An Integrated Ecological Niche Modelling Framework for Risk Mapping of Peste des Petits Ruminants Virus Exposure in African Buffalo (Syncerus caffer) in the Greater Serengeti-Mara Ecosystem.

Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants that threatens livelihoods and food security in developing countries and, in some cases, wild ungulate species conservation. The Greater Serengeti-Mara Ecosystem (GSME) encompasses one of the major wildlife populations of PPR virus (PPRV)-susceptible species left on earth, although no clinical disease has been reported so far. This study aimed to gain further knowledge about PPRV circulation in the GSME by identifying which factors predict PPRV seropositivity in African buffalo (Syncerus caffer). Following an ecological niche modeling framework to map host-pathogen distribution, two models of PPRV exposure and buffalo habitat suitability were performed using serological data and buffalo censuses. Western Maasai Mara National Reserve and Western Serengeti National Park were identified as high-risk areas for PPRV exposure in buffalo. Variables related to wildlife-livestock interaction contributed to the higher risk of PPRV seropositivity in buffalo, providing supportive evidence that buffalo acquire the virus through contact with infected livestock. These findings can guide the design of cost-effective PPRV surveillance using buffalo as a sentinel species at the identified high-risk locations. As more intensive studies have been carried out in Eastern GSME, this study highlights the need for investigating PPRV dynamics in Western GSME.

Peste Des Petits Ruminants in the Middle East: Epidemiological Situation and Status of Control and Eradication Activities after the First Phase of the PPR Global Eradication Program (2017-2021).

Peste des petits ruminants (PPR) is a burdensome viral disease primarily affecting small ruminants, which is currently targeted for eradication by 2030 through the implementation of a Global Control and Eradication Strategy (PPR GCES). The PPR GCES, launched in 2015, has strongly encouraged countries to participate in Regional PPR Roadmaps, designated according to the Food and Agricultural Organization of the United Nations (FAO) and World Organisation for Animal Health (WOAH) regions and epidemiological considerations, with each targeted by dedicated meetings and activities. Following the conclusion of the first phase of the PPR Global Eradication Program (PPR GEP) (2017-2021), the present work focuses on the disease situation and status of the eradication campaign in the fourteen countries of the PPR GCES Middle Eastern Roadmap as well as Egypt. PPR is endemic to or suspected to be present in most of the region, except for Bahrain, which, as of 2021, is preparing to apply for official recognition as being free of PPR. Some substantial shortcomings are observed in surveillance and disease reporting, as well as in the implemented control strategies, most notably vaccination. Since many of these limitations are shared by many of the investigated countries, the international cooperation and harmonization of control efforts appears crucial to making PPR eradication attainable in the Middle East.

Peste des Petits Ruminants in Central and Eastern Asia/West Eurasia: Epidemiological Situation and Status of Control and Eradication Activities after the First Phase of the PPR Global Eradication Programme (2017-2021).

Peste des petits ruminants (PPR) is a highly contagious infectious disease of small ruminants caused by peste des petits ruminants virus (PPRV). PPR poses a significant threat to sheep and goat systems in over 65 endemic countries across Africa, the Middle East and Asia. It is also responsible for devastating outbreaks in susceptible wildlife, threatening biodiversity. For these reasons, PPR is the target of the Global Eradication Programme (PPR GEP), launched in 2016, which is aimed at eradicating the disease by 2030. The end of the first five-year phase of the PPR GEP (2017-2021) provides an ideal opportunity to assess the status of the stepwise control and eradication process. This review analyses 13 countries belonging to Eastern Europe, Transcaucasia, and Central and East Asia. Substantial heterogeneity is apparent in terms of PPR presence and control strategies implemented by different countries. Within this region, one country is officially recognised as PPR-free, seven countries have never reported PPR, and two have had no outbreaks in the last five years. Therefore, there is real potential for countries in this region to move forward in a coordinated manner to secure official PPR freedom status and thus reap the trade and socioeconomic benefits of PPR eradication.

Molecular epidemiology of peste des petits ruminants virus emergence in critically endangered Mongolian saiga antelope and other wild ungulates.

Peste des petits ruminants virus (PPRV) causes disease in domestic and wild ungulates, is the target of a Global Eradication Programme, and threatens biodiversity. Understanding the epidemiology and evolution of PPRV in wildlife is important but hampered by the paucity of wildlife-origin PPRV genomes. In this study, full PPRV genomes were generated from three Mongolian saiga antelope, one Siberian ibex, and one goitered gazelle from the 2016-2017 PPRV outbreak. Phylogenetic analysis showed that for Mongolian and Chinese PPRV since 2013, the wildlife and livestock-origin genomes were closely related and interspersed. There was strong phylogenetic support for a monophyletic group of PPRV from Mongolian wildlife and livestock, belonging to a clade of lineage IV PPRV from livestock and wildlife from China since 2013. Discrete diffusion analysis found strong support for PPRV spread into Mongolia from China, and phylogeographic analysis indicated Xinjiang Province as the most likely origin, although genomic surveillance for PPRV is poor and lack of sampling from other regions could bias this result. Times of most recent common ancestor (TMRCA) were June 2015 (95 per cent highest posterior density (HPD): August 2014 to March 2016) for all Mongolian PPRV genomes and May 2016 (95 per cent HPD: October 2015 to October 2016) for Mongolian wildlife-origin PPRV. This suggests that PPRV was circulating undetected in Mongolia for at least 6 months before the first reported outbreak in August 2016 and that wildlife were likely infected before livestock vaccination began in October 2016. Finally, genetic variation and positively selected sites were identified that might be related to PPRV emergence in Mongolian wildlife. This study is the first to sequence multiple PPRV genomes from a wildlife outbreak, across several host species. Additional full PPRV genomes and associated metadata from the livestock-wildlife interface are needed to enhance the power of molecular epidemiology, support PPRV eradication, and safeguard the health of the whole ungulate community.

RISK FACTORS ASSOCIATED WITH YOLK SAC RETENTION IN CAPTIVE-BRED HUMBOLDT PENGUIN (SPHENISCUS HUMBOLDTI) CHICKS.

Multiple occurrences of yolk sac retention prompted a retrospective investigation in a recently formed colony of captive Humboldt penguins (Spheniscus humboldti). Necropsy reports of 141 parent-reared penguin chicks that died between January 2014 and December 2018 were reviewed for evidence of yolk sac retention, defined as the presence of a yolk sac at postmortem examination of a chick aged 7 d or greater, and analyzed by demographic and pathological variables for identification of risk factors. Fifty-nine (65%) chicks that died at age 7 d or greater had a retained yolk sac at postmortem examination, revealing that this was a common condition in penguins in this population. Chicks that retained their yolk sac were also more likely to present with minimal gut contents (P = 0.02), have a prominent bursa of Fabricius (P < 0.01), and be the first chick hatched of their clutch (P = 0.02). Parental experience and age were not predictive of yolk sac retention, but there was a trend for chicks with retained yolk sacs to present with a poorer body condition, reduced weight, and reduced crown-rump length compared to chicks without a retained yolk sac. Histopathological and bacteriological findings of retained yolk sacs were not significantly different from those of chicks under 7 d of age. Although likely to be multifactorial, the association between yolk sac retention and indicators of suboptimal feed intake and growth (empty gastrointestinal tract, poor body condition score, decreased crown-rump length, and decreased weight at death) is hypothesized to be a result of parental neglect, leading to starvation and absorption arrest of the yolk, as previously indicated in broiler chicks.

Progress towards Eradication of Peste des Petits Ruminants through Vaccination.

Peste des petits ruminants (PPR) is a transboundary viral disease that threatens more than 1.74 billion goats and sheep in approximately 70 countries globally. In 2015, the international community set the goal of eradicating PPR by 2030, and, since then, Food and Agriculture Organization of the United Nations (FAO) and World Organization for Animal Health (OIE) have jointly developed and implemented the Global Control and Eradication Strategy for PPR. Here, data from the United Nations Food and Agriculture Organization Statistical Database (FAOSTAT), the OIE World Animal Health Information System (WAHIS), Regional Roadmap Meetings, and countries' responses to PPR Monitoring and Assessment Tool (PMAT) questionnaires were analyzed to inform on current progress towards PPR eradication. OIE recorded the use of over 333 million doses of vaccine in 12 countries from 2015 to 2018, 41.8% of which were used in Asia and 58.2% in Africa. Between 2015 and 2019, a total of 12,757 PPR outbreaks were reported to OIE: 75.1% in Asia, 24.8% in Africa, and 0.1% in Europe. The number of global outbreaks in 2019 fell to 1218, compared with 3688 in 2015. Analysis of vaccine use and PPR outbreaks in countries indicates that disease control strategies, particularly vaccination campaigns and vaccine distribution strategies, still require scientific evaluation. It is imperative that vaccination is undertaken based on the epidemiology of the disease in a region and is coordinated between neighboring countries to restrict transboundary movements. Strengthening surveillance and post-vaccination sero-monitoring at the national level is also essential. The PPR vaccine stock/bank established by FAO, OIE, and other partners have improved the quality assurance and supply of vaccines. However, to achieve PPR eradication, filling the funding gap for vaccination campaigns and other program activities will be critical.

Eradication of Peste des Petits Ruminants Virus and the Wildlife-Livestock Interface.

Growing evidence suggests that multiple wildlife species can be infected with peste des petits ruminants virus (PPRV), with important consequences for the potential maintenance of PPRV in communities of susceptible hosts, and the threat that PPRV may pose to the conservation of wildlife populations and resilience of ecosystems. Significant knowledge gaps in the epidemiology of PPRV across the ruminant community (wildlife and domestic), and the understanding of infection in wildlife and other atypical host species groups (e.g., camelidae, suidae, and bovinae) hinder our ability to apply necessary integrated disease control and management interventions at the wildlife-livestock interface. Similarly, knowledge gaps limit the inclusion of wildlife in the FAO/OIE Global Strategy for the Control and Eradication of PPR, and the framework of activities in the PPR Global Eradication Programme that lays the foundation for eradicating PPR through national and regional efforts. This article reports on the first international meeting on, "Controlling PPR at the livestock-wildlife interface," held in Rome, Italy, March 27-29, 2019. A large group representing national and international institutions discussed recent advances in our understanding of PPRV in wildlife, identified knowledge gaps and research priorities, and formulated recommendations. The need for a better understanding of PPRV epidemiology at the wildlife-livestock interface to support the integration of wildlife into PPR eradication efforts was highlighted by meeting participants along with the reminder that PPR eradication and wildlife conservation need not be viewed as competing priorities, but instead constitute two requisites of healthy socio-ecological systems.

The Genetics of Life and Death: Virus-Host Interactions Underpinning Resistance to African Swine Fever, a Viral Hemorrhagic Disease.

Pathogen transmission from wildlife hosts to genetically distinct species is a major driver of disease emergence. African swine fever virus (ASFV) persists in sub-Saharan Africa through a sylvatic cycle between warthogs and soft ticks that infest their burrows. The virus does not cause disease in these animals, however transmission of the virus to domestic pigs or wild boar causes a hemorrhagic fever that is invariably fatal. ASFV transmits readily between domestic pigs and causes economic hardship in areas where it is endemic. The virus is also a significant transboundary pathogen that has become established in Eastern Europe, and has recently appeared in China increasing the risk of an introduction of the disease to other pig producing centers. Although a DNA genome mitigates against rapid adaptation of the virus to new hosts, extended epidemics of African swine fever (ASF) can lead to the emergence of viruses with reduced virulence. Attenuation in the field leads to large deletions of genetic material encoding genes involved in modulating host immune responses. Therefore resistance to disease and tolerance of ASFV replication can be dependent on both virus and host factors. Here we describe the different virus-host interfaces and discuss progress toward understanding the genetic determinants of disease outcome after infection with ASFV.

Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses.

IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals - pigs and bats - identified as major reservoirs for emerging viruses.

Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.

Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection.

Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8⁺ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8⁺ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection.

Mapping the IkappaB kinase beta (IKKbeta)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKbeta-mediated activation of nuclear factor kappaB.

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKß subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKß, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKß and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKß. In contrast, a second monomeric mutant (F130K) neither bound IKKß nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKß interaction. Thus, the dimerization and IKKß-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

The cytoplasmic location of chicken mx is not the determining factor for its lack of antiviral activity.

BACKGROUND: Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems. CONCLUSIONS/SIGNIFICANCE: The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.

Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays.

Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.