Cytopathic bovine viral diarrhea viruses (BVDV): emerging pestiviruses doomed to extinction

Bovine viral diarrhea virus (BVDV), a Flaviviridae pestivirus, is arguably one of the most widespread cattle pathogens worldwide. Each of its two genotypes has two biotypes, non-cytopathic (ncp) and cytopathic (cp). Only the ncp biotype of BVDV may establish persistent infection in the fetus when infecting a dam early in gestation, a time point which predates maturity of the adaptive immune system. Such fetuses may develop and be born healthy but remain infected for life. Due to this early initiation of fetal infection and to the expression of interferon antagonistic proteins, persistently infected (PI) animals remain immunotolerant to the infecting viral strain. Although only accounting for some 1% of all animals in regions where BVDV is endemic, PI animals ensure the viral persistence in the host population. These animals may, however, develop the fatal mucosal disease, which is characterized by widespread lesions in the gastrointestinal tract. Cp BVD virus, in addition to the persisting ncp biotype, can be isolated from such animals. The cp viruses are characterized by unrestrained genome replication, and their emergence from the persisting ncp ones is due to mutations that are unique in each virus analyzed. They include recombinations with host cell mRNA, gene translocations and duplications, and point mutations. Cytopathic BVD viruses fail to establish chains of infection and are unable to cause persistent infection. Hence, these viruses illustrate a case of “viral emergence to extinction” – irrelevant for BVDV evolution, but fatal for the PI host.     

Analysis of a pair of END+ and END? viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N^pro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of N^pro determine the difference in characteristics between END-phenomenon-positive (END⁺) and END-phenomenon-negative (END⁻) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END⁺ and END⁻ viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END⁺ and END⁻ viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N^pro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.     

Comparative genetic analyses of Korean bat coronaviruses with SARS-CoV and the newly emerged SARS-CoV-2

BackgroundBats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV).ObjectivesThe objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor.MethodsThe phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA.ResultsPhylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%–67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2.ConclusionsThese results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.     

Mechanisms of viral emergence

A number of virologic and environmental factors are involved in the emergence and re-emergence of viral disease. Viruses do not conservatively occupy a single and permanent ecological niche. Rather, due to their intrinsic capacity for genetic change, and to the evolvability of fitness levels, viruses display a potential to parasitize alternative host species. Mutation, recombination and genome segment reassortment, and combination of these molecular events, produce complex and phenotypically diverse populations of viruses, which constitute the raw material on which selection acts. The majority of emerging viral diseases of humans have a zoonotic origin. Sociologic and ecologic factors produce diverse and changing environments in which viral subpopulations have ample opportunities to be selected from intrinsically heterogeneous viral populations, particularly in the case of RNA viruses. In this manner, new human, animal and plant viruses have emerged periodically and, from all evidence, will continue to emerge. This article reviews some of the mechanisms that have been identified in viral emergence, with a focus on the importance of genetic variation of viruses, and on the general concept of biological complexity.     

Experimental infection of Artibeus intermedius with a vampire bat rabies virus

Experimental infection of Artibeus intermedius, the great fruit-eating bat, was performed with vampire bat rabies isolates. Bats (n = 35) were captured in the wild and quarantined prior to experimental infection. No rabies antibodies were detected by rapid fluorescent focus inhibition test (RFFIT) prior to infection. Three doses of rabies virus (RV) and three different routes of infection were used. One out of 35 bats died without showing any clinical signs at day 14 and was positive for rabies. None of the 34 other bats showed clinical signs for rabies, but high antibody titers were detected post-inoculation, suggesting either innate immune response to the vampire bat rabies virus or possible pre-exposure to RV and inoculation leading to a booster effect. Rabies virus was detected by hemi-nested RT-PCR (hnRT-PCR) in the brain (n = 3), stomach (n = 1) of bats that were negative by immunofluorescence and that survived rabies infection. The bat that died on day 14 was positive by hnRT-PCR on the brain, heart and liver. These results suggest that either previous non-lethal exposure to RV or natural low susceptibility to vampire bat viruses somehow protected Artibeus intermedius from clinical rabies infection leading to a marginal lethality effect on this bats species population in the wild.     

M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). MYXV has a linear double-stranded DNA genome that encodes several factors important for evasion from the host immune system. Among them, four ankyrin (ANK) repeat proteins were identified: M148R, M149R, M150R and M-T5. To date, only M150R and M-T5 were studied and characterized as critical virulence factors. This article presents the first characterization of M148R and M149R. Green Fluorescent Protein (GFP) fusions allowed us to localize them in a viral context. Whereas M149R is only cytoplasmic, interestingly, M148R is in part located in the nucleolus, a unique feature for an ANK repeat poxviral protein. In order to evaluate their implication in viral pathogenicity, targeted M148R, M149R, or both deletions were constructed in the wild type T1 strain of myxoma virus. In vitro infection of rabbit and primate cultured cells as well as primary rabbit cells allowed us to conclude that M148R and M149R are not likely to be implicated in cell tropism or host range functions. However, in vivo experiments revealed that they are virulence factors since after infection of European rabbits with mutant viruses, a delay in the onset of clinical signs, an increase of survival time and a dramatic decrease in mortality rate were observed. Moreover, histological analysis suggests that M148R plays a role in the subversion of host inflammatory response by MYXV.     

Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido,Japan

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5′-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.     

Widespread infection with hemotropic mycoplasmas in bats in Spain,including a hemoplasma closely related to “Candidatus Mycoplasma hemohominis”

Molecular analyses of blood samples revealed infection with hemoplasmas in 97% of 31 cave bats captured in three caves in North-Eastern Spain. The characterization of 1250 bp of the 16S rRNA gene in 29 of the positive bats identified two different groups of sequences. Twenty-two Schreibers’ bats (Miniopterus schreibersii) and one long-eared bat (Myotis capaccinii) shared one group, composed of seven closely related sequences. These sequences showed an identity of about 97% with “Candidatus Mycoplasma hemohominis” and the phylogenetic branch including bat and human sequences showed a 100% bootstrap value, supporting a close phylogenetic relationship between these hemoplasmas. The second group, representing a potentially novel species, was composed of a single sequence shared by six Schreibers’ bats that had 91% identity with the recently reported hemoplasma from little brown bats in North America. Large bat aggregations in roosting caves probably benefits intra and inter-species transmission explaining the high observed prevalence.     

Detection and phylogenetic analysis of Bartonella species from bat flies on eastern bent-wing bats (Miniopterus fuliginosus) in Japan

We examined Bartonella prevalence in 281 bat flies collected from 114 eastern bent-wing bats (Miniopterus fuliginosus) in Japan and phylogenetically analyzed with other bat fly and bat strains. The bat flies were identified as Penicilidia jenynsii (PJ; n = 45), Nycteribia allotopa (NA; n = 157), and novel Nycteribia species (NS; n = 79). Bartonella DNAs were detected in 31.7 % (89/281) of bat flies by PCR targeting the citrate synthase (gltA) gene. The prevalence of Bartonella DNA among the bat flies was 47.1 % (74/157) in NA, 15.2 % (12/79) in NS, and 6.7 % (3/45) in PJ. Bartonella bacteria were also isolated from two NA and one NS. A phylogenetic analysis of the gltA sequences revealed that bat fly-associated strains were classified into three lineages and the same lineages of Bartonella were commonly detected from both Nycteribia bat flies and Miniopterus bats. These results suggest that Nycteribia bat flies are potential vectors for transmitting Bartonella among Miniopterus bats.     

Novel coronaviruses,astroviruses, adenoviruses and circoviruses in insectivorous bats from northern China

Bats are considered as the reservoirs of several emerging infectious disease, and novel viruses are continually found in bats all around the world. Studies conducted in southern China found that bats carried a variety of viruses. However, few studies have been conducted on bats in northern China, which harbours a diversity of endemic insectivorous bats. It is important to understand the prevalence and diversity of viruses circulating in bats in northern China. In this study, a total of 145 insectivorous bats representing six species were collected from northern China and screened with degenerate primers for viruses belonging to six families, including coronaviruses, astroviruses, hantaviruses, paramyxoviruses, adenoviruses and circoviruses. Our study found that four of the viruses screened for were positive and the overall detection rates for astroviruses, coronaviruses, adenoviruses and circoviruses in bats were 21.4%, 15.9%, 20% and 37.2%, respectively. In addition, we found that bats in northern China harboured a diversity of novel viruses. Common Serotine (Eptesicus serotinu), Fringed long‐footed Myotis (Myotis fimriatus) and Peking Myotis (Myotis pequinius) were investigated in China for the first time. Our study provided new information on the ecology and phylogeny of bat‐borne viruses.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Molecular epidemiological analysis of bat rabies viruses in Brazil

A molecular epidemiological analysis was performed in 19 rabies viruses (RVs) isolated from haematophagous, frugivorous and insectivorous bats, in Sao Paulo, Brazil. The authors carried out RT-PCR for amplification of the RV nucleoprotein (N) gene, and determined 1,335 nucleotide sequences of N gene by direct sequencing method. Phylogenetic analysis, which was based on the N gene of Brazilian RV isolates identified presently and previously, revealed that RVs isolated from bats were genetically divided into four lineages had a tendency to depend on the host bat species. The first lineage consisted mainly of haematophagous bat (Desmodus rotundus) isolates, including frugivorous bat (Artibeus spp.) isolates. Other three lineages consisted of insectivorous bat isolates; mainly Eptesicus spp., Molossus spp. and Nyctinomops spp. isolates, respectively. These results indicate a possibility of that there are bat species-specific RV variants in Brazil.     

Sequence and unique phylogeny of G genes of bovine respiratory syncytial viruses circulating in Japan

Bovine respiratory syncytial virus (BRSV) is an etiologic agent of bovine respiratory disease. The rapid evolutionary rate of BRSV contributes to genetic and antigenic heterogeneity of field strains and causes occasional vaccine failure. We conducted molecular epidemiologic characterization of BRSV circulating in Japan to obtain genetic information for vaccine-based disease control. Phylogenetic analysis of G and F gene sequences revealed that all of the isolated Japanese BRSV strains clustered in the same genetic subgroup, which was distinct from the 9 known groups. We assigned the Japanese group to subgenotype X. The Japanese isolates formed 2 temporal clusters: isolates from 2003 to 2005 clustered in lineage A; isolates from 2017 to 2019 formed lineage B. The alignment of the deduced amino acid sequences of the G gene revealed that the central hydrophobic region responsible for viral antigenicity is conserved in all of the isolates; unique amino acid mutations were found mainly in mucin-like regions. Our results suggest that BRSV has evolved uniquely in Japan to form the new subgenotype X; the antigenic homogeneity of the viruses within this group is inferred.     

5种脑炎人兽共患病病毒多重RT-PCR检测方法的建立

为建立同时检测流行性乙型脑炎病毒(JEV)、森林脑炎病毒(TBEV)、东方马脑炎病毒(EEEV)、西方马脑炎病毒(WEEV)和基孔肯雅病毒(CHIKV)5种人兽共患脑炎病病毒的多重RT-PCR方法,本研究根据GenBank登录的相关病毒基因序列设计特异引物,通过优化引物组合及PCR反应条件,建立可同时检测5种病毒的方法,扩增片段长度分别为411 bp(JEV)、945 bp(TBEV)、193 bp(EEEV)、545 bp(WEEV)和769 bp(CHIKV);该方法具有良好的特异性,对病毒核酸最低检测拷贝数分别为7.1×103、3.6×103、2.2×103、5.6×103和5.1×103.该方法具有特异性强、灵敏度高、操作简便等优点,为以上5种人兽共患脑炎病病毒提供快速检测手段.     

Use of S1 nuclease in deep sequencing for detection of double-stranded RNA viruses

Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5–28.4- and 10.1–208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam

Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross‐species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health.     

Neotropical Bats from Costa Rica harbour Diverse Coronaviruses

Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host–species barrier. For analysing coronavirus diversity in a bat species‐rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA‐dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus‐derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α‐CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected.