortality in humans and livestock in Africa and the Arabian Peninsula. In June 2018, 4 alpaca inoculated subcutaneously with live attenuated RVF virus (RVFV) Smithburn strain exhibited pyrexia, aberrant vocalization, anorexia, neurologic signs, and respiratory distress. One animal died the evening of inoculation, and 2 at ~20 d post-inoculation. Concern regarding potential vaccine strain reversion to wild-type RVFV or vaccine-induced disease prompted autopsy of the latter two. Macroscopically, both alpacas had severe pulmonary edema and congestion, myocardial hemorrhages, and cyanotic mucous membranes. Histologically, they had cerebral nonsuppurative encephalomyelitis with perivascular cuffing, multifocal neuronal necrosis, gliosis, and meningitis. Lesions were more severe in the 4-mo-old cria. RVFV antigen and RNA were present in neuronal cytoplasm, by immunohistochemistry and in situ hybridization (ISH) respectively, and cerebrum was also RVFV positive by RT-rtPCR. The virus clustered in lineage K (100% sequence identity), with close association to Smithburn sequences published previously (identity: 99.1–100%). There was neither evidence of an aberrant immune-mediated reaction nor reassortment with wild-type virus. The evidence points to a pure infection with Smithburn vaccine strain as the cause of the animals’ disease.

Rift Valley fever (RVF) is a zoonotic, viral, mosquito‐borne disease that causes considerable morbidity and mortality in ruminants during the rainy season in Africa. In June 2018, 4 alpaca inoculated subcutaneously with Rift Valley Fever (RVF) Smithburn live attenuated vaccine strain exhibited pyrexia, aberrant vocalization, anorexia, neurological signs (tremors, disorientation, overknuckling and ataxia leading to full recumbency), respiratory distress and sudden death (3 animals). One animal died the evening of inoculation, the other two at 2–3 weeks post inoculation. The fourth alpaca fully recovered. Due to significant concern that this was wildtype Rift Valley fever, vaccine strain reversion, or an atypical camelid response to Smithburn vaccination, a full necropsy and further workup were conducted on the latter 2 fatal cases including necropsy, histopathology, immunohistochemistry (IHC), RNAscope in situ hybridization (ISH), reverse transcription real‐time PCR (RT‐qPCR) and confirmatory sequencing. Necropsy and histopathology were conducted per standard procedures at the WCPVL. RT‐qPCR for RVFV L segment and sequencing of each of the three genome segments were conducted per standard procedures at the OVI. Anti‐RVFV glycoprotein IHC and ISH for RVFV L segment RNA were conducted per published methods in Ragan et al., 2019. Macroscopic lesions included cyanotic mucous membranes, severe pulmonary oedema and congestion and multifocal petechiae and ecchymoses throughout the left and right ventricular epi‐, endo‐ and myocardium. The most distinctive microscopic lesions were multifocal neuronal necrosis, gliosis and prominent histiocytic and lymphoplasmacytic perivascular cuffing of blood vessels in the cerebrum as well as a histiocytic and lymphoplasmacytic cerebral meningitis. Multifocally, in the cerebrum, viral antigen and RNA were present in the cytoplasm of neurons, including in the cell body, dendritic arborizations and axons (Figure 1) and brain samples tested positive for RVFV RNA by RT‐qPCR. The liver was negative for RVFV by both IHC and ISH. Additional genetic analysis of the brain tissue revealed that the virus clustered in Lineage K (100% sequence identity) with close association to published Smithburn vaccine sequences (sequence identity ranging from 99.1 – 100%). This is the first report of respiratory distress, neurological signs and sudden death in alpaca post inoculation with the Smithburn strain. It is also the first examination of RVF viral RNA distribution in alpaca tissues. RVF live attenuated Smithburn vaccine can cause meningoencephalitis in alpacas. Support or Funding Information WCPVL and Laboratory of Investigative Pathology, Department of Diagnostic Medicine/Pathobiology, KSU CVM Alpaca Rift Valley fever virus RNA positive encephalitisCerebral encephalitis in an alpaca vaccinated with Smithburn live attenuated vaccine strain labeled with pan‐RVFV (L segment probe‐set) ISH (brown), counterstained with hematoxylin. Bar is 50 microns. Rift Valley Fever Viral RNA Detection by In Situ Hy...