Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.     

Distribution of human rotaviruses, especially G9 strains, in Japan from 1996 to 2000

A 4-year (1996-2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998-1999, and in Saga (78.4%) in 1999-2000. G9 strains isolated from 1998-1999 belonged to the P[8]-NSP4-Wa-group with long RNA pattern, while, G9 strains isolated from 1999-2000 belonged to three groups, the P[8]-NSP4-Wa-group with long RNA pattern, the P[4]-NSP4-KUN-group with short RNA pattern and a mixed-type group (P[4]/P[8]-NSP4-KUN/Wa-group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999-2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998-1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998-1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999-2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P-genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619-628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00-Ad2863VP7 (AB091746), 00-OS2986VP7 (AB091747), 00-SG2509VP7 (AB091748), 00-SG2518VP7 (AB091749), 00-SG2541 (AB091750), 00-SG2864 (AB091751), 00-SP2737VP7 (AB091752), 99-SP1542VP7 (AB091753), 99-SP1904VP7 (AB091754), 99-TK2082VP7 (AB091755) and 99-TK2091VP7 (AB091756).     

Apparent re-emergence of serotype G9 in 1995 among rotaviruses recovered from Japanese children hospitalized with acute gastroenteritis

Serotype G9 rotaviruses have been detected in about 0.5% of the circulating strains worldwide. However, G9 strains emerged globally in the middle of the 90s and thereafter. A rotavirus, contained in stool specimen 95H115, possessing a G9 VP7 emerged in Japan in the 1994-1995 season for the first time after a 9-year interval since prototype G9 strains AU32 and F45 were discovered in the 1985-1986 season. In comparison with other G9 VP7 genes thus far published, the sequencing of the VP7 genes of AU32 and 95H115 revealed that the 95H115 VP7 gene did not directly evolve from the AU32 VP7 gene but was much more closely related to the contemporary G9 VP7 genes found in the United States of America. Thus, recently emerging G9 VP7 genes were not direct descendants of the VP7 genes of the prototype strains in the 80s, rather they evolved independently into 4 phylogenetic clusters from a common ancestor.     

Phylogenetic and genetic characterization of a 2017 clinical isolate of H7N9 virus in Guangzhou,China during the fifth epidemic wave

Pathogenic H7N9 influenza viruses continue to pose a public health concern. The H7N9 virus has caused five outbreak waves of human infections in China since 2013. In the present study, a novel H7N9 strain (A/Guangdong/8H324/2017) was isolated from a female patient with severe respiratory illness during the fifth wave of the 2017 H7N9 epidemic. Phylogenetic analysis showed that the H7N9 viruses collected during the fifth wave belong to two different lineages: the Pearl River Delta lineage and the Yangtze River Delta lineage. The novel isolate is closely related to the Pearl River Delta H7N9 viruses, which were isolated from patients in Guangdong Province. The novel H7N9 isolate has an insertion of three basic amino acids in the cleavage site of hemagglutinin (HA), which may enhance virulence in poultry. The 2017 isolate also possesses an R292K substitution in the neuraminidase (NA) protein, which confers oseltamivir resistance. This study highlights the pandemic potential of the novel H7N9 virus in mammals; thus, future characterization and surveillance is warranted.     

The Distribution of G (VP7) and P (VP4) Serotypes among Human Rotaviruses Recovered from Japanese Children with Diarrhea

Both the G (VP7) and P (VP4) serotypes of human rotaviruses collected over a 10-year period from Japanese children with diarrhea were determined by recently-developed polymerase chain reaction-based typing assays. The combination of G1 and P8 was found in 65.2% and the combination of G2 and P4 was found in 15.2%. For the rest of the specimens, only a few other combinations occurred and their relative frequencies were less than 10%. The viruses carrying P9 were always associated with G3 as is the prototype strain AU-1.     

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.     

Dominance of Emerging G9 and G12 Genotypes and Polymorphism of VP7 and VP4 of Rotaviruses from Bhutanese Children with Severe Diarrhea Prior to the Introduction of Vaccine

A prospective study was performed to determine the molecular characteristics of rotaviruses circulating among children aged <5 years in Bhutan. Stool samples were collected from February 2010 through January 2011 from children who attended two tertiary care hospitals in the capital Thimphu and the eastern regional headquarters, Mongar. The samples positive for rotavirus was mainly comprised genotype G1, followed by G12 and G9. The VP7 and VP4 genes of all genotypes clustered mainly with those of neighboring countries, thereby indicating that they shared common ancestral strains. The VP7 gene of Bhutanese G1 strains belonged to lineage 1c, which differed from the lineages of vaccine strains. Mutations were also identified in the VP7 gene of G1 strains, which may be responsible for neutralization escape strains. Furthermore, we found that lineage 4 of P[8] genotype differed antigenically from the vaccine strains, and mutations were identified in Bhutanese strains of lineage 3. The distribution of rotavirus genotypes varies among years, therefore further research is required to determine the distribution of rotavirus strain genotypes in Bhutan.     

G9型人A组轮状病毒LL52696与LL52727株的主要基因及其编码蛋白特征的分析

人A组轮状病毒(Human rotavirus,HRV)是引起世界范围婴幼儿重症腹泻的最主要病原,也是导致发展中国家婴幼儿死亡的主要病因之一[1-3],世界卫生组织统计每年大约引起611 000婴儿和儿童死亡,特别是发展中国家[4].HRV感染广泛,且改善营养状况和卫生条件对HRV发病危险影响不大,因此在发达国家和发展中国家HRV感染率接近.HRV引起的腹泻至今无特效药,发展疫苗对控制HRV感染的作用就显得特别突出.     

山东省嘉祥县鼠类肠道病毒组分析

鼠类是重要的病毒储存库和多种病毒的自然宿主,是对人类病毒传播极具威胁的野生动物之一,因此对鼠类携带病毒进行研究并挖掘其携带新病毒对于病毒病的防治具有重要意义。本研究于2016年在山东省嘉祥县采集99只褐家鼠、仓鼠和黑线姬鼠肠道内容物,通过高通量测序对其病毒组成进行研究。分析结果显示,采集的鼠类样本携带病毒主要包括冠状病毒科、呼肠孤病毒科、星状病毒科、小RNA病毒科、小双节病毒科、圆环病毒科、细小病毒科等。挑选可能与疾病相关的病毒进行进一步的序列分析和进化分析。分析结果显示,本研究中发现的冠状病毒为甲型冠状病毒属,与中国浙江发现的Lucheng-19病毒株和英国UKRn3病毒株具有共同祖先;本研究还发现包括Rosavirus、Rabovirus和Kobuvirus三个种在内的6株小RNA病毒科病毒,其中Rabovirus包含4株序列差异较大的病毒株,表明小RNA病毒在鼠类中具有丰富的基因多样性。研究发现该地区鼠类中同时流行多株星状病毒,表明极其丰富的基因多样性,系统发育分析显示,其中1株与猪的星状病毒4型聚在一起,提示可能的跨种传播。此外,样本中发现一株G3P[3]型A组轮状病毒,其VP4和VP7基因与猴的病毒株具有90%左右核苷酸同源性,而其余片段与鼠类的片段同源性最高,说明该病毒株可能是一株猴-鼠重配病毒株。本研究的鼠类病毒组数据为我国提供更丰富的本底资料,为我国应对相关的新发传染病提供了基础数据支持。     

Molecular characterization of two strains of porcine group C rotavirus

Group C rotaviruses are an important cause of acute gastroenteritis in humans and animals. Fecal samples were collected from a porcine herd in July, 2009. Group C rotavirus RNA was detected using RT-PCR for the VP6 gene. The identified strain was further characterized by sequencing and phylogenetic analysis of the partial VP4, and complete VP6 and VP7 gene sequences. The partial VP4 and complete VP6 gene sequences of the CUK-5 strain were most closely related to those of the CUK-6 strain of group C rotaviruses. Phylogenetic analysis of the VP7 gene of the 2 strains (CUK-5 and CUK-6) and reference strains of group G rotavirus by the neighbor-joining method also confirmed that CUK-5 and CUK-6 belonged to type G5 and G1 strains, respectively. This study provides useful data for the prediction of newly appearing variants of porcine group C rotaviruses in neighboring countries through comparisons with GCRVs and fundamental research for vaccine development.     

Characterization of H7 Influenza A Virus in Wild and Domestic Birds in Korea

During surveillance programs in Korea between January 2006 and March 2011, 31 H7 avian influenza viruses were isolated from wild birds and domestic ducks and genetically characterized using large-scale sequence data. All Korean H7 viruses belonged to the Eurasian lineage, which showed substantial genetic diversity, in particular in the wild birds. The Korean H7 viruses from poultry were closely related to those of wild birds. Interestingly, two viruses originating in domestic ducks in our study had the same gene constellations in all segment genes as viruses originating in wild birds. The Korean H7 isolates contained avian-type receptors (Q226 and G228), no NA stalk deletion (positions 69–73), no C-terminal deletion (positions 218–230) in NS1, and no substitutions in PB2-627, PB1-368, and M2-31, compared with H7N9 viruses. In pathogenicity experiments, none of the Korean H7 isolates tested induced clinical signs in domestic ducks or mice. Furthermore, while they replicated poorly, with low titers (10 ^0.7–1.3EID₅₀/50 µl) in domestic ducks, all five viruses replicated well (up to 7–10 dpi, 10 ^0.7–4.3EID₅₀/50 µl) in the lungs of mice, without prior adaptation. Our results suggest that domestic Korean viruses were transferred directly from wild birds through at least two independent introductions. Our data did not indicate that wild birds carried poultry viruses between Korea and China, but rather, that wild-type H7 viruses were introduced several times into different poultry populations in eastern Asia.     

Molecular evolution of human echovirus 9 isolated from patients with aseptic meningitis in northern Kyushu during the summer of 1997.

An epidemic of aseptic meningitis caused by human echovirus 9 (E-9) occurred in the summer of 1997 in northern Kyushu, Japan. Sequences of genome position 2504-3358, which encoded a part of VP1, of the nine isolated viruses were determined. An RGD motif and B-C loop were found in all. They were almost identical and closely related to the virulent strain Barty.     

Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999

The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394-4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.     

2018–2020年人轮状病毒锦州地方株VP4及VP7基因特征

【背景】人A组轮状病毒(Rotavirus Group A,RVA)是婴幼儿胃肠炎的主要病原体及发展中国家婴幼儿死亡的重要原因,目前无特效药物治疗,疫苗预防是唯一可行的预防感染方法。外衣壳蛋白VP7和VP4是疫苗设计的主要靶点,针对该基因加强RVA地方株分子流行病学监测十分必要。【目的】对锦州地方流行RVA株VP7和VP4基因进行型别鉴定和序列特征分析。【方法】收集锦州地区2018-2020年RVA感染腹泻患儿的粪便标本,提取病毒RNA,通过RT-PCR扩增VP7、VP4基因片段并测序,得到7株RVA VP7和VP4序列。使用在线基因分型工具Rota C V2.0对测序结果进行分型分析。应用BLAST、DNAStar、MEGA X、Bio Edit等生物软件与临床流行株及疫苗株进行系统发育分析及氨基酸序列比对分析。【结果】分型结果表明7株锦州地方株均为G9P[8]型,系统发育分析证实其VP7和VP4基因分别属于G9-Ⅵ和P[8]-3谱系,核苷酸序列相似性分别为99.32%-100%与99.41%-100%。JZ株VP7与疫苗株Rotavac和Rotasiil相比,在抗原表位区7-1a、7-1b、7-2中分别存在4个和3个氨基酸替换。JZ株VP4与疫苗株Rotarix和Rota Teq VP4氨基酸序列相比,发现7个和4个氨基酸替换,位于抗原表位区8-1和8-3。【结论】2018-2020年在辽宁锦州地区检测到7株G9P[8]型RVA株,VP7和VP4序列相似性高于99%,G9P[8]型可能是辽宁省锦州地区2018-2020年婴幼儿轮状病毒腹泻的主要流行基因型之一。与同基因型疫苗株比较,位于JZ株VP7和VP4抗原表位区的氨基酸位点差异对于野毒株免疫逃逸机制的研究具有意义。     

新成人腹泻轮状病毒J19株VP2、VP3的编码基因序列分析

利用一种改良的非依赖核酸序列的单引物扩增方法,从新成人腹泻轮状病毒J19株的核酸中扩增基因,克隆至pMD18-T中并进行测序和基因序列分析。J19株的VP2、VP3的编码基因为基因2、4,分别长2 969bp、2 204bp,它们分别编码973个氨基酸和719个氨基酸。J19株的VP2蛋白序列对B组人轮状病毒IDIR株的一致性为47.2%;J19株的VP3蛋白序列对C组人轮状病毒Cowden株一致性为25.1%。对J19株VP2的遗传进化分析表明,J19株在进化树上的位置靠近外群蛋白以及A、B和C组轮状病毒分枝的根部,并且它比较偏向于B组轮状病毒的分枝。这与VP6的遗传进化分析结果相一致。根据上述结果推测J19株可能是一个与B组轮状病毒的起源和进化密切相关的毒株之一;同时,这表明VP2在研究轮状病毒的遗传进化上具有重要价值。关于新成人腹泻轮状病毒J19株VP2、VP3的编码基因的序列分析,这是首次报道。