Evidence of coinfection of pigs with African swine fever virus and porcine circovirus 2.

Archival swine DNA samples from Indonesia and Mongolia, some of which were previously shown to be positive for African swine fever virus, were screened for the presence of porcine circovirus 2 (PCV-2) and porcine circovirus 3 (PCV-3) by PCR. Samples from both countries were positive for PCV-2 (three from Mongolia and two from Indonesia), while none were positive for PCV-3. The PCV-2 amplicons were sequenced, and phylogenetic analysis revealed that the PCV-2 strains belonged to four different genotypes: PCV-2a (Mongolia), PCV-2b (Mongolia and Indonesia), PCV-2d (Indonesia), and PCV-2g (Mongolia). This is the first report of ASFV/PCV-2 coinfection in pigs and the first report of the presence of PCV-2 in Mongolia.

Coinfections of African swine fever virus, porcine circovirus 2 and 3, and porcine parvovirus 1 in swine in Nigeria.

As pig production increases in Africa, it is essential to identify the pathogens that are circulating in the swine population to assess pig welfare and implement targeted control measures. For this reason, DNA samples collected from pigs in Nigeria in the context of African swine fever monitoring were further screened by PCR for porcine circovirus 2 (PCV-2), porcine circovirus 3 (PCV-3), and porcine parvovirus 1 (PPV1). Forty-seven (45%) pigs were positive for two or more pathogens. Sequence analysis identified PCV-2 genotypes a, b, and d, while limited genetic heterogenicity was observed among PCV-3 strains. All except one of the PPV1 sequences were genetically distinct from those previously identified in other countries.

First genetic characterization of peste des petits ruminants virus from Mongolia.

Between August and September 2016 pathological samples were collected from sheep and goats following suspected peste des petits ruminants (PPR) outbreaks in western Mongolia. RT-PCR followed by sequencing and phylogenetic analysis of the samples confirmed the presence of a PPR virus belonging to lineage IV. A full genome analysis of the viral RNA from one of the samples revealed a high similarity (99.0-99.5%) with PPR viruses currently circulating in China (2013-2015) indicating a common origin. This is the first genetic characterization of PPR virus in Mongolia and the data generated will have important implications for control and management of the disease in the region.

Highly pathogenic avian influenza H5N1 virus outbreak among Cape cormorants (Phalacrocorax capensis) in Namibia, 2022.

In January 2022, significant mortality was observed among Cape cormorants (Phalacrocorax capensis) on the west coast of Namibia. Samples collected were shown to be positive for H5N1 avian influenza by multiplex RT-qPCR. Full genome analysis and phylogenetic analysis identified the viruses as belonging to clade 2.3.4.4b and that it clustered with similar viruses identified in Lesotho and Botswana in 2021. This is the first genomic characterization of H5N1 viruses in Namibia and has important implications for poultry disease management and wildlife conservation in the region.

A novel multiplex qPCR­HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats.

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe.

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia.

A Phylogeographic Analysis of Porcine Parvovirus 1 in Africa.

Porcine parvovirus 1 (PPV1) is recognized as a major cause of reproductive failure in pigs, leading to several clinical outcomes globally known as SMEDI. Despite being known since the late 1960s its circulation is still of relevance to swine producers. Additionally, the emergence of variants such as the virulent 27a strain, for which lower protection induced by vaccines has been demonstrated, is of increasing concern. Even though constant monitoring of PPV1 using molecular epidemiological approaches is of pivotal importance, viral sequence data are scarce especially in low-income countries. To fill this gap, a collection of 71 partial VP2 sequences originating from eight African countries (Burkina Faso, Côte d'Ivoire, Kenya, Mozambique, Namibia, Nigeria, Senegal, and Tanzania) during the period 2011-2021 were analyzed within the context of global PPV1 variability. The observed pattern largely reflected what has been observed in high-income regions, i.e., 27a-like strains were more frequently detected than less virulent NADL-8-like strains. A phylogeographic analysis supported this observation, highlighting that the African scenario has been largely shaped by multiple PPV1 importation events from other continents, especially Europe and Asia. The existence of such an international movement coupled with the circulation of potential vaccine-escape variants requires the careful evaluation of the control strategies to prevent new strain introduction and persistence.

Viral Co-Infections of Warthogs in Namibia with African Swine Fever Virus and Porcine Parvovirus 1.

Understanding virus circulation in wild animals, particularly those that have contact with domestic animals, is crucial for disease management and control. In Africa, warthogs are known to be asymptomatic carriers of porcine pathogens; a recent study in Namibia has shown them to be positive for Porcine circovirus-2 (PCV-2). In this study, the same samples used for the PCV-2 investigation in Namibia were further screened for the presence of African swine fever virus (ASFV) and porcine parvovirus 1 (PPV1) by PCR. Of the 42 animals tested, 2 (4.8%) and 13 (31%) were positive for AFSV and PPV1, respectively. The two AFSV were also co-infected with PPV1. Combing the results of this study with the results of the previous PCV-2 investigation, four warthogs were shown to be co-infected with both PPV1 and PCV-2. Sequence and phylogenetic analysis revealed that the AFSV belonged to genotype (Ib) but were from different serogroups. Unexpectedly, the ASFVs from the warthogs were genetically distinct to those observed in an outbreak in the same region of Namibia that occurred less than fifteen months prior to the sampling of the warthogs. In fact, a stronger genetic relationship was observed between the warthog viruses and historical Namibian and South African ASFVs identified in 1980, 2004 and 2008. For the PPV1s, the closest relative to the Namibian PPV1 were viruses identified in wild boar in Romania in 2011. This study confirms that warthogs are carriers of porcine pathogens and the data should encourage further studies on larger populations of wild and domestic swine to more fully understand the epidemiology and transmission of viral pathogens from these species.

Comparison of the sensitivity, specificity, correlation and inter-assay agreement of eight diagnostic in vitro assays for the detection of African swine fever virus.

With the recent spread of African swine fever (ASF) in Europe, Asia and the Caribbean region, after being endemic for decades in Africa, PCR-based commercial kits and various master mixes are increasingly being used in addition to the Office International des Epizooties-recommended protocol from King et al. (World Organization for Animal Health). Often, the availability and cost of commercial kits or master mixes can be a limiting factor for diagnostic laboratories, in addition to the requirements for transportation and storage of temperature-sensitive reagents in remote areas. In such cases, alternatives should be ready to maximize surveillance and mining of ASF. To evaluate alternatives, we tested five commercial quantitative real-time PCR (qPCR) master mixes from Applied Biosystems, Bio-Rad, Biotechrabbit, Promega and Qiagen using the same primers and probe mix derived from the King et al.'s protocol for the sensitivity, specificity, correlation and inter-assay agreement. We further included three ad hoc molecular diagnostic kits (VetMax™ African Swine Fever Virus Detection Kit [Applied Biosystems], ID Gene African Swine Fever Duplex [ID-Vet] and Virotype ASF PCR Kit [Qiagen/Indical]). The limit of detection (LOD) was assessed for each assay. The comparative study panel comprised 83 archived DNA samples from ASF virus (ASFV) clinical samples, belonging to five different genotypes from outbreaks in 16 countries in Asia and Africa. The analytical specificity was assessed against a panel of swine pathogens. The LOD ranged from 13 to 41 gene copies per reaction; VetMax ™ African Swine Fever Virus Detection Kit from Applied Biosystems exhibited the lowest detection limit (13 gene copies per reaction) and iQ Supermix from Bio-Rad the highest detection limit (41 gene copies per reaction). Cq values obtained from the lowest dilution, in which all replicates (n = 25) could still be amplified (50 gene copies per reaction), were not significantly different between kits using Kruskal-Wallis test. Inter-assay agreement was assessed using statistical test Fleiss-Kappa and was shown to be excellent in all cases. Agreement using statistical test Bland-Altman was good for samples with Cq values <25 and moderate for Cq values >25. We conclude that all the assays evaluated in this study can be used for the routine detection of ASFV.

Highly pathogenic avian influenza (A/H5N1) virus outbreaks in Lesotho, May 2021.

In May 2021, Lesotho reported its first outbreak of highly pathogenic avian influenza (HPAI) to the OIE. Samples were collected from infected poultry and the virus was confirmed by molecular tests to be of the H5N1 subtype. Full genome sequencing and phylogenetic analysis revealed that the viruses belonged to clade 2.3.4.4b and showed high identity with A/H5N1 viruses identified in Nigeria and Senegal in early 2021. The identification of A/H5N1 HPAI in Lesotho has important implications for disease management and food security in the region.

Identification of porcine circovirus-3 in Mozambique.

Porcine circovirus 3 (PCV-3) has been associated with an assortment of clinical conditions in pigs and has been reported in many countries worldwide. In Africa there is no data on the presence of PCV-3. In this study, DNA samples collected from 91 pigs between 2011 and 2019 in nine of the ten provinces of Mozambique in the context of African swine fever (ASF) monitoring were further screened for the presence of PCV-3. Of these samples, 7 (7.5%) animals were positive for PCV-3. Sequence and phylogenetic analysis of the capsid protein gene (ORF2) of the PCV-3s provided evidence of epidemiological links with PCV-3s identified in North and South America, Asia, and Europe. This is the first identification of PCV-3 in Mozambique (and Africa) and the first evidence of co-infection of PCV-3 and ASF virus. It should provide a starting point for further investigations into the presence and impact of PCV-3 in Africa.

Isolation and Identification of a Highly Pathogenic Avian Influenza H5N6 Virus from Migratory Waterfowl in Western Mongolia.

In April 2020, two Whooper Swans (Cygnus cygnus) and one Swan Goose (Anser cygnoides) were found dead at three different locations in western Mongolia. Virus isolation from organs taken from the carcasses and full genome sequencing revealed that all three birds were positive for highly pathogenic H5N6 avian influenza virus (HPAIV) belonging to subclade 2.3.4.4h. Confirming similar reports from central Mongolia and western China, these findings have important implications for the monitoring, control, and management of HPAIVs in wild bird and commercial poultry populations in Mongolia.

Emergence of High Pathogenicity Avian Influenza Virus H5N1 Clade 2.3.4.4b in Wild Birds and Poultry in Botswana.

Numerous outbreaks of high-pathogenicity avian influenza (HPAI) were reported during 2020-2021. In Africa, H5Nx has been detected in Benin, Burkina Faso, Nigeria, Senegal, Lesotho, Namibia and South Africa in both wild birds and poultry. Botswana reported its first outbreak of HPAI to the World Organisation for Animal Health (WOAH) in 2021. An H5N1 virus was detected in a fish eagle, doves, and chickens. Full genome sequence analysis revealed that the virus belonged to clade 2.3.4.4b and showed high identity within haemagglutinin (HA) and neuraminidase proteins (NA) for viruses identified across a geographically broad range of locations. The detection of H5N1 in Botswana has important implications for disease management, wild bird conservation, tourism, public health, economic empowerment of vulnerable communities and food security in the region.

Porcine circovirus-2 in Africa: Identification of continent-specific clusters and evidence of independent viral introductions from Europe, North America and Asia.

Porcine circovirus-2 (PCV-2) is associated with several disease syndromes in domestic pigs that have a significant impact on global pig production and health. Currently, little is known about the status of PCV-2 in Africa. In this study, a total of 408 archived DNA samples collected from pigs in Burkina Faso, Cameroon, Cape Verde, Ethiopia, the Democratic Republic of the Congo, Mozambique, Nigeria, Senegal, Tanzania and Zambia between 2000 and 2018 were screened by PCR for the presence of PCV-2. Positive amplicons of the gene encoding the viral capsid protein (ORF2) were sequenced to determine the genotypes circulating in each country. Four of the nine currently known genotypes of PCV-2 were identified (i.e. PCV-2a, PCV-2b, PCV-2d and PCV-2 g) with more than one genotype being identified in Burkina Faso, Ethiopia, Nigeria, Mozambique, Senegal and Zambia. Additionally, a phylogeographic analysis which included 38 additional ORF2 gene sequences of PCV-2s previously identified in Mozambique, Namibia and South Africa from 2014 to 2016 and 2019 to 2020 and available in public databases, demonstrated the existence of several African-specific clusters and estimated the approximate time of introduction of PCV-2s into Africa from other continents. This is the first in-depth study of PCV-2 in Africa and it has important implications for pig production at both the small-holder and commercial farm level on the continent.

Comparison of eleven in vitro diagnostic assays for the detection of SARS-CoV-2 RNA.

Transmission mitigation of SARS-CoV-2 requires the availability of accurate and sensitive detection methods. There are several commercial ad hoc molecular diagnostic kits currently on the market, many of which have been evaluated by different groups. However, in low resource settings the availability and cost of these commercial kits can be a limiting factor for many diagnostic laboratories. In such cases alternatives need to be identified. With this in mind, eight commercial reverse transcription quantitative real-time PCR (RT-qPCR) master mixes from Applied Biosystems (Thermo Fisher Scientific), Bio-Rad, Biotech Rabbit, Promega, Qiagen, QuantaBio, Invitrogen (Thermo Fisher Scientific) and Takara using the same commercial primer and probe mix [LightMix® Modular SARS and Wuhan CoV E-gene mix (TIB MolBiol, Germany)] were evaluated. Three ad hoc molecular diagnostic kits [GeneFinder™ COVID-19 Plus RealAmp kit (Osang Healthcare); genesig® Real-Time PCR Coronavirus COVID-19 (Primerdesign); and ViroReal® Kit SARS-CoV-2 & SARS-CoV (Ingenetix)] were also included in the study. The limit of detection was calculated for each assay using serial dilutions of a defined clinical sample. The performances of the assays were compared using a panel of 178 clinical samples and their analytical specificity assessed against a panel of human betacoronaviruses. Inter assay agreement was assessed using statistical tests (Bland-Altman, Fleiss-Kappa and Cohen's Kappa) and was shown to be excellent to good in all cases. We conclude that all of the assays evaluated in this study can be used for the routine detection of SARS-CoV-2 and that the RT-qPCR master mixes are a valid alternative to ad hoc molecular diagnostic kits.

Detection of Lumpy Skin Disease Virus in an Asymptomatic Eland (Taurotragus oryx) in Namibia.

Nasal swabs collected from 40 wild ruminants in Namibia were analyzed by PCR for the presence of lumpy skin disease virus (LSDV) DNA. One sample from an asymptomatic eland (Taurotragus oryx) tested positive, providing the first evidence of the presence of LSDV DNA in an eland.

Transboundary spread of equine influenza viruses (H3N8) in West and Central Africa: Molecular characterization of identified viruses during outbreaks in Niger and Senegal, in 2019.

Since November 2018, several countries in West and Central Africa have reported mortalities in donkeys and horses. Specifically, more than 66,000 horses and donkeys have succumbed to disease in Burkina Faso, Chad, Cameroon, The Gambia, Ghana, Mali, Niger, Nigeria, and Senegal. Strangles caused by Streptococcus equi subsp equi, African Horse Sickness (AHS) virus, and Equine influenza virus (EIV) were all suspected as potential causative agents. This study reports the identification of EIV in field samples collected in Niger and Senegal. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the identified viruses belonged to clade 1 of the Florida sublineage and were very similar to viruses identified in Nigeria in 2019. Interestingly, they were also more similar to EIVs from recent outbreaks in South America than to those in Europe and the USA. This is one of the first reports providing detailed description and characterization of EIVs in West and Central Africa region.

Molecular characterization of African swine fever virus from outbreaks in Namibia in 2018.

Five samples were collected from four suspected outbreaks of African swine fever in Namibia in 2018. Sequencing of the C-terminus of the B646L gene (p72 protein), the central hypervariable region (CVR) of the B602L gene, the E183L gene (p54 protein) and the CD2v (used to determine the serogroup) was performed on DNA isolated from the samples. Phylogenetic analyses of the B646L (p72) revealed that one of the samples belonged to genotype I while the remaining samples could not be assigned to any currently known genotype. In contrast, by using the E183L gene three of the samples were shown to belong to genotype Id and only two were of unknown genotype. Based on the analysis of the partial CD2v amino acid sequences of four of the samples, one of the viruses clustered with serogroup 2 while the other three did not cluster within any of the eight known serogroups. Examination of the CVR identified three variants with 8, 18 and 24 tetrameric tandem repeat sequences. This study indicates that at least three different genetically distinct ASFV are currently present in Namibia.

Molecular characterization of peste-des-petits ruminants virus from Nepal, 2005 to 2016.

Outbreaks of peste-des-petits ruminants (PPR) has been reported regularly in Nepal since 1994. Despite this, there has been limited molecular characterization of circulating virus in the country. In this study a 351 bp segment of the nucleoprotein gene of the PPR virus (PPRV) was amplified and sequenced from ten samples collected between 2005 and 2016. Phylogenetic trees were estimated from these sequences using the maximum likelihood method confirming that all of the PPRV from the samples analysed belonged to the sub-clade IV of clade I of lineage IV and that they shared a common origin with other PPRV isolates in the region.