Search

Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays' target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays. Findings Oligonucleotides of the published real-time RT-PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 x 104 RNA copies/mL of blood and 2 x [10.sup.4] RNA coples/mL of urine. Conclusion We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results. Objectif Etudier la performance diagnostique des tests bases sur l'amplification en chaine par polymerase (PCR) en temps reel apres transcription inverse (RT) pour detecter le virus Zika. Methodes Nous avons compare sept tests publies utilisant la RT-PCR en temps reel et deux nouveaux tests developpes par nos soins. Afin de determiner la sensibilite analytique de chaque test, nous avons concu un acide ribonucleique synthetique de controle universel (ARNcun) contenant toutes les regions ciblees par les tests sur une helice ARN et enrichi de l'urine ou du sang humain de quantites connues de souches africaines ou asiatiques du virus Zika. Les charges virales dans 33 echantillons provenant de patients infectes par le virus Zika ont ete determinees a l'aide de l'un des nouveaux tests. Resultats Les oligonucleotides des tests publies utilisant la RT-PCR en temps reel ont presente jusqu'a TOmauvais apparlements potentiels avec la lignee asiatique provoquant l'epidemie actuelle, alors qu'on a constate de 0 a 4 mauvais appariements dans le cas des nouveaux tests. La limite de detection inferieure a 95% des sept tests les plus sensibles variait de 2,1 a 12,1 copies dARNcun/reaction. Deux tests presentaient des sensibilites plus basses de 17,0 et 1373,3 copies d'ARNcun/reaction et montraient une sensibilite similaire lorsque des echantillons enrichis etaient utilises. Les charges virales moyennes dans les echantillons provenant de patients infectes par le virus Zika etaient de 5 x 104 copies d'ARN/mL de sang et de 2 x 104 copies dARN/mL d'urine. Conclusion Nous proposons pour detecter le virus Zika des reactifs et des protocoles actualises, adaptes aux souches responsables de la flambee actuelle.Tous les tests publies ne permettent pas de detecter le virus Zika en raison d'une sensibilite limitee et d'une incompatibilite potentielle avec certaines souches. Les concentrations virales dans les echantillons cliniques etaient proches de la limite de detection technique, ce qui laisse penser que l'utilisation de tests insensibles donnera des resultats faussement negatifs. Objetivo Examinar el rendimiento del diagnostico de las pruebas de reaccion en cadena de la polimerasa de transcriptasa inversa (RT-PCR) en tiempo real para la deteccion del virus de Zika. Metodos Se compararon siete pruebas de RT-PCR en tiempo real publicadas y dos pruebas nuevas que se han desarrollado. Con el fin de determinar la sensibilidad analitica de cada prueba, se construyo un acido ribonucleico sintetico de control universal (uncRNA) que contenia todas las regiones objetivo de las pruebas en una hebra de RNA y se anadio a sangre u orina humana con cantidades conocidas de cepas de virus de Zika de Asia o Africa. Se determinaron las cargas viricas en 33 muestras procedentes de pacientes infectados por el virus de Zika a traves de una de las pruebas nuevas. Resultados Los oligonucleotidos de las pruebas de RT-PCR en tiempo real publicadas mostraron hasta 10 posibles discordancias con el linaje de Asia causante de los brotes actuales, en comparacion con 0 de 4 discordancias en el caso de las pruebas nuevas. El limite de deteccion inferior del 95% de las siete pruebas mas sensibles abarco de 2,1 a 12,1 copias/reaccion de uncRNA. Dos pruebas mostraron sensibilidades inferiores de 17,0 y 1373,3 copias/reaccion de uncRNA y presentaron una sensibilidad similar al usar muestras Infectadas. Las cargas viricas medias en las muestras procedentes de pacientes infectados por el virus de Zika fueron de 5 x 104 copias de RNA/mL de sangre y de 2 x [10.sup.4] coplas de RNA/ml de orina. Conclusion Se proporcionan reactivos y protocolos actualizados para la deteccion adecuada del virus de Zika en el caso de las cepas de brotes actuales. Algunas pruebas publicadas pueden no ser adecuadas para la deteccion del virus de Zika, debido a la limitada sensibilidad y a la posible incompatibilidad con algunas cepas. Las concentraciones viricas en las muestras clinicas se acercaron al limite de deteccion tecnico, lo que sugeria que el uso de pruebas Intensivas causaria resultados falsos negativos., Introduction The Zika virus is a mosquito-borne flavivirus with an approximately 11 kilobase ribonucleic acid (RNA) genome. (1) The virus usually causes a mild infection in adults, symptoms include fever, [...]

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide (1). Four HEV genotypes belonging to the species Orthohepevirus A are commonly found in humans (HEV-1 through HEV-4). [...]

Astroviruses (AstVs) infect numerous mammalian species including reservoirs such as bats. Peptides encoded by the genes of the highly polymorphic Major Histocompatibility Complex (MHC) form the first line of host defence against pathogens. Aside from direct involvement in mounting adaptive immune responses, MHC class II genes are hypothesized to regulate gut commensal diversity and shape the production of immune‐modulatory substances by microbes, indirectly affecting host susceptibility. Despite initial empirical evidence for the link between host MHC and the microbiota, associations among these factors remain largely unknown. To fill this gap, we examined MHC allelic diversity and constitution, the gut bacterial community and abundance pattern of a wild population of a neotropical bat (Artibeus jamaicensis) challenged by AstV infections. First, we show an age‐dependent relationship between the host MHC class II diversity and constitution and the gut microbiota in AstV‐uninfected bats. Crucially, these associations changed in AstV‐infected bats. Additionally, we identify changes in the abundance of specific bacterial taxa linked to the presence of certain MHC supertypes and AstV infection. We suggest changes in the microbiota to be either a result of AstV infection or the MHC‐mediated modulation of microbial communities. The latter could subsequently affect microbe‐mediated immunity and resistance against AstV infection. Our results emphasize that the reciprocal nature of host immune genetics, gut microbial diversity and pathogen infection require attention, which are particularly important given their repercussions for disease susceptibility and severity in wild animal populations with a history of zoonotic spillover and frequent human contact. [ABSTRACT FROM AUTHOR]

In humans, co‐infection of hepatitis B and C viruses (HBV, HCV) is common and aggravates disease outcome. Infection‐mediated disease aggravation is poorly understood, partly due to lack of suitable animal models. Carnivores are understudied for hepatitis virus homologues. We investigated Mexican carnivores (ringtails, Bassariscus astutus) for HBV and HCV homologues. Three out of eight animals were infected with a divergent HBV termed ringtail HBV (RtHBV) at high viral loads of 5 × 109 –1.4 × 1010 copies/ml serum. Two of the RtHBV‐infected animals were co‐infected with a divergent hepacivirus termed ringtail hepacivirus (RtHV) at 4 × 106–7.5 × 107 copies/ml in strain‐specific qRT‐PCR assays. Immunofluorescence assays relying on HBV core and RtHV NS3/4a proteins indicated that none of the animals had detectable hepadnavirus core‐specific antibodies, whereas one RtHV‐infected animal had concomitant RtHV‐specific antibodies at 1:800 end‐point titre. RtHBV and RtHV complete genomes showed typical HBV and HCV structure and length. All RtHBV genomes were identical, whereas RtHV genomes showed four amino acid substitutions located predominantly in the E1/E2‐encoding genomic regions. Both RtHBV (>28% genomic nucleotide sequence distance) and RtHV (>30% partial NS3/NS5B amino acid sequence distance) formed new species within their virus families. Evolutionary analyses showed that RtHBV grouped with HBV homologues from different laurasiatherian hosts (carnivores, bats, and ungulates), whereas RtHV grouped predominantly with rodent‐borne viruses. Ancestral state reconstructions showed that RtHV, but not RtHBV, likely emerged via a non‐recent host switch involving rodent‐borne hepacivirus ancestors. Conserved hepatitis virus infection patterns in naturally infected ringtails indicate that carnivores may be promising animal models to understand HBV/HCV co‐infection. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) causing coronavirus disease‐2019 (COVID‐19) likely has evolutionary origins in other animals than humans based on genetically related viruses existing in rhinolophid bats and pangolins. Similar to other animal coronaviruses, SARS‐CoV‐2 contains a functional furin cleavage site in its spike protein, which may broaden the SARS‐CoV‐2 host range and affect pathogenesis. Whether ongoing zoonotic infections are possible in addition to efficient human‐to‐human transmission remains unclear. In contrast, human‐to‐animal transmission can occur based on evidence provided from natural and experimental settings. Carnivores, including domestic cats, ferrets and minks, appear to be particularly susceptible to SARS‐CoV‐2 in contrast to poultry and other animals reared as livestock such as cattle and swine. Epidemiologic evidence supported by genomic sequencing corroborated mink‐to‐human transmission events in farm settings. Airborne transmission of SARS‐CoV‐2 between experimentally infected cats additionally substantiates the possibility of cat‐to‐human transmission. To evaluate the COVID‐19 risk represented by domestic and farmed carnivores, experimental assessments should include surveillance and health assessment of domestic and farmed carnivores, characterization of the immune interplay between SARS‐CoV‐2 and carnivore coronaviruses, determination of the SARS‐CoV‐2 host range beyond carnivores and identification of human risk groups such as veterinarians and farm workers. Strategies to mitigate the risk of zoonotic SARS‐CoV‐2 infections may have to be developed in a One Health framework and non‐pharmaceutical interventions may have to consider free‐roaming animals and the animal farming industry. [ABSTRACT FROM AUTHOR]

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection. [ABSTRACT FROM AUTHOR]

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod‐borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two‐toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two‐toed (Choloepus hoffmanni, n = 65) and three‐toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT‐PCR. A clinically healthy adult Hoffmann's two‐toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 107 RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non‐synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro‐evolutionary processes associated with immune evasion during the genealogy of sloth‐associated phleboviruses. However, gene‐wide mean dN/dS ratios were low at 0.02–0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth‐associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely‐related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV‐related phleboviruses with the two extant species of two‐toed sloths. [ABSTRACT FROM AUTHOR]

Hepatitis A virus (HAV) is an ancient and ubiquitous human pathogen recovered previously only from primates. The sole species of the genus Hepatovirus, existing in both enveloped and nonenveloped forms, and with a capsid structure intermediate between that of insect viruses and mammalian picornaviruses, HAV is enigmatic in its origins. We conducted a targeted search for hepatoviruses in 15,987 specimens collected from 209 small mammal species globally and discovered highly diversified viruses in bats, rodents, hedgehogs, and shrews, which by pairwise sequence distance comprise 13 novel Hepatovirus species. Near-complete genomes from nine of these species show conservation of unique hepatovirus features, including predicted internal ribosome entry site structure, a truncated VP4 capsid protein lacking N-terminal myristoylation, a carboxyl-terminal pX extension of VP1, VP2 late domains involved in membrane envelopment, and a cis-acting replication element within the 3Dpol sequence. Antibodies in some bat sera immunoprecipitated and neutralized human HAV, suggesting conservation of critical antigenic determinants. Limited phylogenetic cosegregation among hepatoviruses and their hosts and recombination patterns are indicative of major hepatovirus host shifts in the past. Ancestral state reconstructions suggest a Hepatovirus origin in small insectivorous mammals and a rodent origin of human HAV. Patterns of infection in small mammals mimicked those of human HAV in hepatotropism, fecal shedding, acute nature, and extinction of the virus in a closed host population. The evolutionary conservation of hepatovirus structure and pathogenesis provide novel insight into the origins of HAV and highlight the utility of analyzing animal reservoirs for risk assessment of emerging viruses. [ABSTRACT FROM AUTHOR]

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod-borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two-toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two-toed (Choloepus hoffmanni, n = 65) and three-toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT-PCR. A clinically healthy adult Hoffmann's two-toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 10 7  RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non-synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro-evolutionary processes associated with immune evasion during the genealogy of sloth-associated phleboviruses. However, gene-wide mean dN/dS ratios were low at 0.02-0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth-associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely-related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV-related phleboviruses with the two extant species of two-toed sloths., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV., Importance: The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV., (Copyright © 2016 American Society for Microbiology.)