High Prevalence of Co-Infection with Multiple Torque Teno Sus Virus Species in Italian Pig Herds

Torque teno viruses (TTVs) are a large group of vertebrate-infecting small viruses with circular single-stranded DNA, classified in the Anelloviridae family. In swine, two genetically distinct species, Torque teno sus virus 1a (TTSuV1a) and 1b (TTSuV1b) are currently grouped into the genus Iotatorquevirus. More recently, a novel Torque teno sus virus species, named Torque teno sus virus k2b (TTSuVk2b), has been included with Torque teno sus virus k2a (TTSuVk2a) into the genus Kappatorquevirus. In the present study, TTSuV1 (TTSuV1a and TTSuV1b), TTSuVk2a and TTSuVk2b prevalence was evaluated in 721 serum samples of healthy pigs from Sardinian farms, insular Italy. This is the largest study to date on the presence of TTSuV in healthy pigs in Italy. The global prevalence of infection was 83.2% (600/721), being 62.3% (449/721), 60.6% (437/721), and 11.5% (83/721) the prevalence of TTSuV1, TTSuVk2a and TTSuVk2b, respectively. The rate of co-infection with two and/or three species was also calculated, and data show that co-infections were significantly more frequent than infections with single species, and that TTSuV1+TTSuVk2a double infection was the prevalent combination (35.4%). Quantitative results obtained using species-specific real time-qPCR evidenced the highest mean levels of viremia in the TTSuV1 subgroup, and the lowest in the TTSuVk2b subgroup. Interestingly, multiple infections with distinct TTSuV species seemed to significantly affect the DNA load and specifically, data highlighted that double infection with TTSuVk2a increased the viral titers of TTSuV1, likewise the co-infection with TTSuVk2b increased the titers of TTSuVk2a.     

Prevalence and age distribution of porcine torque teno sus virus (TTSuV) in the Czech Republic

Torque teno sus viruses (TTSuV) were detected in the pig population in the Czech Republic by a nested PCR technique. The prevalence of individual TTSuV was found to be 42.9% (TTSuV1) and 46.7% (TTSuV2). The prevalence for TTSuV 1 and TTSuV2 was determined to be 52.7% and 60.6% in piglets at weaning, 90.9% and 63.6% in gilts, and 75% and 87.5% in sows, respectively. Both virus species were detected in 12% of newborn piglets, which suggests possible transplacental transmission. Sequencing of several virus strains showed that the sequenced TTSuV strains belong to two different species of viruses. The average similarity on the nucleotide levels between these two species was 43.3%.     

Torque teno sus virus (TTSuV) in cell cultures and trypsin

Torque teno sus virus (TTSuV), a member of the family Anelloviridae, is a single-stranded, circular DNA virus, widely distributed in swine populations. Presently, two TTSuV genogroups are recognized: Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). TTSuV genomes have been found in commercial vaccines for swine, enzyme preparations and other drugs containing components of porcine origin. However, no studies have been made looking for TTSuV in cell cultures. In the present study, a search for TTSuV genomes was carried out in cell culture lineages, in sera used as supplement for cell culture media as well as in trypsin used for cell disaggregation. DNA obtained from twenty-five cell lineages (ten from cultures in routine multiplication and fifteen from frozen ampoules), nine samples of sera used in cell culture media and five batches of trypsin were examined for the presence of TTSuV DNA. Fifteen cell lineages, originated from thirteen different species contained amplifiable TTSuV genomes, including an ampoule with a cell lineage frozen in 1985. Three cell lineages of swine origin were co-infected with both TTSuV1 and TTSuV2. One batch of trypsin contained two distinct TTSuV1 plus one TTSuV2 genome, suggesting that this might have been the source of contamination, as supported by phylogenetic analyses of sequenced amplicons. Samples of fetal bovine and calf sera used in cell culture media did not contain amplifiable TTSuV DNA. This is the first report on the presence of TTSuV as contaminants in cell lineages. In addition, detection of the viral genome in an ampoule frozen in 1985 provides evidence that TTSuV contamination is not a recent event. These findings highlight the risks of TTSuV contamination in cell cultures, what may be source for contamination of biological products or compromise results of studies involving in vitro multiplied cells.     

Synonymous Codon Usage in TTSuV2: Analysis and Comparison with TTSuV1

Two species of the DNA virus Torque teno sus virus (TTSuV), TTSuV1 and TTSuV2, have become widely distributed in pig-farming countries in recent years. In this study, we performed a comprehensive analysis of synonymous codon usage bias in 41 available TTSuV2 coding sequences (CDS), and compared the codon usage patterns of TTSuV2 and TTSuV1. TTSuV codon usage patterns were found to be phylogenetically conserved. Values for the effective number of codons (ENC) indicated that the overall extent of codon usage bias in both TTSuV2 and TTSuV1 was not significant, the most frequently occurring codons had an A or C at the third codon position. Correspondence analysis (COA) was performed and TTSuV2 and TTSuV1 sequences were located in different quadrants of the first two major axes. A plot of the ENC revealed that compositional constraint was the major factor determining the codon usage bias for TTSuV2. In addition, hierarchical cluster analysis of 41 TTSuV2 isolates based on relative synonymous codon usage (RSCU) values suggested that there was no association between geographic distribution and codon bias of TTSuV2 sequences. Finally, the comparison of RSCU for TTSuV2, TTSuV1 and the corresponding host sequence indicated that the codon usage pattern of TTSuV2 was similar to that of TTSuV1. However the similarity was low for each virus and its host. These conclusions provide important insight into the synonymous codon usage pattern of TTSuV2, as well as better understangding of the molecular evolution of TTSuV2 genomes.     

Serological Profile of Torque Teno Sus Virus Species 1 (TTSuV1) in Pigs and Antigenic Relationships between Two TTSuV1 Genotypes (1a and 1b), between Two Species (TTSuV1 and -2), and between Porcine and Human Anelloviruses

The family Anelloviridae includes human and animal torque teno viruses (TTVs) with extensive genetic diversity. The antigenic diversity among anelloviruses has never been assessed. Using torque teno sus virus (TTSuV) as a model, we describe here the first investigation of the antigenic relationships among different anelloviruses. Using a TTSuV genotype 1a (TTSuV1a) or TTSuV1b enzyme-linked immunosorbent assay (ELISA) based on the respective putative ORF1 capsid antigen and TTSuV1-specific real-time PCR, the combined serological and virological profile of TTSuV1 infection in pigs was determined and compared with that of TTSuV2. TTSuV1 is likely not associated with porcine circovirus-associated disease (PCVAD), because both the viral loads and antibody levels were not different between affected and unaffected pigs and because there was no synergistic effect of concurrent PCV2/TTSuV1 infections. We did observe a higher correlation of IgG antibody levels between anti-TTSuV1a and -TTSuV1b than between anti-TTSuV1a or -1b and anti-TTSuV2 antibodies in these sera, implying potential antigenic cross-reactivity. To confirm this, rabbit antisera against the putative capsid proteins of TTSuV1a, TTSuV1b, or TTSuV2 were generated, and the antigenic relationships among these TTSuVs were analyzed by an ELISA and by an immunofluorescence assay (IFA) using PK-15 cells transfected with one of the three TTSuV ORF1 constructs. The results demonstrate antigenic cross-reactivity between the two genotypes TTSuV1a and TTSuV1b but not between the two species TTSuV1a or -1b and TTSuV2. Furthermore, an anti-genogroup 1 human TTV antiserum did not react with any of the three TTSuV antigens. These results have important implications for an understanding of the diversity of anelloviruses as well as for the classification and vaccine development of TTSuVs.     

Rescue of a porcine anellovirus (torque teno sus virus 2) from cloned genomic DNA in pigs

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.     

Genetic diversity of the genus Cosavirus in the family Picornaviridae: a new species, recombination, and 26 new genotypes

The proposed viral genus human Cosavirus (HCoSV) consists of diverse picornaviruses found at high prevalence in the feces of children from developing countries. We sequenced four near-full length genomes and 45 partial VP1 region from HCoSV in human feces from healthy children and children with acute flaccid paralysis in Pakistan, Nigeria and Tunisia and from healthy and diarrhetic adults in Nepal. Genetic analyses of the near-full length genomes revealed presence of a new candidate cosavirus species provisionally labelled as species F (HCoSV-F). A HCoSV genome showed evidence of recombination between species D and E viruses at the P1/P2 junction indicating that these viruses may be reclassified as a single highly diverse species. Based on genetic distance criteria for assigning genotypes corresponding to neutralization serotypes in enteroviruses we identified 26 new HCoSV genotypes belonging to species A, D, and E. The detection of a large number of HCoSV genotypes based on still limited geographic sampling indicates that the phenotypic effects of cosaviruses on infected subjects are likely to be as highly diverse as those of human enteroviruses.     

Novel reassortment of Eurasian avian-like and pandemic/2009 influenza viruses in swine: infectious potential for humans

Pigs are considered to be intermediate hosts and "mixing vessels," facilitating the genesis of pandemic influenza viruses, as demonstrated by the emergence of the 2009 H1N1 pandemic (pdm/09) virus. The prevalence and repeated introduction of the pdm/09 virus into pigs raises the possibility of generating novel swine influenza viruses with the potential to infect humans. To address this, an active influenza surveillance program was conducted with slaughtered pigs in abattoirs in southern China. Over 50% of the pigs tested were found to be seropositive for one or more H1 influenza viruses, most commonly pdm/09-like viruses. Out of 36 virus isolates detected, one group of novel reassortants had Eurasian avian-like swine H1N1 surface genes and pdm/09 internal genes. Animal experiments showed that this virus transmitted effectively from pig to pig and from pig to ferret, and it could also replicate in ex vivo human lung tissue. Immunization against the 2009 pandemic virus gave only partial protection to ferrets. The continuing prevalence of the pdm/09 virus in pigs could lead to the genesis of novel swine reassortant viruses with the potential to infect humans.     

Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing

Background

Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure.

Results

The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pig virus.

Conclusion

The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures comparability of the method and may be used for further method optimization.     

Genomic characterization and high prevalence of bocaviruses in swine

Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ～2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45-75% and 55-70% respectively, with 30-47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses.     

Ancient and modern retroviruses

Retroviruses are transmitted in two distinct ways: as infectious particles and as 'endogenous' proviral DNA integrated in the germ line of the host. Modern infectious viruses such as HIV-1 and HIV-2 recently infected mankind from chimpanzee and simian hosts, whereas human endogenous retroviral genomes have been present throughout old world primate evolution. Human T-cell leukemia viruses (HTLV-1 and II) have a much older human provenance than HIV, although new zoonoses from simians may also occur. We have recently characterized new retroviruses in pigs and humans. Porcine endogenous retroviral (PERV) genomes are carried in chromosomal DNA but can be activated to produce virions that are infectious for human cells, which has raised concern over human xenotransplantation using pig tissues. Human retrovirus 5 (HRV-5) is detected as an exogenous genome in association with arthritis and systemic lupus erythematosus.     

Genome-wide profile of oxidoreductases in viruses, prokaryotes, and eukaryotes

Enzymes that utilize nicotinamide adenine dinucleotide (NAD) or its 2'-phosphate derivative (NADP) are found throughout the kingdoms of life. These enzymes are fundamental to many biochemical pathways, including central intermediary metabolism and mechanisms for cell survival and defense. The complete genomes of 25 organisms representing bacteria, protists, fungi, plants, and animals, and 811 viruses, were mined to identify and classify NAD(P)-dependent enzymes. An average of 3.4% of the proteins in these genomes was categorized as NAD(P)-utilizing proteins, with highest prevalence in the medium-chain oxidoreductase and short-chain oxidoreductase families. In general, the distribution of these enzymes by oxidoreductase family was correlated to the number of different catalytic mechanisms in each family. Organisms with smaller genomes encoded a larger proportion of NAD(P)-dependent enzymes in their proteome (approximately 6%) as compared to the larger genomes of eukaryotes (approximately 3%). Among viruses, those with large, double-strand DNA genomes were shown to encode oxidoreductases. Gram-positive and gram-negative bacteria showed some differences in the distribution of NAD(P)-dependent proteins. Several organisms such as M. tuberculosis, P. falciparum, and A. thaliana showed unique distributions of oxidoreductases corresponding to some phenotypic features.     

Mechanisms for RNA Capture by ssDNA Viruses: Grand Theft RNA

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA–DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA–DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.     

Terminally Repeated Sequences on a Herpesvirus Genome Are Deleted following Circularization but Are Reconstituted by Duplication during Cleavage and Packaging of Concatemeric DNA

The mechanisms underlying cleavage of herpesvirus genomes from replicative concatemers are unknown. Evidence from herpes simplex virus type 1 suggests that cleavage occurs by a nonduplicative process; however, additional evidence suggests that terminal repeats may also be duplicated during the cleavage process. This issue has been difficult to resolve due to the variable numbers of reiterated terminal repeats that the herpes simplex virus type 1 genome can contain. Guinea pig cytomegalovirus is a herpesvirus with a simple terminal repeat arrangement that defines two genome types. Type II genomes have a single copy of a 1-kb terminal repeat at both their left and right termini, whereas type I genomes have only one copy at their left termini and lack the repeat at their right termini. In a previous study, we constructed a recombinant guinea pig cytomegalovirus in which certain cis elements were disrupted such that only type II genomes were produced. Here we show that double repeats that are formed by circularization of infecting genomes are rapidly converted to single repeats, such that the junctions between genomes within replicative concatemers formed late in infection almost exclusively contain single copies of the terminal repeat. Therefore, for the recombinant virus, each cleavage event begins with a single repeat within a concatemer yet produces two repeats, one at each of the resulting termini, demonstrating that terminal repeat duplication occurs in conjunction with cleavage. For wild-type guinea pig cytomegalovirus, the formation of type I genomes further suggests that cleavage can also occur by a nonduplicative process and that duplicative and nonduplicative cleavage can occur concurrently. Other herpesviruses having terminal repeats, such as the herpes simplex viruses and human cytomegalovirus, may also utilize repeat duplication and deletion; however, the biological importance of these events remains unknown.     

浙江地区鼬獾和犬源狂犬病病毒分离株全基因组测序与分析

测定浙江地区狂犬病病毒分离株(鼬獾和犬)全基因组序列,从分子水平对病毒进行遗传变异特征分析,了解狂犬病病毒在浙江的流行和变异情况以及目前浙江流行株的遗传学背景,以丰富中国狂犬病病毒街毒流行株的全基因组信息。脑内接种1~2日乳鼠分离狂犬病病毒,RT-PCR反应测定浙江地区狂犬病病毒分离株全基因组核苷酸序列,并进行编码蛋白和序列相似性比较及种系发生分析。测序获得狂犬病病毒浙江淳安鼬獾分离株F02、F04和松阳犬分离株D01、D02全基因组核苷酸序列信息:基因组全长11 923和11 925 nts,前导序列Leader长58nts,5个ORF为:NP(1 353 nts);PP(894 nts);MP(609 nts);GP(1 575 nts);LP(6 386 nts),N-P-M-G间隔序列长2、5、5 nts;G-L基因间的伪基因Ψ长423 nts;Trailer尾长70 nts。核酸BLAST及多重序列比对分析显示浙江地区4个狂犬病病毒分离株的全基因组序列的组成和结构符合弹状病毒科狂犬病病毒属的特征;中国毒株之间特别是浙江同种动物狂犬病病毒之间各个基因区域核苷酸与氨基酸序列相似性最高,浙江病毒全基因组序列编码蛋白氨基酸序列相似性高于核苷酸序列相似性,说明蛋白质编码基因的核苷酸变异大多属于同义突变;浙江病毒负链RNA基因组5个基因编码氨基酸的长度没有变异,5个编码蛋白仅表现较少的序列变化;浙江病毒与本研究选择的代表性引用街毒株或者来自街毒的减毒株的变异位点和变异类型相似,多重序列相似性的比较和种系发生分析显示所分离的狂犬病病毒浙江街毒株均属于基因1型,具有较独特的中国地域性特点,故本研究中的浙江地区分离株极有可能是自然界中固有的街毒株。     

Detection and Genetic Characteristics of H9N2 Avian Influenza Viruses from Live Poultry Markets in Hunan Province,China

H9N2 avian influenza viruses (AIVs) are highly prevalent and of low pathogenicity in domestic poultry. These viruses show a high genetic compatibility with other subtypes of AIVs and have been involved in the genesis of H5N1, H7N9 and H10N8 viruses causing severe infection in humans. The first case of human infection with H9N2 viruses in Hunan province of China have been confirmed in November 2013 and identified that H9N2 viruses from live poultry markets (LPMs) near the patient’s house could be the source of infection. However, the prevalence, distribution and genetic characteristics of H9N2 viruses in LPMs all over the province are not clear. We collected and tested 3943 environmental samples from 380 LPMs covering all 122 counties/districts of Hunan province from February to April, 2014. A total of 618 (15.7%) samples were H9 subtype positive and 200 (52.6%) markets in 98 (80.3%) counties/districts were contaminated with H9 subtype AIVs. We sequenced the entire coding sequences of the genomes of eleven H9N2 isolates from environmental samples. Phylogenetic analysis showed that the gene sequences of the H9N2 AIVs exhibited high homology (94.3%-100%). All eleven viruses were in a same branch in the phylogenetic trees and belonged to a same genotype. No gene reassortment had been found. Molecular analysis demonstrated that all the viruses had typical molecular characteristics of contemporary avian H9N2 influenza viruses. Continued surveillance of AIVs in LPMs is warranted for identification of further viral evolution and novel reassortants with pandemic potential.     

Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney

Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles in order to investigate the prevalence of virus pathogens. Reoviruses were isolated in 11 of the 13 winter moth populations sampled, with 3 novel species being detected. Two species of Cypoviridae (CPV) were isolated, Operophtera brumata CPV18 and O. brumata CPV19, with one host population suffering 46% infection prevalence of OpbuCPV19. A third virus, O. brumata Reovirus (OpbuRV), was isolated from both winter moth and a hymenopteran parasitoid wasp, Phobocampe tempestiva, which is abundant in these populations. This was identified as a non-occluded reovirus, which was clearly able to infect and persist in both the lepidopteran and the hymenopteran host. The genomes of the three viruses were characterised using gel electrophoresis and the virus structure was investigated using transmission electron microscopy. The relationship of these viruses with a baculovirus that also infects winter moth, OpbuNPV, was investigated, as well as the association of OpbuRV with P. tempestiva. The detection of such viruses is discussed with reference to studies of similar viruses in other lepidopteran and hymenopteran host systems.     

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12–April 1 2020) and 31 from later time-points ( 25–27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5–99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.     

Long-Term Inactivation Study of Three Enteroviruses in Artificial Surface and Groundwaters, Using PCR and Cell Culture

Since the transmission of pathogenic viruses via water is indistinguishable from the transmission via other routes and since the levels in drinking water, although significant for health, may be too low for detection, quantitative viral risk assessment is a useful tool for assessing disease risk due to consumption of drinking water. Quantitative viral risk assessment requires information concerning the ability of viruses detected in drinking water to infect their host. To obtain insight into the infectivity of viruses in relation to the presence of virus genomes, inactivation of three different enteroviruses in artificial ground and surface waters under different controlled pH, temperature, and salt conditions was studied by using both PCR and cell culture over time. In salt-peptone medium, the estimated ratio of RNA genomes to infectious poliovirus 1 in freshly prepared suspensions was about 10⁰. At 4°C this ratio was 10³ after 600 days, and at 22°C it was 10⁴ after 200 days. For poliovirus 1 and 2 the RNA/infectious virus ratio was higher in artificial groundwater than in artificial surface water, but this was not the case for coxsackievirus B4. When molecular detection is used for virus enumeration, it is important that the fraction of infectious virus (based on all virus genomes detected) decays with time, especially at temperatures near 22°C.     

Genetic and phenotypic variation of foot-and-mouth disease virus during serial passages in a natural host

Foot-and-mouth disease virus (FMDV), like other RNA viruses, exhibits high mutation rates during replication that have been suggested to be of adaptive value. However, even though genetic variation in RNA viruses and, more specifically, FMDV has been extensively examined during virus replication in a wide variety of in vitro cell cultures, very little is known regarding the generation and effects of genetic variability of virus replication in the natural host under experimental conditions and no genetic data are available regarding the effects of serial passage in natural hosts. Here, we present the results of 20 serial contact transmissions of the highly pathogenic, pig-adapted O Taiwan 97 (O Tw97) isolate of FMDV in swine. We examined the virus genomic consensus sequences for a total of 37 full-length viral genomes recovered from 20 in vivo passages. The characteristics and distributions of changes in the sequences during the series of pig infections were analyzed in comparison to the O Tw97 genomes recovered from serially infected BHK-21 cell cultures. Unexpectedly, a significant reduction of virulence upon pig passages was observed, and finally, interruption of the viral transmission chain occurred after the14th pig passage (T14). Virus was, however, isolated from the tonsils and nasal swabs of the asymptomatic T15 pigs at 26 days postcontact, consistent with a natural establishment of the carrier state previously described only for ruminants. Surprisingly, the region encoding the capsid protein VP1 (1D) did not show amino acid changes during in vivo passages. These data demonstrate that contact transmission of FMDV O Tw97 in pigs mimics the fitness loss induced by the bottleneck effect, which was previously observed by others during plaque-to-plaque FMDV passage in vitro, suggesting that unknown mechanisms of virulence recovery might be necessary during the evolution and perpetuation of FMDV in nature.