Increase human metapneumovirus mediated morbidity following pandemic influenza infection

Human metapneumovirus (hMPV) is a recently discovered respiratory pathogen, infecting mainly young children. The infected patients suffer from influenza like symptoms (ILS). In Israel the virus is mainly circulating in February to March. Here we report on an increased rate of hMPV infection in the winter season of 2009-10. The 2009-10 infection had several unique characteristics when compared to previous seasons; it started around January and a large number of infants were infected by the virus. Genetic analysis based on the viral L and F genes of hMPV showed that only subtypes A2 and B2 circulated in Israel. Additionally, we have identified a novel variant of hMPV within subgroup A2b, which subdivide it into A2b1 and A2b2. Finally, we showed that the hMPV infection was detected in the country soon after the infection with the pandemic influenza virus had declined, that infection with the pandemic influenza virus was dominant and that it interfered with the infection of other respiratory viruses. Thus, we suggest that the unusual increase in hMPV infection observed in 2009-10 was due to the appearance of the pandemic influenza virus in the winter season prior to 2009-10.     

Genetic variability and circulation pattern of human metapneumovirus isolated in Italy over five epidemic seasons

Human metapneumovirus (hMPV) is an important aetiological agent of respiratory tract infection (RTI) in infants. Based on genetic differences, hMPVs are separated into two main groups, A and B, and two subgroups 1 and 2. To better understand the genetic diversity of hMPV, we analyzed 435 bp fragments of the F gene from 49 isolates obtained from a paediatric clinical centre in Northern Italy from 2005 to 2009. The phylogenetic analysis showed that our hMPV sequences clustered into five main clades (A1, A2a, A2b, B1, B2) statistically supported. Most of the strains belong to A2 (49%) and to B1 (28.5%) lineages. The intermixing in the phylogenetic tree showed no seasonal distribution between samples collected over a 5 year period. Mean genetic distances showed that clade A2 was more heterogeneous than others. In our F glycoprotein dataset we observed only two positively selected sites with an w value of 1.408 and 1.429, respectively, and 27 negatively selected sites. The two positively selected sites could be considered evolutionary "hot spots" because they were under positive selection and/or relaxed selective constraints. The abundant negatively selected sites reflect a high degree of conservation of F protein, probably necessary for viral infection.     

The Global Spread of Hepatitis C Virus 1a and 1b: A Phylodynamic and Phylogeographic Analysis

Background

Hepatitis C virus (HCV) is estimated to affect 130–180 million people worldwide. Although its origin is unknown, patterns of viral diversity suggest that HCV genotype 1 probably originated from West Africa. Previous attempts to estimate the spatiotemporal parameters of the virus, both globally and regionally, have suggested that epidemic HCV transmission began in 1900 and grew steadily until the late 1980s. However, epidemiological data suggest that the expansion of HCV may have occurred after the Second World War. The aim of our study was to elucidate the timescale and route of the global spread of HCV.

Methods and Findings

We show that the rarely sequenced HCV region (E2P7NS2) is more informative for molecular epidemiology studies than the more commonly used NS5B region. We applied phylodynamic methods to a substantial set of new E2P7NS2 and NS5B sequences, together with all available global HCV sequences with information in both of these genomic regions, in order to estimate the timescale and nature of the global expansion of the most prevalent HCV subtypes, 1a and 1b. We showed that transmission of subtypes 1a and 1b “exploded” between 1940 and 1980, with the spread of 1b preceding that of 1a by at least 16 y (95% confidence interval 15–17). Phylogeographic analysis of all available NS5B sequences suggests that HCV subtypes 1a and 1b disseminated from the developed world to the developing countries.

Conclusions

The evolutionary rate of HCV appears faster than previously suggested. The global spread of HCV coincided with the widespread use of transfused blood and blood products and with the expansion of intravenous drug use but slowed prior to the wide implementation of anti-HCV screening. Differences in the transmission routes associated with subtypes 1a and 1b provide an explanation of the relatively earlier expansion of 1b. Our data show that the most plausible route of the HCV dispersal was from developed countries to the developing world. Please see later in the article for the Editors'' Summary     

Exploring the Molecular Epidemiology and Evolutionary Dynamics of Influenza A Virus in Taiwan

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.     

我国牛病毒性腹泻流行现状与防控策略

我国牛病毒性腹泻病(Bovine viral diarrhea,BVD)的流行比较复杂,其病原BVDV (BVDV-1和BVDV-2)不仅仅局限于已知易感动物牛群感染,其他动物种群中感染BVDV-1和BVDV-2的现象也值得注意,如猪群中BVDV感染很大程度上混淆了猪瘟等病原的监测,从而加剧病程发展。牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)可致持续感染(Persistent infection,PI),这一特性导致该病的净化面临巨大困难,对整个养殖场的健康发展形成了严峻威胁。BVDV抗原变异速率非常快,目前BVDV-1已有22个亚型,BVDV-2有4个亚型,鉴于病原在自然界的适应和演进特性,对该病的防控措施迟后其病原的变异速度。因此,定期摸清BVDV-1和BVDV-2在我国的流行现状是实施疫病净化的第一步和关键步骤,进一步借鉴国外BVD净化成功经验,综合考虑我国国情,采取适宜的防控策略,逐步净化该病原感染,有助于促进国内养殖业的健康发展。     

Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region

Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, approximately 90 degrees C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of approximately 50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors.     

戊型肝炎病毒遗传多样性和进化机制的研究进展

戊型肝炎病毒(hepatitis E virus, HEV)是一种引发全球急性病毒性肝炎的人兽共患病病原。HEV具有丰富的遗传多样性,不同基因型或基因亚型的流行与地理位置、宿主物种以及防控策略等密切相关。欧洲和美洲HEV流行株为HEV-3,包括3a-i亚型,而亚洲流行株含HEV-3和HEV-4;我国的流行毒株已从HEV-1进化到HEV-4。近年来,研究发现HEV进化的影响机制,包括同义密码子使用模式、氨基酸突变和基因重组等,其中氨基酸突变是病毒持续流行的主要驱动力。因此,本文就HEV的分类、全球流行特征、进化机制等进行综述,以期为戊型肝炎的有效防控以及疫苗开发提供参考。     

The molecular epidemiological aspects of hiv-infection spreading in Perm region

Retrospective analysis of HIV-infection spreading in Perm region in conjunction with the genetic characterization of viral subtypes circulated on this territory from 1988 (when 1st case of infection was detected) until 2005 was performed. Analysis of epidemic process allowed to determine three periods of its development basing on both epidemic intensity and nature of circulating HIV-1 subtypes. During 1988 - 1996 (first period), when viral population was heterogenous (simultaneous circulation of three HIV-1 subtypes) with multiple routes of transmission, the epidemic process was characterized by low intensity. High incidence of HIV-infection among injection drug users and high homogeneity of circulated HIV-1 variants (98% of isolated variants belonged to HIV-1 subtype A with low level of genetic variability) were characteristics of the second period lasted from 1997 to 2001. Decrease in HIV-infection incidence in 2002-2005 was accompanied by the increase of HIV-1 transmission through heterosexual contacts and continuation of subtype A predominance between isolates. However increase in heterogeneity of viral population during this period, which manifested as increase of env and pol genes polymorphism, was detected.     

HCV subtype characterization among injection drug users: implication for a crucial role of Zhenjiang in HCV transmission in China

Background

HCV transmission is closely associated with drug-trafficking routes in China. However, the transmission route of HCV in Eastern China remains unclear. Here, we investigate the role of Zhenjiang city of Jiangsu province, an important transportation hub linking Shanghai with other regions of China, in HCV transmission.

Methodology/Principal Findings

A total of 141 whole blood samples were collected from injection drug users (IDUs) in Zhenjiang and then tested for HCV infection. Of them, 115 HCV positive plasmas were subjected to RNA extraction, RT-PCR amplification, and sequencing. The subtype characterization and the evolutionary origin of HCV strains circulating in Zhenjiang were determined using polygenetic or phylogeographic analyses. Seven HCV subtypes 1b, 2a, 3a, 3b, 6a, 6e and 6n were detected among Zhenjiang IDUs, showing a complex HCV epidemic. The most predominant subtypes were 3a (38%) and 1b (26.8%). Among these subtypes, subtypes 3b, 6n and 6e originated from Southwestern China (i.e., Yunnan and/or Guangxi), subtypes 2a and 6a from Southern China (i.e., Guangdong), subtype 1b from Central (i.e., Henan) and Northwestern (i.e., Xinjiang) China, and subtype 3a from Southwestern (i.e., Yunnan) and Northwestern (i.e., Xinjiang) China. From Zhenjiang, subtypes 1b and 2a were further spread to Eastern (i.e., Shanghai) and Northern (i.e., Beijing) China, respectively.

Conclusions/Significance

The mixing of seven HCV subtypes in Zhenjiang from all quarters of China indicates that as an important middle station, Zhenjiang plays a crucial role in HCV transmission, just as it is important in population migration between other regions of China and Eastern China.     

The Current Hepatitis C Virus Prevalence in China May Have Resulted Mainly from an Officially Encouraged Plasma Campaign in the 1990s: a Coalescence Inference with Genetic Sequences

In this study, we investigated hepatitis C virus (HCV) molecular epidemiology and evolutionary dynamics. Both E1 and NS5B sequences were characterized in 379 of 433 patients in southern China and classified into five major subtypes: 1b in 256 patients, 6a in 67 patients, 2a in 29 patients, 3a in 14 patients, and 3b in 13 patients. Using the E1 sequences obtained, along with those from other studies using samples from China, we inferred the HCV epidemic history by means of coalescence strategies. Five Bayesian skyline plots (BSPs) were estimated for the five subtypes. They concurrently highlighted the rapid growth in the HCV-infected population size from 1993 to 2000, followed by an abrupt slowing. Although flanked on both sides by variable population sizes, the plots showed distinct patterns of rapid HCV growth. Coincidently, 1993 to 2000 was a period when contaminated blood transfusions were common in China due to a procedural error in an officially encouraged plasma campaign. The abrupt slowing in 1998 to 2000 corresponded to the central government outlawing paid blood donations in 1998. Using a parametric model, the HCV population growth rates were estimated during 1993 to 2000. It was revealed that the 6a rate was the highest, followed by those of 1b, 2a, 3b, and 3a. Because these rates differed significantly (P < 1e−9) from each other, they may help explain why 6a is increasingly prevalent in southern China and 1b is predominant nationwide. These rates are approximately 10-fold higher than those reported elsewhere. These findings suggested that during the plasma campaign, certain barriers to efficient viral transmission were removed, allowing wide HCV dissemination.     

Y Fuse? Sex Chromosome Fusions in Fishes and Reptiles

Chromosomal fusion plays a recurring role in the evolution of adaptations and reproductive isolation among species, yet little is known of the evolutionary drivers of chromosomal fusions. Because sex chromosomes (X and Y in male heterogametic systems, Z and W in female heterogametic systems) differ in their selective, mutational, and demographic environments, those differences provide a unique opportunity to dissect the evolutionary forces that drive chromosomal fusions. We estimate the rate at which fusions between sex chromosomes and autosomes become established across the phylogenies of both fishes and squamate reptiles. Both the incidence among extant species and the establishment rate of Y-autosome fusions is much higher than for X-autosome, Z-autosome, or W-autosome fusions. Using population genetic models, we show that this pattern cannot be reconciled with many standard explanations for the spread of fusions. In particular, direct selection acting on fusions or sexually antagonistic selection cannot, on their own, account for the predominance of Y-autosome fusions. The most plausible explanation for the observed data seems to be (a) that fusions are slightly deleterious, and (b) that the mutation rate is male-biased or the reproductive sex ratio is female-biased. We identify other combinations of evolutionary forces that might in principle account for the data although they appear less likely. Our results shed light on the processes that drive structural changes throughout the genome.     

北京地区成人呼吸道感染病人中人偏肺病毒感染情况的初步分析

目的:了解北京地区成人呼吸道感染患者中人偏肺病毒(hMPV)的感染情况、流行分布和临床表现特点。方法:采集2010年5月至2011年4月北京地区成人呼吸道感染患者鼻咽拭子标本413份,利用巢式PCR法进行hMPV筛查,对PCR阳性片段进行核酸序列测定,确定hMPV感染基因型别;同时,分析hMPV感染的流行病学特点,并对感染阳性患者进行临床表现的初步分析。结果:413份鼻咽拭子标本中检出hMPV阳性4份(0.97%),分别存在于2010年8月、10月和2011年2月、4月,其中1例合并鼻病毒感染;感染患者临床表现主要是发热、鼻塞、流涕、头痛、咳嗽,有1例出现呕吐和腹泻;hMPV阳性片段系统进化分析发现这4例感染中的3例为B2型,1例为A2b型。结论:北京地区成人呼吸道感染患者中存在hMPV感染,其基因型为B2和A2b型。     

Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of Listeria monocytogenes Strains Representing Clinically Important Serotypes

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone. Received: 18 June 1997 / Accepted: 4 December 1997     

A stochastic evolutionary model of molecular sequences.

A stochastic evolutionary model of molecular sequences is proposed. The basic forces in evolution are supposed to be mutation and selection. The concept is somewhat similar to Kauffman-Levin's concept of adaptive walks and corresponding analytical expressions have been developed. The selective force is divided into two parts: a slowly-varying part and a rapidly-changing fluctuation. The latter influences the distribution of sequences and results in an equation of motion along the flow line. The former plays a more important role in the emergence of evolutionary order. It is demonstrated that the asymmetry of selective forces would lead to a definite order of the system.     

T lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4⁺ and CD8⁺ T cells cooperate synergistically in hMPV eradication during primary infection, but CD4⁺ more than CD8⁺ T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4⁺ and CD8⁺ T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4⁺ T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8⁺ T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.     

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.     

The mid-depth method and HIV-1: a practical approach for testing hypotheses of viral epidemic history.

We introduce the mid-depth method, a practical approach for testing hypotheses of demographic history using genealogies reconstructed from sequence data. The relative positions of internal nodes within a genealogy contain information about past population dynamics. We explain how this information can be used to (1) test the null hypothesis of constant population size and (2) estimate the growth rate and current population size of an exponentially growing population. Simulation tests indicate that, as expected, estimates of exponential growth rates are sometimes biased. The mid-depth method is computationally rapid and does not require knowledge of the sample's mutation rate. However, it does assume that the reconstructed genealogy is correct and is therefore best suited to the analysis of variation-rich viral data sets. When applied to HIV-1 sequence data, the mid-depth method provides phylogenetic evidence of different exponential growth rates for subtypes A and B. We posit that this difference in growth rate reflects the different transmission routes and epidemiological histories of the two subtypes.     

Recombination confounds the early evolutionary history of human immunodeficiency virus type 1: subtype G is a circulating recombinant form

Human immunodeficiency virus type 1 (HIV-1) is classified in nine subtypes (A to D, F, G, H, J, and K), a number of subsubtypes, and several circulating recombinant forms (CRFs). Due to the high level of genetic diversity within HIV-1 and to its worldwide distribution, this classification system is widely used in fields as diverse as vaccine development, evolution, epidemiology, viral fitness, and drug resistance. Here, we demonstrate how the high recombination rates of HIV-1 may confound the study of its evolutionary history and classification. Our data show that subtype G, currently classified as a pure subtype, has in fact a recombinant history, having evolved following recombination between subtypes A and J and a putative subtype G parent. In addition, we find no evidence for recombination within one of the lineages currently classified as a CRF, CRF02_AG. Our analysis indicates that CRF02_AG was the parent of the recombinant subtype G, rather than the two having the opposite evolutionary relationship, as is currently proposed. Our results imply that the current classification of HIV-1 subtypes and CRFs is an artifact of sampling history, rather than reflecting the evolutionary history of the virus. We suggest a reanalysis of all pure subtypes and CRFs in order to better understand how high rates of recombination have influenced HIV-1 evolutionary history.