Clinical and molecular epidemiology of enterovirus D68 from 2013 to 2020 in Shanghai.

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

A novel astrovirus identified in wild rhesus monkey feces in China.

The discovery and analysis of pathogens carried by non-human primates are important for understanding zoonotic infections in humans. We identified a highly divergent astrovirus (AstV) from fecal matter from a rhesus monkey in China, which has been tentatively named "monkey-feces-associated AstV" (MkAstV). The full-length genome of MkAstV was determined to be 7377 nt in length. It exhibits the standard genomic AstV organization of three open reading frames (ORFs) and is most closely related to duck AstV (28%, 49%, and 35% amino acid sequence identity in ORF1a, ORF1b, and ORF2, respectively). Coincidentally, while this report was being prepared, an astrovirus sequence from Hainan black-spectacled toad became available in the GenBank database, showing 95%, 94% and 92% aa sequence identity in ORF1a, ORF1b and ORF2, respectively, to the corresponding ORFs of MkAstV. Phylogenetic analysis of ORF1a, ORF1b, and ORF2 indicated that MkAstV and the amphibian-related astroviruses formed an independent cluster in the genus Avastrovirus. The host of MkAstV remains unknown. Epidemiological and serological studies of this novel virus should be undertaken in primates, including humans.

Experimental infection of Marmota monax with a novel hepatitis A virus.

To establish an animal model for the newly identified Marmota Himalayana hepatovirus, MHHAV, so as to develop a better understanding of the infection of hepatitis A viruses. Five experimental woodchucks (Marmota monax) were inoculated intravenously with the purified MHHAV from wild woodchuck feces. One animal injected with PBS was defined as a control. Feces and blood were routinely collected. After the animals were subjected to necropsy, different tissues were collected. The presence of viral RNA and negative sense viral RNA was analyzed in all the samples and histopathological and in situ hybridization analysis was performed for the tissues. MHHAV infection caused fever but no severe symptoms or death. Virus was shed in feces beginning at 2 dpi, and MHHAV RNA persisted in feces for ~2 months, with a biphasic increase, and in blood for ~30 days. Viral RNA was detected in all the tissues, with high levels in the liver and spleen. Negative-strand viral RNA was detected only in the liver. Furthermore, the animals showed histological signs of hepatitis at 45 dpi. MHHAV can infect M. monax and is associated with hepatic disease. Therefore, this animal can be used as a model of HAV pathogenesis and to evaluate antiviral and anticancer therapeutics.

Diverse novel astroviruses identified in wild Himalayan marmots.

With advances in viral surveillance and next-generation sequencing, highly diverse novel astroviruses (AstVs) and different animal hosts had been discovered in recent years. However, the existence of AstVs in marmots had yet to be shown. Here, we identified two highly divergent strains of AstVs (tentatively named Qinghai Himalayanmarmot AstVs, HHMAstV1 and HHMAstV2), by viral metagenomic analysis in liver tissues isolated from wild Marmota himalayana in China. Overall, 12 of 99 (12.1 %) M. himalayana faecal samples were positive for the presence of genetically diverse AstVs, while only HHMAstV1 and HHMAstV2 were identified in 300 liver samples. The complete genomic sequences of HHMAstV1 and HHMAstV2 were 6681 and 6610 nt in length, respectively, with the typical genomic organization of AstVs. Analysis of the complete ORF 2 sequence showed that these novel AstVs are most closely related to the rabbit AstV, mamastrovirus 23 (with 31.0 and 48.0 % shared amino acid identity, respectively). Phylogenetic analysis of the amino acid sequences of ORF1a, ORF1b and ORF2 indicated that HHMAstV1 and HHMAstV2 form two distinct clusters among the mamastroviruses, and may share a common ancestor with the rabbit-specific mamastrovirus 23. These results suggest that HHMAstV1 and HHMAstV2 are two novel species of the genus Mamastrovirus in the Astroviridae. The remarkable diversity of these novel AstVs will contribute to a greater understanding of the evolution and ecology of AstVs, although additional studies will be needed to understand the clinical significance of these novel AstVs in marmots, as well as in humans.

Analysis of Aichi virus and Saffold virus association with pediatric acute gastroenteritis.

BACKGROUND: Aichi virus (AiV) and Saffold virus (SAFV) have been reported in children with acute gastroenteritis and respiratory disease worldwide; however, their causative role in acute gastroenteritis remains ambiguous. OBJECTIVES: To assess the clinical association of AiV and SAFV with acute gastroenteritis in the pediatric population. STUDY DESIGN: A case-control study involving 461 paired stool samples from pediatric cases with diarrhea and healthy controls was conducted in China. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to screen AiV and SAFV. RESULTS: In the 461 paired samples, AiV and SAFV were more prevalent among asymptomatic children than children with acute gastroenteritis (0.87% vs. 0.43% and 2.8% vs. 1.5%, respectively), with no significant differences between groups (p=0.142 and p=0.478, respectively). Cox regression model analysis revealed no correlation between AiV (odds ratio, OR=2.24; 95% confidence interval, CI, 0.76-6.54) or SAFV infection (OR=1.36; 95% CI, 0.86-2.15) and diarrhea. High viral loads were found in both AiV- and SAFV-positive groups, with no significant difference in viral load between the groups (p=0.507 and p=0.677, respectively). No other known enteric pathogens were found in the AiV-positive samples but common in SAFV-positive cases. Phylogenetic analysis revealed that all 6 AiV subjects clustered with genotype B. All 7 SAFV-positive cases and 8 of 13 SAFV-positive controls were genotyped successfully; the genotypes identified included SAFV-1, SAFV-2 SAFV-3, and SAFV-6. CONCLUSION: Our study revealed no association of these viruses in acute gastroenteritis in children. These viruses may have the ability to replicate in humans; however, the infections are usually asymptomatic.

Identification of a Novel Enterovirus Species in Rhesus Macaque in China.

Recent studies of Enterovirus (EV) in nonhuman primates (NHPs), which could act as a source of future emerging human viral diseases, have boosted interest in the search for novel EVs. Here, a highly divergent strain of EV, tentatively named SEV-gx, was identified by viral metagenomic analysis from stool samples of rhesus macaques in China. In total, 27 of 280 (9.6%) faecal samples from rhesus macaques were positive for SEV-gx. Its complete genomic sequence is 7,367 nucleotide (nt). Genomic analyses showed that it has a standard genomic organisation for EVs, being more closely related to EV-J strains (approximately 54.0%, 43.0-44.1%, 52.3-55.2%, 61.1-62.7% and 64.0% amino acids identity in polyprotein, P1, P2 and P3 and combined 2C/3CD regions, respectively). It was also shown to have genome characteristics typical of EVs. Phylogenetic analysis of P1, 2C and 3CD aa indicated that SEV-gx can be classified as a distinct cluster in the EVs. All of this evidence demonstrates SEV-gx is a novel species (tentatively named EV-K) in the EV genus, which contributes to our understanding of the genetic diversity and evolution of EVs. Further studies are needed to investigate the potential pathogenicity of SEV-gx in NHPs and humans.

A novel hepatovirus identified in wild woodchuck Marmota himalayana.

Hepatitis A virus (HAV) is a hepatotropic picornavirus that causes acute liver disease worldwide. Here, we report on the identification of a novel hepatovirus tentatively named Marmota Himalayana hepatovirus (MHHAV) in wild woodchucks (Marmota Himalayana) in China. The genomic and molecular characterization of MHHAV indicated that it is most closely related genetically to HAV. MHHAV has wide tissue distribution but shows tropism for the liver. The virus is morphologically and structurally similar to HAV. The pattern of its codon usage bias is also consistent with that of HAV. Phylogenetic analysis indicated that MHHAV groups with known HAVs but forms an independent branch, and represents a new species in the genus Hepatovirus within the family Picornaviridae. Antigenic site analysis suggested MHHAV has a new antigenic property to other HAVs. Further evolutionary analysis of MHHAV and primate HAVs led to a most recent common ancestor estimate of 1,000 years ago, while the common ancestor of all HAV-related viruses including phopivirus can be traced back to 1800 years ago. The discovery of MHHAV may provide new insights into the origin and evolution of HAV and a model system with which to explore the pathogenesis of HAV infection.

Salivirus in Children and Its Association with Childhood Acute Gastroenteritis: A Paired Case-Control Study.

Salivirus was recently discovered in children with gastroenteritis and in sewage. Though a causative role for salivirus in childhood gastroenteritis was suggested in the previous study, the relationship between salivirus and acute gastroenteritis has not yet been clearly clarified. The sewage strain reported by Ng, although represented by incomplete genome sequencing data, was distinct from previously reported saliviruses, and had not previously been detected in humans. A case-control study examining 461 paired stool samples from children with diarrhea and healthy controls (1:1) was conducted in this study. Also, common diarrheal viruses were detected and complete genome of a salivirus was determined. Results showed that salivirus was detected in 16 (3.5%) and 13 (2.8%) of the case and control samples, respectively; no differences in detection rates (p=0.571) or mean values of viral loads (p=0.400) were observed between the groups. Multivariate Cox regression revealed no association between salivirus and gastroenteritis (p=0.774). The data also demonstrated that salivirus infection did not exacerbate clinical symptoms of gastroenteritis in children. Furthermore, complete genome sequence of a salivirus recovered from the feces of a child with diarrhea (i.e., SaliV-FHB) shared a 99% nucleotide identity with the sewage strain. In conclusion, a paired case-control study did not support a causative role for salivirus strains detected in this study with pediatric gastroenteritis. This study also demonstrated that all known saliviruses can be detected in the feces of children with or without gastroenteritis.

Complete genome sequence of a novel human papillomavirus identified by metagenomic analysis from a child with diarrhea in China.

A highly divergent human papillomavirus, named HPV-CH2, was identified in fecal samples from children with diarrhea in China by 454 high-throughput sequencing. Here, we report the complete genome sequence and genetic organization of the virus. The full-length nucleotide sequence of HPV-CH2 shares the highest sequence similarity with HPV-156, with 72 % nucleotide sequence identity. The L1 gene of the HPV-CH2 shared <70 % nucleotide identity with previously reported HPVs, suggesting HPV-CH2 as a new type of papillomavirus. Phylogenetic analysis revealed that the HPV-CH2 belongs to the genus Gammapapillomavirus. No HPV-CH2 was detected by PCR in samples from children with both gastroenteritis and respiratory infection.

Detection of human norovirus GIV.1 in China: a case report.

Noroviruses (NoVs) are a common cause of acute gastroenteritis (AGE) around the world; however, reports of genogroup IV (GIV) NoVs are rare. Here we report a human GIV genotype 1 (GIV.1) NoV strain (named CHNNGIV2011) identified by 454 high-throughput sequencing from stool samples of children with diarrhea. This is the first documented human GIV.1 NoVs infection in China. The complete genome of the virus is 7525 nucleotides in length. Sequencing and phylogenetic analyses showed that CHNNGIV2011 shared high sequence similarity to other GIV.1 NoVs from all over the world, especially to the recently reported NoV GIV.1 strain Lake Macquarie genome (99.0%). By seminested PCR and real-time PCR, a total of 2 out of 466 samples were positive for GIV.1 CHNNGIV2011 in Lulong County, Hebei Province, China, which supported a low prevalence of GIV.1 NoVs. The positive samples contained 7.2×10(7) and 2.6×10(8)copies/g in feces. In addition, one positive sample was found coinfection with strains NoV GII and salivirus. These findings suggest more study is needed to address the worldwide prevalence and role of GIV NoVs in AGE.

Identification of a novel strain of human papillomavirus from children with diarrhea in china.

A highly divergent human papillomavirus (HPV) strain, HPV-L55, was identified in fecal samples from children hospitalized with diarrhea in China. The L1 gene of HPV-L55 shares <75% identity with previously reported HPVs, indicating that this virus represents a novel type of HPV. Phylogenetic analysis classified this virus as a member of the gammapapillomaviruses.

Identification of a novel picornavirus in healthy piglets and seroepidemiological evidence of its presence in humans.

In this study, we describe a novel porcine parechovirus-like virus (tentatively named PLV-CHN) from healthy piglets in China using 454 high-throughput sequencing. The complete genome of the virus comprises 6832 bp, encoding a predicted polyprotein of 2132 amino acids that is most similar to Ljungan virus (32% identity). A similar virus that belongs to a novel Picornaviridae genus, named swine pasivirus 1 (SPaV-1), was reported during the preparation of this paper. Sequence analysis revealed that PLV-CHN and SPaV1 shared 82% nucleotide identity and 89% amino acid identity. Further genomic and phylogenetic analyses suggested that both SPaV1 and PLV-CHN shared similar genomic characteristics and belong to the same novel Picornaviridae genus. A total of 36 (20.0%) fecal samples from 180 healthy piglets were positive for PLV-CHN by RT-PCR, while no fecal samples from 100 healthy children and 100 children with diarrhea, and no cerebrospinal fluid samples from 196 children with suspected viral encephalitis, was positive for the virus. However, Western blot and enzyme-linked immunosorbent assays using recombinant PLV-CHN VP1 polypeptide as an antigen showed a high seroprevalence of 63.5% in the healthy population. When grouped by age, the antibody-positivity rates showed that the majority of children under 12 years of age have been infected by the virus. It was suggested that PLV-CHN, SPaV1, or an as-yet-uncharacterized virus can infect humans early in life. Thus, investigation of the role of this novel virus is vital.

Detection of novel viruses in porcine fecal samples from China.

BACKGROUND: Pigs are well known source of human infectious disease. To better understand the spectrum of viruses present in pigs, we utilized the 454 Life Sciences GS-FLX high-throughput sequencing platform to sequence stool samples from healthy pigs. FINDINGS: Total nucleic acid was extracted from stool samples of healthy piglets and randomly amplified. The amplified materials were pooled and processed using a high-throughput pyrosequencing technique. The raw sequences were deconvoluted on the basis of the barcode and then processed through a standardized bioinformatics pipeline. The unique reads (348, 70 and 13) had limited similarity to known astroviruses, bocaviruses and parechoviruses. Specific primers were synthesized to assess the prevalence of the viruses in healthy piglets. Our results indicate extremely high rates of positivity. CONCLUSIONS: Several novel astroviruses, bocaviruses and Ljungan-like viruses were identified in stool samples from healthy pigs. The rates of isolation for the new viruses were high. The high detection rate, diverse sequences and categories indicate that pigs are well-established reservoirs for and likely sources of different enteric viruses.

[Optimizing expression of the capsid protein VP2 from human Bocavirus and establish it's seroepidemiology assying methord].

OBJECTIVE: To obtain sufficient recombinant VP2 protein of human Bocavirus and establish it's seroepidemiology assying metbord. METHORD: Tbe capsid protein VP2 DNA genes of HBoV1 and 2 were optimized in accordance with tbe usage of the favorite codons in K coil so as to enhance its protein expression in prokaryotic expressing system. The protein was purified by Ni-NTA column, and its antigenicity was determined by Western Blot. Then establish ELISA to detect the specific anti-VP2 IgG antibodies against HBoV1 and 2 in healthy children aged 3-6 years in Nanjing, China. RESULTS: The recombinant protein 6 x His-VP2 was produced in a larger quantity at 25 degrees C induced by IPTG (1 mmol/L) over night and purified by Ni-NTA column. Seropositive rates of HBoV1 and 2 were 62.2% and 55.5% and their mixed seropositivity was 37%. CONCLUSION: The optimizing expression of the capsid protein VP2 from human Bocavirus constructed successfully and get a high yield under certain conditions. The established ELISA could be used to further analyze seroepidemiology of HBoV in China.