Nonstructural Gene Region Research Articles

Acute bee paralysis virus field isolates from apiaries suffering colony losses in Türkiye

Multiple viral infections are one of the main drivers of honey bee colony losses and threaten the sustainability of colony health. The Acute bee paralysis virus (ABPV) is one of the common honey bee viruses that can induce overt and/or covert infections in bee colonies. Globally more knowledge is needed about its effects leading to colony losses. ABPV-related pathology includes paralytic, trembling bees, unable to fly and, in some cases, ABPV causes the death of the bee colonies. The molecular phylogenetic of ABPV isolates obtained from apiaries suffering colony losses and/or with CCD-like complaints in Türkiye was performed in this study. The isolates’ structural and non-structural gene regions were analyzed phylogenetically and compared according to sampling years and apiaries. According to the results, the nucleotide identity of the RNA-dependent RNA polymerase (RdRp) gene in Turkish ABPV field isolates is more conserved than the capsid gene. The evaluation of molecular differentiation among the ABPV field isolates can be used for determining the origin and relatedness of ABPV strains and periodic monitoring of ABPV infections in apiaries.

Molecular Characterization of Megalocytiviruses from Diseased Fishes in Korean Aquatic Farms from 2013 to 2017

We identified structural and non-structural gene regions encoding major capsid protein (MCP) and DNA polymerase (DPOL) of megalocytiviruses collected from infected cultured fishes in RBIVD outbreak farms in 2013-2017 in Korea. With the two PCRs using 1-F/1-R and 4-F/4-R primer sets of the Manual of Diagnosis Tests for Aquatic Animals of the World Organization for Animal Health (OIE), amplicons were generated from the spleen and kidney tissue from approxmately ~30 fishes, including rock bream (Oplegnathus fasciatus), red sea bream (Pagrus major), and rock fish (Sebastes schlegeli), from 15 outbreak regions in the aquatic farms of the South Sea and Jeju Island. In phylogenetic analysis, complete MCP and partial DPOL genes belonged to RSIV type-subgroup2. Interestingly, these genes formed a cluster indicating closer relatedness to GSIV-K1, RIE12-1, and RBIV-C1, which were previously isolated from Japan and China, than with RBIV-KOR-TY1 isolated from Korea. However, the nucleotide sequence identities of the MCP and DPOL genes of these viruses were high, at 99.8% and 99.7%, respectively, compared with RBIV-KOR-TY1. Comparisons of nucleotide and amino acid sequences showed minimal differences between the obtained strains in the MCP gene, however, one or two nucleotide sequences substitutions of the DPOL gene were detected in nine strains, including a silent mutation detected in five strains. These findings suggest a slow rate of evolution of megalocytiviruses in this region, but the potential for mutations and new pathogenic strains warrants continuous surveillance.

Characteristics of the mosaic genome of a human parechovirus type 1 strain isolated from an infant with pneumonia in China

Human parechoviruses (HPeVs) belong to the Parechovirus genus of the large and growing family of Picornaviridae with a non-enveloped, single-stranded and positive-sense RNA. An HPeV strain was isolated from the nasopharyngeal aspirate specimen of a 2months old infant hospitalized with pneumonia in Beijing, China and nominated as BJ-37359 followed the code of the specimen. Strain BJ-37359 was identified as HPeV1 by whole genome sequencing. The full genome of strain BJ-37359 consisted of 7336 nucleotides (nt), excluding a poly (A) tail and contained an ORF of 6537 nt flanked by 5′UTR of 709 nt and 3′UTR of 90nt. Phylogenetic analyses revealed that strain BJ-37359 were clustered together with HPeV1 strains in the structural capsid protein region, while uncoupling in the non-structural gene regions. Analyses with Simplot and Bootscan indicated that multiple recombination events occurred in the non-structural region and VP0 region of strain BJ-37359 with other HPeV1, and other types might have contributed to the recombination, especially HPeV6 and HPeV7 strains. Recombination analyses indicated that strain BJ-37359 may have a mosaic genome with new genomic recombination breakpoints.

High rates of infection with novel enterovirus variants in wild populations of mandrills and other old world monkey species.

Enteroviruses (EVs) are a genetically and antigenically diverse group of viruses infecting humans. A mostly distinct set of EV variants have additionally been documented to infect wild apes and several, primarily captive, Old World monkey (OWM) species. To investigate the prevalence and genetic characteristics of EVs infecting OWMs in the wild, fecal samples from mandrills (Mandrillus sphinx) and other species collected in remote regions of southern Cameroon were screened for EV RNA. Remarkably high rates of EV positivity were detected in M. sphinx (100 of 102 screened), Cercocebus torquatus (7/7), and Cercopithecus cephus (2/4), with high viral loads indicative of active infection. Genetic characterization in VP4/VP2 and VP1 regions allowed EV variants to be assigned to simian species H (EV-H) and EV-J (including one or more new types), while seven matched simian EV-B variants, SA5 and EV110 (chimpanzee). Sequences from the remaining 70 formed a new genetic group distinct in VP4/2 and VP1 region from all currently recognized human or simian EV species. Complete genome sequences were obtained from three to determine their species assignment. In common with EV-J and the EV-A A13 isolate, new group sequences were chimeric, being most closely related to EV-A in capsid genes and to EV-B in the nonstructural gene region. Further recombination events created different groupings in 5' and 3' untranslated regions. While clearly a distinct EV group, the hybrid nature of new variants prevented their unambiguous classification as either members of a new species or as divergent members of EV-A using current International Committee on Taxonomy of Viruses (ICTV) assignment criteria. This study is the first large-scale investigation of the frequency of infection and diversity of enteroviruses (EVs) infecting monkeys (primarily mandrills) in the wild. Our findings demonstrate extremely high frequencies of active infection (95%) among mandrills and other Old World monkey species inhabiting remote regions of Cameroon without human contact. EV variants detected were distinct from those infecting human populations, comprising members of enterovirus species B, J, and H and a large novel group of viruses most closely related to species A in the P1 region. The viral sequences obtained contribute substantially to our growing understanding of the genetic diversity of EVs and the existence of interspecies chimerism that characterizes the novel variants in the current study, as well as in previously characterized species A and J viruses infecting monkeys. The latter findings will contribute to future development of consensus criteria for species assignments in enteroviruses and other picornavirus genera.

Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice

Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3′-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.

Live attenuated rubella viral vectors stably express HIV and SIV vaccine antigens while reaching high titers

Live attenuated viruses make potent and effective vaccines. Despite the urgent need for an HIV vaccine, this approach has not been feasible, since it has not been possible to attenuate the virus reliably and guarantee vaccine safety. Instead, live viral vectors have been proposed that could present HIV vaccine antigens in the most immunogenic way, in the context of an active infection. We have adapted the rubella vaccine strain RA27/3 as a vector to express HIV and SIV antigens, and tested the effect of insert size and composition on vector stability and viral titer. We have identified an acceptor site in the rubella nonstructural gene region, where foreign genes can be expressed as a fusion protein with the nonstructural protein P150 without affecting essential viral functions. The inserts were expressed as early genes of rubella, under control of the rubella genomic promoter. At this site, HIV and SIV antigens were expressed stably for at least seven passages, as the rubella vectors reached high titers. Rubella readily infects rhesus macaques, and these animals will provide an ideal model for testing the new vectors for replication in vivo, immunogenicity, and protection against SIV or SHIV challenge.

Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.

Recombination in the Nonstructural Gene Region in Type 2 Dengue Viruses

In view of the rising prevalence of dengue virus and mixing of host and vector populations, complete full-length sequence determination of dengue strains isolated from different epidemic areas is important for the study of virus evolution, pathogenicity, vaccine efficiency and diagnosis. Based on the genomic analysis of 51 complete dengue virus sequences, all of which cocirculated in Thailand between 1974 and 2001, we report here the occurrence of homologous recombination in the NS5 nonstructural gene region of dengue virus type 2 (DENV-2) strains. In order to analyze those 51 virus sequences at one time, we chose to use a highly sensitive recombination detection program called RDP. When RDP detects a possible recombination event, further bootscanning and phylogenetic tree analyses are applied to these candidate sequences to identify this recombination event. We found that within the DENV-2 subfamily, the strain ThNH63/93 is the evolutionary product of a recombination event between ThNH62/93 and ThD2_0284_90 strains. The strain ThNH62/93 was identified as the major parent, while the strain ThD2_0284_90 was the minor parent. The recombination site was determined to localize between positions 7967 (±36 nt) and 8283 (±36 nt) with a significance level of p < 0.001. Our results showed, for the first time, that an intraserotype recombination event occurred between DENV-2 strains in the nonstructural gene region; by contrast, an interserotype recombination between different serotypes of dengue strains was not identified. This study thus supports the theory that homologous recombination plays a key role in dengue virus evolution.

P227 Seroprevalence of human cytomegalovirus infection in Singapore

Human parechoviruses (HPeVs) belong to the Parechovirus genus of the large and growing family of Picornaviridae with a non-enveloped, single-stranded and positive-sense RNA. An HPeV strain was isolated from the nasopharyngeal aspirate specimen of a 2 months old infant hospitalized with pneumonia in Beijing, China and nominated as BJ-37359 followed the code of the specimen. Strain BJ-37359 was identified as HPeV1 by whole genome sequencing. The full genome of strain BJ-37359 consisted of 7336 nucleotides (nt), excluding a poly (A) tail and contained an ORF of 6537 nt flanked by 5′UTR of 709 nt and 3′UTR of 90 nt. Phylogenetic analyses revealed that strain BJ-37359 were clustered together with HPeV1 strains in the structural capsid protein region, while uncoupling in the non-structural gene regions. Analyses with Simplot and Bootscan indicated that multiple recombination events occurred in the non-structural region and VP0 region of strain BJ-37359 with other HPeV1, and other types might have contributed to the recombination, especially HPeV6 and HPeV7 strains. Recombination analyses indicated that strain BJ-37359 may have a mosaic genome with new genomic recombination breakpoints.

The complete genome of Enterovirus 71 China strain

Five overlapping clones covering the full genome of Enterovirus 71 China strain SHZH98 were obtained and then the sequences were determined by the chain termination method. It showed that the full length of EV71 SHZH98 genome (not including Poly A tail) is 7408 bp. There are some diversities on the lengths and sequences of 5' UTR and 3' UTR between SHZH98 and the other EV71 strains. In P1 capsid region, which is closely associated with viral immunogenicity, EV71 strain SHZH98 shares the highest homology with Taiwan strains; but in P2 and P3 non-structural gene regions there are higher identities with Coxsakievirus A16 and EV71 strains MS, BrCr than with Taiwan strains. Phylogenetic tree constructed by structural gene region indicates that China strain SHZH98 has a closer relationship with Taiwan strains, however, in the non-coding region it has a closer relationship with Coxsakievirus A16, EV71 strains MS and BrCr. EV71 China strain was analyzed at the molecular level. The results will contribute to the basic study on enteroviruses and the EV71 prevention in China.

Complete nucleotide sequence and cell-line multiplication pattern of the attenuated variant CH2195LA of Japanese encephalitis virus

Strain-specific determinant of Japanese encephalitis virus (JEV) can be different among each virulence phenotype. We reported previously that the attenuated variant CH2195LA compared with the non-attenuated variant CH2195SA had four amino acid differences in E protein (E-85, E-306, E-331, and E-387) (Wu, S.-C., Lian, W.-C., Hsu, L.-C., Liau, M., 1997. Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res. 51, 173–181). Our present study determined the complete nucleotide sequences of these two variants and found another five amino acid changes in the nonstructural gene regions, including NS2A-215, NS3-350, NS4B-196, NS4B-197, and NS4B-198. The complete nucleotide sequences of the attenuated variant CH2195LA were compared with the non-attenuated variant CH2195SA and other 13 JEV strains. CH2195LA was mostly close to CH2195SA and JaOArS982, and phylogenetically distant to SA14-14-2, SA14-2-8, and RP-2ms of the attenuated JEV strains. The multiplication patterns for CH2195LA compared with CH2195SA in other seven cell lines were different from Vero cells. Relative fitness vector analysis based on a mixture of both variants during cell passage indicated the attenuated variant CH2195LA showed increased viral fitness in Vero cell adaptation. These results revealed that the attenuated variant CH2195LA was unique to culture in Vero cells.

Mapping of genetic determinants of rubella virus associated with growth in joint tissue.

Rubella virus (RV) strains vary in their abilities to replicate and persist in cell cultures derived from human joint tissue (synovial cells [SC]), and this arthrotropism appears to be linked to their association with joint symptoms in vivo. In order to map the genetic determinants of arthrotropism, an infectious clone of the Cendehill vaccine strain of RV was constructed, as well as two chimeric clones containing cDNAs from both Cendehill and Therien (wild-type) strains. Replacement of the entire structural gene region of Therien in the infectious clone pROBO302 with the corresponding region of Cendehill did not affect growth in SC. A further observation that Cendehill bound equally well to SC and the permissive Vero cell line indicated that restriction was not at the level of receptor binding, a function of the envelope proteins. Mutations that affected growth in joint cells were mapped to two locations in the nonstructural gene region. The first of these (nucleotides 2803 and 6416) resulted in a 10-fold decrease in yield of progeny virus from SC. This region contained five mutations, at nucleotides 2829, 3060, 3164, and 3528 (near the carboxy terminus of P150 where the protease domain is located) and at nucleotide 4350 in p90. Further substitution of the sequence representing nucleotides 1 to 2803 to give a complete Cendehill infectious clone restricted growth in SC by a further 100-fold to less than 10 PFU/ml. This region contains three mutations, at nucleotides 34, 37, and 55, within the 5' stem-loop structure. In conclusion, the Cendehill-specific mutations believed to be determinants of joint cell growth are located in two regions, the 5' nontranslated region and in a sequence that encodes the carboxy-terminal region of p150 extending into the helicase domain of p90.

Evaluation of nucleic acid amplification methods for the detection of hog cholera virus.

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

Hepatitis E virus (HEV): strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site.

Hepatitis is transmitted by a number of infectious agents. The epidemiological characterization of waterborne or enterically transmitted non-A, non-B hepatitis (ET-NANBH) is unique when compared with other known hepatitides. We have reported on the molecular cloning of a cDNA clone derived from the etiologic agent associated with ET-NANBH, the hepatitis E virus (HEV). The complete sequence of these first molecular clones, isolated from an HEV-infected human after passage in Macaca fascicularis (cynomolgus macaques), illustrates a distant relationship to other known positive-strand RNA viruses of plants and animals. The translated major open reading frame (ORF-1) from these clones indicates that this portion of the genome encodes a polyprotein with consensus sequences found in RNA-dependent RNA polymerase and ATP/GTP binding domains. The latter activity has been associated with putative helicases of positive-strand RNA viruses. These viral-encoded enzymatic activities identify this region and ORF-1 as containing at least two different nonstructural genes involved in HEV replication. Molecular clones obtained from two other geographically distinct HEV isolates demonstrated sequence heterogeneity in this nonstructural gene region. Further study will be required to elucidate the pathogenic significance (if any) of this observed divergence in the nonstructural region.

Transient expression of B19 parvovirus gene products in COS-7 cells transfected with B19-SV40 hybrid vectors

Hybrid B19 parvovirus-SV40 origin vectors were transfected into COS-7 cells and replication of these plasmids studied. Plasmids that have a frameshift mutation within the nonstructural gene region replicated to high level (copy number ∼10,000/transfected cell) although somewhat lower than pSVOd, the SV40 origin vector without B19 sequence (copy number ≈100,000/transfected cell). However, hybrid B19 parvovirus-SV40 origin vectors that do not contain these frameshift mutations replicated to a much lower level (copy number ≈ 1000/transfected cell). Although the hybrid vectors studied replicated at different efficiencies in COS-7 cells, they are transcribed at approximately the same level, resulting in RNA species that are indistinguishable from those seen in B19 virus-infected erythroid bone marrow cells. Western blot analysis demonstrated that the mRNAs are translated into polypeptides of the same size and, in the case of viral structural proteins, in same relative abundance as seen in a B19-infected clinical sample.