Wide distribution of Mediterranean and African spotted fever agents and the first identification of Israeli spotted fever agent in ticks in Uganda.

Rickettsia microorganisms are causative agents of several neglected emerging infectious diseases in humans transmitted by arthropods including ticks. In this study, ticks were collected from four geographical regions of Uganda and pooled in sizes of 1-179 ticks based on location, tick species, life stage, host, and time of collection. Then, they were tested by real-time PCR for Rickettsia species with primers targeting gltA, 17kDa and ompA genes, followed by Sanger sequencing of the 17kDa and ompA genes. Of the 471 tick pools tested, 116 (24.6%) were positive for Rickettsia spp. by the gltA primers. The prevalence of Rickettsia varied by district with Gulu recording the highest (30.1%) followed by Luwero (28.1%) and Kasese had the lowest (14%). Tick pools from livestock (cattle, goats, sheep, and pigs) had the highest positivity rate, 26.9%, followed by vegetation, 23.1%, and pets (dogs and cats), 19.7%. Of 116 gltA-positive tick pools, 86 pools were positive using 17kDa primers of which 48 purified PCR products were successfully sequenced. The predominant Rickettsia spp. identified was R. africae (n = 15) in four tick species, followed by R. conorii (n = 5) in three tick species (Haemaphysalis elliptica, Rhipicephalus appendiculatus, and Rh. decoloratus). Rickettsia conorii subsp. israelensis was detected in one tick pool. These findings indicate that multiple Rickettsia spp. capable of causing human illness are circulating in the four diverse geographical regions of Uganda including new strains previously known to occur in the Mediterranean region. Physicians should be informed about Rickettsia spp. as potential causes of acute febrile illnesses in these regions. Continued and expanded surveillance is essential to further identify and locate potential hotspots with Rickettsia spp. of concern.

Molecular characterisation of human adenoviruses associated with respiratory infections in Uganda.

Human adenoviruses (HAdV) are a diverse group of viruses causing a broad range of infections of the respiratory, urogenital and gastrointestinal tracts and keratoconjunctivitis. There are seven species of human adenoviruses with 113 genotypes which may contain multiple genetic variants. This study characterised respiratory human adenoviruses and associated factors in samples collected from selected hospitals in Uganda. A total of 2,298 nasopharyngeal samples were collected between the period of 2008 to 2016 from patients seeking health care at tertiary hospitals for influenza-like illness. They were screened by polymerase chain reaction (PCR) to determine the prevalence of HAdV. HAdV was cultured in A549 cell lines and the hexon gene was sequenced for genotyping. Of the 2,298 samples tested, 225 (9.8%) were adenovirus-positive by PCR. Age was found to be significantly associated with HAdV infections (p = 0.028) with 98% (220/225) of the positives in children aged 5 years and below and none in adults above 25 years of age. The sequenced isolates belonged to species HAdV-B and HAdV-C with most isolates identified as genotype B3. The results showed a high prevalence and genetic diversity in respiratory HAdV circulating in Ugandan population. Deeper genomic characterization based on whole genome sequencing may be necessary to further elucidate possible transmission and impact of current adenovirus-vectored vaccines in Africa.

Genetic Evolution of Avian Influenza A (H9N2) Viruses Isolated from Domestic Poultry in Uganda Reveals Evidence of Mammalian Host Adaptation, Increased Virulence and Reduced Sensitivity to Baloxavir.

A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.

Serological Surveillance of Influenza D Virus in Ruminants and Swine in West and East Africa, 2017-2020.

Influenza D virus (IDV) was first isolated in 2011 in Oklahoma, USA from pigs presenting with influenza-like symptoms. IDV is known to mainly circulate in ruminants, especially cattle. In Africa, there is limited information on the epidemiology of IDV, although the virus has likely circulated in the region since 2012. In the present study, we investigated the seropositivity of IDV among domestic ruminants and swine in West and East Africa from 2017 to 2020. Serum samples were analyzed using the hemagglutination inhibition (HI) assay. Our study demonstrated that IDV is still circulating in Africa, with variations in seropositivity among countries and species. The highest seropositivity was detected in cattle (3.9 to 20.9%). Our data highlights a need for extensive surveillance of IDV in Africa in order to better understand the epidemiology of the virus in the region.

Molecular Characterization of Closely Related H6N2 Avian Influenza Viruses Isolated from Turkey, Egypt, and Uganda.

Genetic analysis of circulating avian influenza viruses (AIVs) in wild birds at different geographical regions during the same period could improve our knowledge about virus transmission dynamics in natural hosts, virus evolution as well as zoonotic potential. Here, we report the genetic and molecular characterization of H6N2 influenza viruses isolated from migratory birds in Turkey, Egypt, and Uganda during 2017-2018. The Egyptian and Turkish isolates were genetically closer to each other than they were to the virus isolated from Uganda. Our results also suggest that multiple reassortment events were involved in the genesis of the isolated viruses. All viruses contained molecular markers previously associated with increased replication and/or pathogenicity in mammals. The results of this study indicate that H6N2 viruses carried by migratory birds on the West Asian/East African and Mediterranean/Black Sea flyways have the potential to transmit to mammals including humans. Additionally, adaptation markers in these viruses indicate the potential risk for poultry, which also increases the possibility of human exposure to these viruses.

Antigenic and molecular characterization of low pathogenic avian influenza A(H9N2) viruses in sub-Saharan Africa from 2017 through 2019.

Sub-Saharan Africa was historically considered an animal influenza cold spot, with only sporadic highly pathogenic H5 outbreaks detected over the last 20 years. However, in 2017, low pathogenic avian influenza A(H9N2) viruses were detected in poultry in Sub-Saharan Africa. Molecular, phylogenetic, and antigenic characterization of isolates from Benin, Togo, and Uganda showed that they belonged to the G1 lineage. Isolates from Benin and Togo clustered with viruses previously described in Western Africa, whereas viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Viruses from Benin exhibited decreased cross-reactivity with those from Togo and Uganda, suggesting antigenic drift associated with reduced replication in Calu-3 cells. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2). Therefore, viral genetic and antigenic surveillance in Africa is of paramount importance to detect further evolution or emergence of new zoonotic strains.