Molecular basis of hippopotamus ACE2 binding to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide range of hosts, including hippopotami, which are semi-aquatic mammals and phylogenetically closely related to Cetacea. In this study, we characterized the binding properties of hippopotamus angiotensin-converting enzyme 2 (hiACE2) to the spike (S) protein receptor binding domains (RBDs) of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs). Furthermore, the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 PT S protein complexed with hiACE2 was resolved. Structural and mutational analyses revealed that L30 and F83, which are specific to hiACE2, played a crucial role in the hiACE2/SARS-CoV-2 RBD interaction. In addition, comparative and structural analysis of ACE2 orthologs suggested that the cetaceans may have the potential to be infected by SARS-CoV-2. These results provide crucial molecular insights into the susceptibility of hippopotami to SARS-CoV-2 and suggest the potential risk of SARS-CoV-2 VOCs spillover and the necessity for surveillance. IMPORTANCE: The hippopotami are the first semi-aquatic artiodactyl mammals wherein SARS-CoV-2 infection has been reported. Exploration of the invasion mechanism of SARS-CoV-2 will provide important information for the surveillance of SARS-CoV-2 in hippopotami, as well as other semi-aquatic mammals and cetaceans. Here, we found that hippopotamus ACE2 (hiACE2) could efficiently bind to the RBDs of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs) and facilitate the transduction of SARS-CoV-2 PT and VOCs pseudoviruses into hiACE2-expressing cells. The cryo-EM structure of the SARS-CoV-2 PT S protein complexed with hiACE2 elucidated a few critical residues in the RBD/hiACE2 interface, especially L30 and F83 of hiACE2 which are unique to hiACE2 and contributed to the decreased binding affinity to PT RBD compared to human ACE2. Our work provides insight into cross-species transmission and highlights the necessity for monitoring host jumps and spillover events on SARS-CoV-2 in semi-aquatic/aquatic mammals.

Genomic Characterization of a Novel Alphacoronavirus Isolated from Bats, Korea, 2020.

Coronavirus, an important zoonotic disease, raises concerns of future pandemics. The bat is considered a source of noticeable viruses resulting in human and livestock infections, especially the coronavirus. Therefore, surveillance and genetic analysis of coronaviruses in bats are essential in order to prevent the risk of future diseases. In this study, the genome of HCQD-2020, a novel alphacoronavirus detected in a bat (Eptesicus serotinus), was assembled and described using next-generation sequencing and bioinformatics analysis. The comparison of the whole-genome sequence and the conserved amino acid sequence of replicated proteins revealed that the new strain was distantly related with other known species in the Alphacoronavirus genus. Phylogenetic construction indicated that this strain formed a separated branch with other species, suggesting a new species of Alphacoronavirus. Additionally, in silico prediction also revealed the risk of cross-species infection of this strain, especially in the order Artiodactyla. In summary, this study provided the genetic characteristics of a possible new species belonging to Alphacoronavirus.

Mammalian Orthoreovirus (MRV) Is Widespread in Wild Ungulates of Northern Italy.

Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR; confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.

A novel Betacoronavirus characterised in collared peccaries from the Rio de Janeiro Zoo (Brazil) killed by unknown disease.

In an enclosure with nine collared peccaries (Pecari tajacu) from the Rio de Janeiro city Zoo, Brazil, one specimen was found dead and two others developed prostration, apathy and dehydration, resulting on its death. Necropsy of two animals pointed to pulmonary and renal damage. Histological examination revealed vasculitis in spleen from both P. tajacu, suggesting a systemic viral infection. Lungs from one specimen showed fibrinoid vasculitis, alveolar damage with hyaline membrane, and interstitial lymphocytes infiltration. Virome analysis in anal wash samples from the latter two animals revealed a new type of Betacoronavirus, lineage A, provisionally named Ptajacu-CoV.

A novel Betacoronavirus characterised in collared peccaries from the Rio de Janeiro Zoo (Brazil) killed by unknown disease

In an enclosure with nine collared peccaries (Pecari tajacu) from the Rio de Janeiro city Zoo, Brazil, one specimen was found dead and two others developed prostration, apathy and dehydration, resulting on its death. Necropsy of two animals pointed to pulmonary and renal damage. Histological examination revealed vasculitis in spleen from both P. tajacu, suggesting a systemic viral infection. Lungs from one specimen showed fibrinoid vasculitis, alveolar damage with hyaline membrane, and interstitial lymphocytes infiltration. Virome analysis in anal wash samples from the latter two animals revealed a new type of Betacoronavirus, lineage A, provisionally named Ptajacu-CoV.

Development and validation of a pen side test for Rift Valley fever.

BACKGROUND: Rift Valley fever (RVF) is one of the main vector borne zoonotic diseases that affects a wide range of ruminants and human beings in Africa and the Arabian Peninsula. A rapid and specific test for RVF diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. METHODOLOGY/PRINCIPAL FINDINGS: A first-line lateral flow immunochromatographic strip test (LFT) was developed for the detection of the nucleoprotein (N) of the RVF virus (RVFV). Its diagnostic performance characteristics were evaluated using reference stocks isolates recovered from different hosts and in geographic regions mimicking clinical specimens and from known RVF negative serum samples. A high level of diagnostic accuracy (DSe (35/35), DSp (167/169)) was observed, including the absence of cross-reactivity with viruses belonging to different genera. CONCLUSION/SIGNIFICANCE: The fact no specialized reagents and laboratory equipment are needed, make this assay a valuable, first-line diagnostic tool in resource-poor diagnostic territories for on-site RVFV detection, however the staff require training.

Determination of the optimal method for the concentration and purification of 146S particles for foot-and-mouth disease vaccine production.

After the severe outbreak of foot-and-mouth disease (FMD) in South Korea in 2010, the Korean government implemented a vaccination policy and set out to develop an FMD vaccine using a local FMD virus (FMDV) strain. As a part of the basic research for domestic FMD vaccine development, three methods commonly used for the concentration and purification of FMDV to produce FMD vaccine antigens were compared. Among common concentration methods, including polyethylene glycol (PEG) precipitation, ammonium sulfate precipitation, and ultrafiltration, the most effective method both for concentrating 146S particles and eliminating non-structural proteins (NSPs) was found to be PEG precipitation. Classical PEG precipitation showed the highest recovery of 146S particles (85.4%) with removing 99.8% of the other proteins, including NSPs. To the author's knowledge, this is the first study to compare the current three methods with regard to quantifying intact virus particles (146S). These findings may provide important insights for the development of new FMD vaccines using a local FMDV strain in the near future.

Collared peccary (Pecari tajacu) are susceptible to porcine reproductive and respiratory syndrome virus (PRRSV).

Collared peccary (Pecari tajacu) and pigs (Sus scrofa) are two members of superfamily Suoidea that coexist in the Americas and share some of the same viral infections. Although porcine reproductive and respiratory syndrome virus (PRRSV) is among the most impactful pathogens of swine on a worldwide basis, the susceptibility of peccaries to PRRSV has not been investigated. In this study, three peccaries were intramuscularly inoculated with a PRRSV-2 field virus. One PRRSV-inoculated pig served as a positive control and two pigs and one peccary as negative controls. Serum samples were collected at regular intervals over a 23-day observation period and tested by PRRSV rtRT-PCR and isotype-specific (IgM, IgA, IgG) PRRSV ELISAs. The detection of viremia (DPI 3-23) and a PRRSV-specific humoural immune response (≥DPI 10) supported the conclusion that collared peccary are susceptible to PRRSV. The results raise questions regarding the natural history of PRRSV in non-Sus members of superfamily Suoidea and, more broadly, their role in the evolution and ecology of PRRSV.

Detection of persistent pestivirus infection in pudú (Pudu puda) in a captive population of artiodactyls in Chile.

BACKGROUND: Bovine Viral Diarrhea Virus (BVDV) is the viral agent causing the most important economic losses in livestock throughout the world. Infection of fetuses before their immunological maturity causes the birth of animals persistently infected with BVDV (PI), which are the main source of infection and maintenance of this pathogen in a herd. There is evidence of susceptibility to infection with BVDV in more than 50 species of the order Artiodactyla, and the ability to establish persistent infection in wild cervid species of South America could represent an important risk in control and eradication programs of BVDV in cattle, and a threat to conservation of these wild species. In this study, a serological and virological study was performed to detect BVDV infection in a captive population of non-bovine artiodactyl species in a Chilean zoo with antecedents of abortions whose pathology suggests an infectious etiology. RESULTS: Detection of neutralizing antibodies against BVDV was performed in 112 artiodactyl animals from a zoo in Chile. Three alpacas (Vicugna pacos), one guanaco (Lama guanicoe) and seven pudús (Pudu puda) resulted seropositive, and the only seronegative pudú was suspected to be persistently infected with BVDV. Then two blood samples nine months apart were analyzed by a viral neutralization test and RT-PCR. Non-cytopathogenic BVDVs were isolated in both samples. A phylogenetic analysis showed that the virus was highly related to BVDV-1b strains circulating among Chilean cattle. CONCLUSIONS: This is the first report of a South American deer persistently infected with Bovine Viral Diarrhea Virus. Further studies are needed to determine the possible role of BVDV as a pathogen in pudús and as a threat to their conservation.

Role of Food Insecurity in Outbreak of Anthrax Infections among Humans and Hippopotamuses Living in a Game Reserve Area, Rural Zambia.

In September 2011, a total of 511 human cases of anthrax (Bacillus anthracis) infection and 5 deaths were reported in a game management area in the district of Chama, Zambia, near where 85 hippopotamuses (Hippopotamus amphibious) had recently died of suspected anthrax. The human infections generally responded to antibiotics. To clarify transmission, we conducted a cross-sectional, interviewer-administered household survey in villages where human anthrax cases and hippopotamuses deaths were reported. Among 284 respondents, 84% ate hippopotamus meat before the outbreak. Eating, carrying, and preparing meat were associated with anthrax infection. Despite the risk, 23% of respondents reported they would eat meat from hippopotamuses found dead again because of food shortage (73%), lack of meat (12%), hunger (7%), and protein shortage (5%). Chronic food insecurity can lead to consumption of unsafe foods, leaving communities susceptible to zoonotic infection. Interagency cooperation is necessary to prevent outbreaks by addressing the root cause of exposure, such as food insecurity.

Exposure of Asian Elephants and Other Exotic Ungulates to Schmallenberg Virus.

Schmallenberg virus (SBV) is an emerging Orthobunyavirus, first described in 2011 in cattle in Germany and subsequently spread throughout Europe, affecting mainly ruminant livestock through the induction of foetal malformations. To gain a better understanding of the spectrum of susceptible species and to assess the value of current SBV serological assays, screening of serum samples from exotic artiodactyls and perissodactyls collected at the Living Collections from the Zoological Society of London (Whipsnade and London Zoos) and Chester Zoo was carried out. There was compelling evidence of SBV infection in both zoological collections. The competitive ELISA has proved to be applicable for the detection of SBV in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in endangered Asian elephants (Elephas maximus), but unreliable for the screening of Camelidae, for which the plaque reduction neutralisation test was considered the assay of choice.

Malignant catarrhal fever: inching toward understanding.

Malignant catarrhal fever (MCF) is an often lethal infection of many species in the order Artiodactyla. It is caused by members of the MCF virus group within Gammaherpesvirinae. MCF is a worldwide problem and has a significant economic impact on highly disease-susceptible hosts, such as cattle, bison, and deer. Several epidemiologic forms of MCF, defined by the reservoir ruminant species from which the causative virus arises, are recognized. Wildebeest-associated MCF (WA-MCF) and sheep-associated MCF (SA-MCF) are the most prevalent and well-studied forms of the disease. Historical understanding of MCF is largely based on WA-MCF, in which the causative virus can be propagated in vitro. Characterization of SA-MCF has been constrained because the causative agent has never been successfully propagated in vitro. Development of molecular tools has enabled more definitive studies on SA-MCF. The current understanding of MCF, including its etiological agents, epidemiology, pathogenesis, and prevention, is the subject of the present review.

Complete genomic sequence analyses of the first group A giraffe rotavirus reveals close evolutionary relationship with rotaviruses infecting other members of the Artiodactyla.

Group A Rotaviruses (RVA) have been established as significant contributory agents of acute gastroenteritis in young children and many animal species. In 2008, we described the first RVA strain detected in a giraffe calf (RVA/Giraffe-wt/IRL/GirRV/2008/G10P[11]), presenting with acute diarrhoea. Molecular characterisation of the VP7 and VP4 genes revealed the bovine-like genotypes G10 and P[11], respectively. To further investigate the origin of this giraffe RVA strain, the 9 remaining gene segments were sequenced and analysed, revealing the following genotype constellation: G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H3. This genotype constellation is very similar to RVA strains isolated from cattle or other members of the artiodactyls. Phylogenetic analyses confirmed the close relationship between GirRV and RVA strains with a bovine-like genotype constellation detected from several host species, including humans. These results suggest that RVA strain GirRV was the result of an interspecies transmission from a bovine host to the giraffe calf. However, we cannot rule out completely that this bovine-like RVA genotype constellation may be enzootic in giraffes. Future RVA surveillance in giraffes may answer this intriguing question.

Rift Valley fever risk map model and seroprevalence in selected wild ungulates and camels from Kenya.

Since the first isolation of Rift Valley fever virus (RVFV) in the 1930s, there have been multiple epizootics and epidemics in animals and humans in sub-Saharan Africa. Prospective climate-based models have recently been developed that flag areas at risk of RVFV transmission in endemic regions based on key environmental indicators that precede Rift Valley fever (RVF) epizootics and epidemics. Although the timing and locations of human case data from the 2006-2007 RVF outbreak in Kenya have been compared to risk zones flagged by the model, seroprevalence of RVF antibodies in wildlife has not yet been analyzed in light of temporal and spatial predictions of RVF activity. Primarily wild ungulate serum samples from periods before, during, and after the 2006-2007 RVF epizootic were analyzed for the presence of RVFV IgM and/or IgG antibody. Results show an increase in RVF seropositivity from samples collected in 2007 (31.8%), compared to antibody prevalence observed from 2000-2006 (3.3%). After the epizootic, average RVF seropositivity diminished to 5% in samples collected from 2008-2009. Overlaying maps of modeled RVF risk assessments with sampling locations indicated positive RVF serology in several species of wild ungulate in or near areas flagged as being at risk for RVF. Our results establish the need to continue and expand sero-surveillance of wildlife species Kenya and elsewhere in the Horn of Africa to further calibrate and improve the RVF risk model, and better understand the dynamics of RVFV transmission.

Detection and characterisation of papillomavirus in skin lesions of giraffe and sable antelope in South Africa.

Papillomavirus was detected electron microscopically in cutaneous fibropapillomas of a giraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particles measured 45 nm in diameter. Histopathologically, the lesions showed histopathological features similar to those of equine sarcoid as well as positive immunoperoxidase-staining of tissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detected bovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised by real-time PCR in the sable and giraffe, and cloning and sequencing of the PCR product revealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealed locally malignant pleomorphism, possibly indicating the lesional end-point of papilloma infection. Neither virus particles nor positively staining papillomavirus antigen could be demonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCR which corresponded with BPV-1 and BPV-2.

Pathology and immunohistochemistry of papillomavirus-associated cutaneous lesions in Cape mountain zebra, giraffe, sable antelope and African buffalo in South Africa.

Skin lesions associated with papillomaviruses have been reported in many animal species and man. Bovine papillomavirus (BVP) affects mainly the epidermis, but also the dermis in several species including bovine, the best-known example being equine sarcoid, which is associated with BVP types 1 and 2. This publication describes and illustrates the macroscopic and histological appearance of BPV-associated papillomatous, fibropapillomatous or sarcoid-like lesions in Cape mountain zebra (Equus zebra zebra) from the Gariep Dam Nature Reserve, 2 giraffes (Giraffa camelopardalis) from the Kruger National Park, and a sable antelope (Hippotragus niger) from the Kimberley area of South Africa. An African buffalo (Syncerus caffer) cow from Kruger National Park also had papillomatous lesions but molecular characterisation of lesional virus was not done. Immunohistochemical staining using polyclonal rabbit antiserum to chemically disrupted BPV-1, which cross-reacts with the L1 capsid of most known papillomaviruses, was positive in cells of the stratum granulosum of lesions in Giraffe 1, the sable and the buffalo and negative in those of the zebra and Giraffe 2. Fibropapillomatous and sarcoid-like lesions from an adult bovine were used as positive control for the immunohistochemistry and are described and the immunohistochemistry illustrated for comparison. Macroscopically, both adult female giraffe had severely thickened multifocal to coalescing nodular and occasionally ulcerated lesions of the head, neck and trunk with local poorly-circumscribed invasion into the subcutis. Necropsy performed on the 2nd giraffe revealed neither internal metastases nor serious underlying disease. Giraffe 1 had scattered, and Giraffe 2 numerous, large, anaplastic, at times indistinctly multinucleated dermal fibroblasts with bizarre nuclei within the sarcoid-like lesions, which were BPV-1 positive in Giraffe 1 and BPV-1 and -2 positive in Giraffe 2 by RT-PCR. The sable antelope presented with a solitary large lesion just proximal to the right hind hoof, which recurred after excision, and was BPV-1 positive by RT-PCR. Other wart-like growths were present elsewhere on the body. The Cape mountain zebra either succumbed from their massive lesions or were euthanased or removed from the herd because of them. The lesions were BPV-1 and/or -2 positive by RT-PCR. The buffalo lesions were wart-like papillomatous projections in the inguinal and udder region. Stratum granulosum cells that stained immunohistochemically positive in the various species appeared koilocyte-like, as described in human papillomaviral lesions.

Full genomic analysis of a G8P[1] rotavirus strain isolated from an asymptomatic infant in Kenya provides evidence for an artiodactyl-to-human interspecies transmission event.

Group A rotavirus (GAR) G8P[1] strains, found sometimes in cattle, have been reported rarely from humans. Therefore, analysis of the full genomes of human G8P[1] strains are of significance in the context of studies on interspecies transmission of rotaviruses. However, to date, only partial-length nucleotide sequences are available for the 11 genes of a single human G8P[1] strain, while the partial sequences of two other strains have been reported. The present study reports the first complete genome sequence of a human G8P[1] strain, B12, detected from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the full-length nucleotide sequences of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of strain B12 were assigned to G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotypes, respectively. Each of the 11 genes of strain B12 appeared to be more related to cognate genes of artiodactyl (ruminant and/or camelid) and/or artiodactyl-derived human GAR strains than those of most other rotaviruses. Strain B12 exhibited low levels of genetic relatedness to canonical human GAR strains, such as Wa and DS-1, ruling out the possibility of its origin from reassortment events between artiodactyl-like human and true human strains. These observations suggest that strain B12 might have been directly transmitted from artiodactyls to humans. Unhygienic conditions and close proximity of humans to livestock at the sampling site might have facilitated this rare event. This is the first report on a full genomic analysis of a rotavirus strain from Kenya. To our knowledge, strain B12 might be the oldest G8 strain characterized molecularly from the Africa continent.

Ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses.

Understanding viral factors that promote cross-species transmission is important for evaluating the risk of zoonotic emergence. We constructed a database of viruses of domestic artiodactyls and examined the correlation between traits linked in the literature to cross-species transmission and the ability of viruses to infect humans. Among these traits--genomic material, genome segmentation, and replication without nuclear entry--the last is the strongest predictor of cross-species transmission. This finding highlights nuclear entry as a barrier to transmission and suggests that the ability to complete replication in the cytoplasm may prove to be a useful indicator of the threat of cross-species transmission.

Evolutionary origin of the Jiv90 gene of Pestivirus.

Phylogenetic analyses were used to test the hypothesis that the Jiv90 insert in the genomes of Pestivirus species originated by horizontal transfer from their hosts (mammals in the order Artiodactyla). The results supported this hypothesis because the Jiv90 insert clustered with the corresponding domains of mammalian Jiv proteins and closer to artiodactyl than to rodent or primate genes. A phylogeny of Pestivirus genotypes showed that the Jiv90 insert occurred only in one clade (Pestivirus Type 1), although numerous members of this clade lacked the insert. This pattern is probably most easily explained on the hypothesis that the insert occurred in the common ancestor of the Type 1 clade and that it has been subsequently lost independently by several members of the clade.

Novel herpesviruses of Suidae: indicators for a second genogroup of artiodactyl gammaherpesviruses.

Five novel herpesviruses were identified in suid species from Africa (common warthog, Phacochoerus africanus) and South-East Asia (bearded pig, Sus barbatus; babirusa, Babyrousa babyrussa) by detection and analysis of their DNA polymerase genes. Three of the novel species, P. africanus cytomegalovirus 1, P. africanus lymphotropic herpesvirus 1 (PafrLHV-1) and S. barbatus lymphotropic herpesvirus 1 (SbarLHV-1), were closely related to known beta- (porcine cytomegalovirus) and gammaherpesviruses [porcine lymphotropic herpesvirus (PLHV) 1 and 3] of domestic pigs. In contrast, two novel species, S. barbatus rhadinovirus 1 (SbarRHV-1) and Babyrousa babyrussa rhadinovirus 1 (BbabRHV-1), were more closely related to a ruminant gammaherpesvirus, bovine herpesvirus 4 (BoHV-4), than to the porcine gammaherpesviruses PLHV-1, -2, -3, PafrLHV-1 and SbarLHV-1. SbarRHV-1, BbabRHV-1 and BoHV-4 were therefore tentatively assigned to a novel genogroup of artiodactyl gammaherpesviruses. This latter genogroup may also contain an as yet undiscovered gammaherpesvirus of domestic pigs, thereby adding a concern to their use in xenotransplantation.