GIII Strains Research Articles

Genomic Evolution and Selective Pressure Analysis of a Novel Porcine Sapovirus in Shanghai, China.

Porcine sapovirus (PoSaV) is one of the most significant pathogens causing piglet diarrhea, and one with limited genetic characterization. In this study, the prevalence, infection pattern, and genetic evolution of porcine sapovirus were elucidated in detail. The positive rate of PoSaV was 10.1% (20/198), with dual, triple, and quadruple infections of 45%, 40%, and 5%, respectively. To further explore the viral composition in the PoSaV-positive diarrhea feces, metagenomic sequencing was carried out. The results confirmed that RNA viruses accounted for a higher proportion (55.47%), including the two primary viruses of PoSaV (21.78%) and porcine astrovirus (PAstV) (24.54%) in the tested diarrhea feces samples. Afterward, a full-length sequence of the PoSaV isolate was amplified and named SHCM/Mega2023, and also given the identifier of GenBank No. PP388958. Phylogenetic analysis identified the prevalent PoSaV strain SHCM/Mega2023 in the GIII genogroup, involving a recombinant event with MK962338 and KT922089, with the breakpoint at 2969-5132 nucleotides (nt). The time tree revealed that the GIII genogroup exhibits the widest divergence time span, indicating a high likelihood of viral recombination. Moreover, SHCM/Mega2023 had three nucleotide "RPL" insertions at the 151-153 nt site in the VP2 gene, compared to the other GIII strains. Further selective pressure calculations demonstrate that the whole genome of the SHCM/Mega2023 strain was under purifying selection (dN/dS < 1), with seven positively selected sites in the VP1 protein, which might be related to antigenicity. In conclusion, this study presents a novel genomic evolution of PoSaV, offering valuable insights into antigenicity and for vaccine research.

Attenuated Chimeric GI/GIII Vaccine Candidate against Japanese Encephalitis Virus.

Japanese encephalitis (JE) is a very severe disease characterized by high fatality rates and the development of permanent behavioral, psychiatric, and neurological sequelae among survivors. Japanese encephalitis virus (JEV), a flavivirus, is responsible for JE. In Asia, Genotype I (GI) has emerged as the dominant strain, replacing Genotype III (GIII). However, no clinically approved drug is available to treat JEV infection, and currently available commercial vaccines derived from JEV GIII strains provide only partial protection against GI. Utilizing a reverse genetics system, this study attempted to produce a novel chimeric JEV strain with high efficacy against JEV GI. Accordingly, a GI/GIII intertypic recombinant strain, namely SA14-GI env, was generated by substituting the E region of the GIII SA14-14-2 strain with that of the GI strain, K05GS. The neurovirulence of the mutant virus was significantly reduced in mice. Analysis of the immunogenicity of the chimeric virus revealed that it induced neutralizing antibodies against JEV GI in mice, and the protective efficacy of SA14-GI env was higher than that of SA14-14-2. These findings suggest that SA14-GI env may be a safe and effective live-attenuated vaccine candidate against JEV GI.

Virulence and Cross-Protection Conferred by an Attenuated Genotype I-Based Chimeric Japanese Encephalitis Virus Strain Harboring the E Protein of Genotype V in Mice.

Japanese encephalitis virus (JEV) genotype V (GV) emerged in China in 2009, then South Korea, and has since spread to other regions in Asia and beyond, raising concern about its pathogenicity and the cross-protection offered by JEV vaccines against different genotypes. In this study, we replaced the structural proteins (C-prM-E) of an attenuated genotype I (GI) SD12-F120 strain with those of a virulent GV XZ0934 strain to construct a recombinant chimeric GI-GV JEV (JEV-GI/V) strain to determine the role of the structural proteins in virulence and cross-protection. The recombinant chimeric virus was highly neurovirulent and neuroinvasive in mice. This demonstrated the determinant role of the structural proteins in the virulence of the GV strain. Intracerebral or intraperitoneal inoculation of mice with JEV-GI/V-E5 harboring a combination of substitutions (N47K, L107F, E138K, H123R, and I176R) in E protein, but not mutants containing single substitution of these residues, resulted in decreased or disappeared mortality, suggesting that these residues synergistically, but not individually, played a role in determining the neurovirulence and neuroinvasiveness of the GV strain. Immunization of mice with attenuated strain JEV-GI/V-E5 provided complete protection and induced high neutralizing antibody titers against parental strain JEV-GI/V, but partial cross-protection and low cross-neutralizing antibodies titers against the heterologous GI and GIII strains in mice, suggesting the reduced cross-protection of JEV vaccines among different genotypes. Overall, these findings suggested the essential role of the structural proteins in determination of the virulence of GV strain, and highlighted the need for a novel vaccine against this newly emerged strain. IMPORTANCE The GV JEV showed an increase in epidemic areas, which exhibited higher pathogenicity in mice than the prevalent GI and GIII strains. We replaced a recombinant chimeric GI-GV JEV (JEV-GI/V) strain to determine the role of the structural proteins in virulence and cross-protection. It was found that the essential role of the structural proteins is to determinethe virulence of the GV strain. It is also suggested that there is reduced cross-protection of JEV vaccines among different genotypes, which provides basic data for subsequent JEV prevention, control, and new vaccine development.

High Genetic Diversity of Porcine Sapovirus From Diarrheic Piglets in Yunnan Province, China

As one of the most important enteric viruses, sapovirus (SaV) can infect humans and a variety of animals. Until now, 19 SaV genogroups have been identified, among which 4 from human (GI, GII, GIV, and GV) and 8 from swine (GIII, GV–GXI). Porcine sapovirus (PoSaV) GIII has been prevalent in China; however, the status of PoSaV infection in Yunnan province remains unknown. In this study, 202 fecal samples were collected from piglets associated with outbreaks of acute diarrhea in Yunnan between January and May 2020. PoSaV detection revealed that the total PoSaV infection rate in Yunnan was 35.2%, with 21 PoSaV strains determined and phylogenetically analyzed. The phylogenetic tree analyses demonstrated that twenty PoSaV strains belonged to GIII and fell into five genotypes, whereas one PoSaV strain (YNQB) belonged to GV. Sequence alignments revealed deletions in VP2 region in 10 of the 20 GIII strains, as well as deletions and insertions in VP1 region of the GV strain (YNQB). Furthermore, genomic recombination analyses showed that two GIII strains (YNAN and YNJD) were recombinants, closely related to reference sequences MK965898 and LC215880, MK965898 and FJ387164, respectively. In summary, PoSaV-GIII strains were identified in Yunnan in 2020, and for the first time, a PoSaV-GV strain was identified from China, whereas the comprehensive analyses illustrated high genetic diversity of Yunnan PoSaV strains. This study may shed new light on the current PoSaV infections in Yunnan and pave the way toward further control of the PoSaV infections in China.

NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/β induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings.

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and β production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and β induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and β production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and β induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and β production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and β production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.

Genetically divergent porcine sapovirus identified in pigs, United States.

Porcine sapoviruses (SaVs) are genetically diverse and widely distributed in pig-producing countries. Eight genogroups of porcine SaV have been identified, and genogroup III is the predominant type. Most of the eight genogroups of porcine SaV are circulating in the United States. In the present study, we report detection of porcine SaVs in pigs at different ages with clinical diarrhoea using next-generation sequencing and genetic characterization. All seven cases have porcine SaV GIII strains detected and one pooled case was found to have a porcine SaV GVI strain IA27912-B-2018. Sequence analysis showed that seven GIII isolates were genetically divergent and formed four different lineages on the trees of complete genome, RdRP, VP1 and VP2. In addition, these seven GIII isolates had three different deletion/insertion patterns in an identified variable region close to the 3' end of VP2. The GVI strain IA27912-B-2018 was closely related to strains previously detected in the United States and Japan. A 3-nt deletion in VP1 region of GVI IA27912-B-2018 was identified. Our study showed that genetically divergent SaVs of different genogroups are co-circulating in pigs in the United States. Future studies comparing the virulence of these different genogroups in pigs are needed to better understand this virus and to determine if surveillance and vaccine development are needed to monitor and control porcine SaVs.

Partial cross-protection between Japanese encephalitis virus genotype I and III in mice.

Genotype III (GIII) Japanese encephalitis virus (JEV) predominance has gradually been replaced by genotype I (GI) over the last 20 years in many Asian countries. This genotype shift raises concerns about the protective efficacy of Japanese encephalitis (JE) vaccines, as all of the currently licensed JE vaccines are derived from GIII strains. In this study, we conducted vaccination-challenge protection assays to evaluate the cross-protective efficacy of GI- or GIII-derived vaccines against the challenge of a heterologous genotype using a mouse challenge model. Titration of the neutralizing antibodies elicited by SA14-14-2 live-attenuated JE vaccine (SA14-14-2 vaccine), a GIII-derived vaccine, indicated that the titer of neutralizing antibodies specific to heterologous genotype GI stain was significantly lower than that specific to homologous genotype GIII strain in both pigs and mice immunized with the SA14-14-2 vaccine. Vaccination of mice with SA14-14-2 vaccine or a GIII-inactivated vaccine at high and medium doses completely protected vaccinated mice against challenge with the homologous genotype GIII strains, but failed to provide the vaccinated mice complete protection against the challenge of heterologous genotype GI strains. The protection rates against GI strain challenge were 60%–80%, showing that these vaccines were partially protective against GI strain challenge. Additionally, vaccination of mice with a GI-inactivated vaccine conferred 100% protection against the challenge of homologous genotype GI strains, but 50%–90% protection against the challenge of heterologous genotype GIII strains, showing a reduced protective efficacy of a GI-derived vaccine against GIII strain challenge. Overall, these observations demonstrated a partial cross-protection between GI and GIII strains and suggested a potential need for new JE vaccine strategies, including options like a bivalent vaccine, to control both genotype infection.

Neutralization Potency of Sera from Vietnamese Patients with Japanese Encephalitis (JE) against Genotypes I and V JE Viruses.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.

Differential Infectivities among Different Japanese Encephalitis Virus Genotypes in Culex quinquefasciatus Mosquitoes.

During the last 20 years, the epidemiology of Japanese encephalitis virus (JEV) has changed significantly in its endemic regions due to the gradual displacement of the previously dominant genotype III (GIII) with clade b of GI (GI-b). Whilst there is only limited genetic difference distinguishing the two GI clades (GI-a and GI-b), GI-b has shown a significantly wider and more rapid dispersal pattern in several regions in Asia than the GI-a clade, which remains restricted in its geographic distribution since its emergence. Although previously published molecular epidemiological evidence has shown distinct phylodynamic patterns, characterization of the two GI clades has only been limited to in vitro studies. In this study, Culex quinquefasciatus, a known competent JEV mosquito vector species, was orally challenged with three JEV strains each representing GI-a, GI-b, and GIII, respectively. Infection and dissemination were determined based on the detection of infectious viruses in homogenized mosquitoes. Detection of JEV RNA in mosquito saliva at 14 days post infection indicated that Cx. quinquefasciatus can be a competent vector species for both GI and GIII strains. Significantly higher infection rates in mosquitoes exposed to the GI-b and GIII strains than the GI-a strain suggest infectivity in arthropod vectors may lead to the selective advantage of previously and currently dominant genotypes. It could thus play a role in enzootic transmission cycles for the maintenance of JEV if this virus were ever to be introduced into North America.

Molecular Epidemiology of Japanese Encephalitis Virus in Mosquitoes during 2013- 2014

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Japanese encephalitis is widespread in Asia and endemic in Taiwan. Virological surveillance in mosquitoes showed that the genotype III (GIII) strains of JEV were the predominant epidemic strains circulating in Asia before 1990; however, the genotype I (GI) strains have been introduced into China, Japan, Vietnam, South Korea, and Thailand, and cocirculated with or replaced the GIII strains in Asia. Previous studies showed that in Taiwan, all of the JEV strains obtained during 2005- 2007 belonged to GIII of JEV, but the predominant genotype has shifted from GIII to GI during 2008-2012. This study continued to report the genotype distribution, genetic variation, and mosquito species potentially involved in the transmission of JEV during 2013-2014. Overall, 37,637 mosquitoes were collected; 96 pools were tested JEV positive by RT-PCR, including Culex tritaeniorhynchus (94 pools) and Cx. annulus (2 pools). Phylogenetic analysis of envelope gene sequences of JEV isolated from mosquitoes demostrated that GI was the predominant genotype of JEV between 2013 and 2014, whilst only a few GIII strains of JEV were found in Eastern Taiwan. We will continue the monitoring of endemic and newly introduced JEV in Taiwan.

Transmission and Demographic Dynamics of Coxsackievirus B1.

The infectious activity of coxsackievirus B1 (CV-B1) in Taiwan was high from 2008 to 2010, following an alarming increase in severe neonate disease in the United States (US). To examine the relationship between CV-B1 strains isolated in Taiwan and those from other parts of the world, we performed a phylodynamic study using VP1 and partial 3Dpol (414 nt) sequences from 22 strains of CV-B1 isolated in Taiwan (1989–2010) and compared them to sequences from strains isolated worldwide. Phylogenetic trees were constructed by neighbor-joining, maximum likelihood, and Bayesian Monte Carlo Markov Chain methods. Four genotypes (GI–IV) in the VP1 region of CV-B1 and three genotypes (GA–C) in the 3Dpol region of enterovirus B were identified and had high support values. The phylogenetic analysis indicates that the GI and GIII strains in VP1 were geographically distributed in Taiwan (1993–1994) and in India (2007–2009). On the other hand, the GII and GIV strains appear to have a wider spatiotemporal distribution and ladder-like topology A stair-like phylogeny was observed in the VP1 region indicating that the phylogeny of the virus may be affected by different selection pressures in the specified regions. Further, most of the GI and GII strains in the VP1 tree were clustered together in GA in the 3D tree, while the GIV strains diverged into GB and GC. Taken together, these data provide important insights into the population dynamics of CV-B1 and indicate that incongruencies in specific gene regions may contribute to spatiotemporal patterns of epidemicity for this virus.

In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain.

The characteristics of genotype V Japanese encephalitis virus (GV JEV) remain poorly understood as only two strains have been isolated to date. In this study, we examined the effects of the GV JEV Muar strain on in vitro growth and pathogenicity in mice; we also evaluated the efficacy of inactivated JEV vaccines against the Muar strain. Although growth of the Muar strain in mouse neuroblastoma N18 cells was clearly worse than that of the GIII Beijing-1 and GI Mie/41/2002 strains, neuroinvasiveness of the Muar strain was similar to that of the Beijing-1 strain and significantly higher than that of the Mie/41/2002 strain. The results of a plaque reduction neutralization test suggested that the neutralization ability of the JEV vaccines against the Muar strain was reduced compared with the GI and GIII strains. However, the protection potency of the JEV vaccine against the Muar strain was similar to that for the Beijing-1 strain in mice. Our data indicate that GV JEV has unique growth, virulence and antigenicity features.

Molecular epidemiology of Japanese encephalitis in northern Vietnam, 1964-2011: genotype replacement.

BackgroundJapanese encephalitis virus (JEV) is an arthropod-borne virus causing serious public health issues in Asia. JEV consists of five genotypes and recent studies have shown the emergence of JEV genotype I (GI) and its replacement of genotype III (GIII). Using an archival JEV collection, we investigated the molecular evolution of JEV in Vietnam over the last 48 years (1964–2012) in humans, mosquitoes, and pigs, within the global context.MethodsThe nine JEV isolates from humans, pigs, and mosquitoes sequenced in this study and 29 sequences available in GenBank were used to analyze the envelope (E) protein of the Vietnamese JEVs. A collection of 225 cerebrospinal fluid specimens from patients with suspected Japanese encephalitis (JE) was also tested and genotyped with real-time RT–PCR.ResultsThe 38 E genes identified with sequencing and nine Vietnamese JEV strains genotyped with real-time RT–PCR, belonging to two lineages, evolved in accordance with those in the rest of the world. The first GIII strain was detected in humans in Vietnam in 1964, and in mosquitoes in 1979, whereas GI strains were first detected in humans and mosquitoes in 1990 and 1994, respectively. After 2004, GI was the only genotype detected in Vietnam, demonstrating that the GIIII strains had been displaced by GI strains. Five haplotypes were identified in the Vietnamese JEVs, with SKSS predominant. The S123N and S123R substitutions in the E protein were already present in the Vietnamese JEVs.ConclusionThis study describes the long evolutionary history of JEV in Vietnam over 34 years, which correlates well with the global evolution of JEV. The Vietnamese GIII strains have been replaced by GI strains in mosquitoes, pigs, and humans. The predominant haplotypes of the Vietnamese strains support this genotype displacement in Vietnam. Further surveillance is required to confirm the disappearance of the GIII strains in nature and the emergence of new pathogens causing encephalitis in Vietnam, after the long-term use of JEV vaccines in that country.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0278-4) contains supplementary material, which is available to authorized users.

Molecular characterization of Japanese encephalitis virus strains prevalent in Chinese swine herds

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia and domestic pigs serve as the amplifying hosts. In the present study, the full genomic sequences of two JEV strains (HEN0701 and SH0601) isolated from pigs in China were determined and compared with other 12 JEV strains deposited in GenBank. These two strains had an 88.8% nucleotide sequence similarity and 97.9% deduced amino acid sequence homology. HEN0701 had high nucleotide sequence and high amino acid sequence identity with genotype I (GI) strains, while SH0601 had high nucleotide sequence and high amino acid sequence identity with GIII strains at both the gene and full genome levels. Further phylogenetic analysis showed that HEN0701 belonged to the JEV GI group and SH0601 was classified as a GIII strain. Analysis of codon usage showed there were a few differences between the GI and GIII strains in nucleotide composition and codon usage for the open reading frames.

Introduction of Japanese Encephalitis Virus Genotype I, India

Introduction of Japanese Encephalitis Virus Genotype I, India

Identification and isolation of Genotype-I Japanese Encephalitis virus from encephalitis patients

Historically, Japanese Encephalitis virus (JEV) genotype III (GIII) has been responsible for human diseases. In recent years, JEV genotype I (GI) has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF) of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM) and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.

Bovine Norovirus: Carbohydrate Ligand, Environmental Contamination, and Potential Cross-Species Transmission via Oysters

Noroviruses (NoV) are major agents of acute gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Previous studies showed that some human strains bind to oyster tissues through carbohydrate ligands that are similar to their human receptors. Thus, based on presentation of shared norovirus carbohydrate ligands, oysters could selectively concentrate animal strains with increased ability to overcome species barriers. In comparison with human GI and GII strains, bovine GIII NoV strains, although frequently detected in bovine feces and waters of two estuaries of Brittany, were seldom detected in oysters grown in these estuaries. Characterization of the carbohydrate ligand from a new GIII strain indicated recognition of the alpha-galactosidase (α-Gal) epitope not expressed by humans, similar to the GIII.2 Newbury2 strain. This ligand was not detectable on oyster tissues, suggesting that oysters may not be able to accumulate substantial amounts of GIII strains due to the lack of shared carbohydrate ligand and that they should be unable to contribute to select GIII strains with an increased ability to recognize humans.

Molecular Epidemiology of Japanese Encephalitis Virus, Taiwan

Molecular Epidemiology of Japanese Encephalitis Virus, Taiwan

Molecular evolution of Japanese encephalitis virus isolates from swine in Oita, Japan during 1980–2009

In order to identify the patterns of genetic change of Japanese encephalitis virus (JEV) strains circulating in Oita, the complete envelope (E) gene has been sequenced for 35 isolates from swine in a 30-year span. Based on nucleotide and deduced amino acid sequences, the genetic variation was examined, phylogeny was estimated and selection pressures were also analyzed. This study demonstrated that the major genotype (G) of JEV isolates had shifted from GIII to GI in the mid-1990s in Oita. The intensities of selection acting on the Oita GIII and GI strains were found to be almost same. It suggests that the intensity of selection might not be the reason for such a genotype shift observed in Oita. Pairwise comparisons revealed the high conservation of the E gene at the protein level. Compared with the Oita GIII strains, all the Oita GI strains shared four amino acid changes at E129 (T–M), E222 (A–S), E327 (S–T) and E366 (A–S). Among all 70 JEV isolates involved in this paper, the GI strains shared only one amino acid change at E222 (A–S) in comparison with the GIII strains. No strong evidence for positive selection was found, the JEV evolution has generally been subject to strong purifying selection, but one ongoing evolutionary pathway was found to be under relaxed purifying selection in Oita. This study is a localized example of JEV molecular evolution in nature.

Expression of sapovirus virus-like particles in mammalian cells

Sapovirus (SaV) is an etiological agent of acute gastroenteritis in human and swine. SaV can be divided into five genogroups, GI to GV. Virus-like particles (VLPs) morphologically similar to native SaV have been expressed for GI, GII, GIII and GV strains in insect cells, although only low expression levels were observed for GII strains. In this study, we report the successful expression of SaV GII VLPs using cultured mammalian COS-7 and 293T cells. Our results demonstrated that this mammalian expression system was able to express and form SaV VLPs.