Investigation of arenavirus and filovirus infections in rodents and non-human primates in the United Kingdom

Recent years have witnessed virus disease outbreaks that have caused considerable morbidity and mortality. As 80% of human viruses are zoonotic, outbreaks can be prevented by active surveillance of wildlife for pathogens with a potential for cross-species transmission. Rodents serve as important reservoirs for many zoonoses and epizooses. In this thesis, a cDNA library of 976 rodents, shrews, moles and birds was constructed. Molecular methods using inosine-containing degenerate primers were developed for the screening of novel viruses of the families, Arenaviridae and Filoviridae. Filovirus RNA was absent in all specimen screened. Lymphocytic choriomeningitis mammarenavirus (LCMV) RNA was detected in 4 Mus musculus (19%). Further screening of specimens obtained from a zoo outbreak resulted in the detection of LCMV RNA in a total of 20 M musculus, 2 Geoffroy’s marmoset, 1 black and white colobus and 1 Black-crested gibbon from two separate sampling sample sets. Viral RNA was detected in non-human primates (NHP) not previously reported. Rodents screened at the zoo after the outbreak were negative for LCMV RNA. Phylogenetic analyses of full-length glycoprotein precursor (GPC) of 4 NHP-derived and 4 rodent-derived LCMV revealed a clustering of the former with other lineage-I LCMV GPC sequences. Molecular characterisation of the novel LCMV strains uncovered a consistency in amino acid substitutions across primate-derived and rodent-derived LCMV strains at position 211 of GPC which may be a possible determinant for cross-species transmission. Analysis of the alpha-dystroglycan receptor of rodents and NHPs revealed residues that might influence GPC binding affinity and therefore drive virus evolution. Pseudoviruses constructed from GPC of rodent-derived LCMV displayed higher infectivities in hepatocellular carcinoma cells than their NHP-derived LCMV counterpart which is indicative of the existence of another unidentified host receptor. Together, the results establish a framework for further investigation of the molecular basis for cross-species transmission of LCMV between rodent and primate hosts

Retrieval of the Complete Coding Sequence of the UK-Endemic Tatenale Orthohantavirus Reveals Extensive Strain Variation and Supports Its Classification as a Novel Species

©2020 by the authors. Licensee MDPI, Basel, Switzerland. Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus

Air and surface sampling for monkeypox virus in a UK hospital: an observational study

Background An outbreak of monkeypox virus infections in non-endemic countries was recognised on May 12, 2022. As of September 29, more than 67 000 infections have been reported globally, with more than 3400 confirmed cases in the UK by September 26. Monkeypox virus is believed to be predominantly transmitted through direct contact with lesions or infected body fluids, with possible involvement of fomites and large respiratory droplets. A case of monkeypox in a health-care worker in the UK in 2018 was suspected to be due to virus exposure while changing bedding. We aimed to measure the extent of environmental contamination in the isolation rooms of patients with symptomatic monkeypox. Methods We investigated environmental contamination with monkeypox virus from infected patients admitted to isolation rooms at the Royal Free Hospital (London, UK) between May 24 and June 17, 2022. Surface swabs of high-touch areas in five isolation rooms, of the personal protective equipment (PPE) of health-care workers in doffing areas in three rooms, and from air samples collected before and during bedding changes in five rooms were analysed using quantitative PCR to assess monkeypox virus contamination levels. Virus isolation was performed to confirm presence of infectious virus in selected positive samples. Findings We identified widespread surface contamination (56 [93%] of 60 samples were positive) in occupied patient rooms (monkeypox DNA cycle threshold [Ct] values 24·7–37·4), on health-care worker PPE after use (Ct 26·1–35·6), and in PPE doffing areas (Ct 26·3–36·8). Of 20 air samples taken, five (25%) were positive. Three (75%) of four air samples collected before and during a bedding change in one patient's room were positive (Ct 32·7–36·2). Replication-competent virus was identified in two (50%) of four samples selected for viral isolation, including from air samples collected during bedding change. Interpretation These data show contamination in isolation facilities and potential for suspension of monkeypox virus into the air during specific activities. PPE contamination was observed after clinical contact and changing of bedding. Contamination of hard surfaces in doffing areas supports the importance of cleaning protocols, PPE use, and doffing procedures. Fundin

Shared common ancestry of rodent alphacoronaviruses sampled globally

The recent discovery of novel alphacoronaviruses (alpha-CoVs) in European and Asian rodents revealed that rodent coronaviruses (CoVs) sampled worldwide formed a discrete phylogenetic group within this genus. To determine the evolutionary history of rodent CoVs in more detail, particularly the relative frequencies of virus-host co-divergence and cross-species transmission, we recovered longer fragments of CoV genomes from previously discovered European rodent alpha-CoVs using a combination of PCR and high-throughput sequencing. Accordingly, the full genome sequence was retrieved from the UK rat coronavirus, along with partial genome sequences from the UK field vole and Poland-resident bank vole CoVs, and a short conserved ORF1b fragment from the French rabbit CoV. Genome and phylogenetic analysis showed that despite their diverse geographic origins, all rodent alpha-CoVs formed a single monophyletic group and shared similar features such as the same gene constellations, a recombinant beta-CoV spike gene, and similar core transcriptional regulatory sequences (TRS). These data suggest that all rodent alpha CoVs sampled so far originate from a single common ancestor, and that there has likely been a long-term association between alpha CoVs and rodents. Despite this likely antiquity, the phylogenetic pattern of the alpha-CoVs was also suggestive of relatively frequent host-jumping among the different rodent species

Investigation of arenavirus and filovirus infections in rodents and non-human primates in the United Kingdom

Recent years have witnessed virus disease outbreaks that have caused considerable morbidity and mortality. As 80% of human viruses are zoonotic, outbreaks can be prevented by active surveillance of wildlife for pathogens with a potential for cross-species transmission. Rodents serve as important reservoirs for many zoonoses and epizooses. In this thesis, a cDNA library of 976 rodents, shrews, moles and birds was constructed. Molecular methods using inosine-containing degenerate primers were developed for the screening of novel viruses of the families, Arenaviridae and Filoviridae. Filovirus RNA was absent in all specimen screened. Lymphocytic choriomeningitis mammarenavirus (LCMV) RNA was detected in 4 Mus musculus (19%). Further screening of specimens obtained from a zoo outbreak resulted in the detection of LCMV RNA in a total of 20 M musculus, 2 Geoffroy’s marmoset, 1 black and white colobus and 1 Black-crested gibbon from two separate sampling sample sets. Viral RNA was detected in non-human primates (NHP) not previously reported. Rodents screened at the zoo after the outbreak were negative for LCMV RNA. Phylogenetic analyses of full-length glycoprotein precursor (GPC) of 4 NHP-derived and 4 rodent-derived LCMV revealed a clustering of the former with other lineage-I LCMV GPC sequences. Molecular characterisation of the novel LCMV strains uncovered a consistency in amino acid substitutions across primate-derived and rodent-derived LCMV strains at position 211 of GPC which may be a possible determinant for cross-species transmission. Analysis of the alpha-dystroglycan receptor of rodents and NHPs revealed residues that might influence GPC binding affinity and therefore drive virus evolution. Pseudoviruses constructed from GPC of rodent-derived LCMV displayed higher infectivities in hepatocellular carcinoma cells than their NHP-derived LCMV counterpart which is indicative of the existence of another unidentified host receptor. Together, the results establish a framework for further investigation of the molecular basis for cross-species transmission of LCMV between rodent and primate hosts

Longitudinal mpox virus surface sampling in an outpatient setting.

Letter to the Edito

Shared Common Ancestry of Rodent Alphacoronaviruses Sampled Globally

The recent discovery of novel alphacoronaviruses (alpha-CoVs) in European and Asian rodents revealed that rodent coronaviruses (CoVs) sampled worldwide formed a discrete phylogenetic group within this genus. To determine the evolutionary history of rodent CoVs in more detail, particularly the relative frequencies of virus-host co-divergence and cross-species transmission, we recovered longer fragments of CoV genomes from previously discovered European rodent alpha-CoVs using a combination of PCR and high-throughput sequencing. Accordingly, the full genome sequence was retrieved from the UK rat coronavirus, along with partial genome sequences from the UK field vole and Poland-resident bank vole CoVs, and a short conserved ORF1b fragment from the French rabbit CoV. Genome and phylogenetic analysis showed that despite their diverse geographic origins, all rodent alpha-CoVs formed a single monophyletic group and shared similar features, such as the same gene constellations, a recombinant beta-CoV spike gene, and similar core transcriptional regulatory sequences (TRS). These data suggest that all rodent alpha CoVs sampled so far originate from a single common ancestor, and that there has likely been a long-term association between alpha CoVs and rodents. Despite this likely antiquity, the phylogenetic pattern of the alpha-CoVs was also suggestive of relatively frequent host-jumping among the different rodent species

Air and surface sampling for monkeypox virus in a UK hospital: an observational study

Background An outbreak of monkeypox virus infections in non-endemic countries was recognised on May 12, 2022. As of September 29, more than 67 000 infections have been reported globally, with more than 3400 confirmed cases in the UK by September 26. Monkeypox virus is believed to be predominantly transmitted through direct contact with lesions or infected body fluids, with possible involvement of fomites and large respiratory droplets. A case of monkeypox in a health-care worker in the UK in 2018 was suspected to be due to virus exposure while changing bedding. We aimed to measure the extent of environmental contamination in the isolation rooms of patients with symptomatic monkeypox. Methods We investigated environmental contamination with monkeypox virus from infected patients admitted to isolation rooms at the Royal Free Hospital (London, UK) between May 24 and June 17, 2022. Surface swabs of high-touch areas in five isolation rooms, of the personal protective equipment (PPE) of health-care workers in doffing areas in three rooms, and from air samples collected before and during bedding changes in five rooms were analysed using quantitative PCR to assess monkeypox virus contamination levels. Virus isolation was performed to confirm presence of infectious virus in selected positive samples. Findings We identified widespread surface contamination (56 [93%] of 60 samples were positive) in occupied patient rooms (monkeypox DNA cycle threshold [Ct] values 24·7–37·4), on health-care worker PPE after use (Ct 26·1–35·6), and in PPE doffing areas (Ct 26·3–36·8). Of 20 air samples taken, five (25%) were positive. Three (75%) of four air samples collected before and during a bedding change in one patient's room were positive (Ct 32·7–36·2). Replication-competent virus was identified in two (50%) of four samples selected for viral isolation, including from air samples collected during bedding change. Interpretation These data show contamination in isolation facilities and potential for suspension of monkeypox virus into the air during specific activities. PPE contamination was observed after clinical contact and changing of bedding. Contamination of hard surfaces in doffing areas supports the importance of cleaning protocols, PPE use, and doffing procedures. Funding None