Molecular evolution and genomic characteristics of genotype H1 of measles virus.

Measles is one of the most infectious diseases of humans. It is caused by the measles virus (MeV) and can lead to serious illness, lifelong complications, and even death. Whole-genome sequencing (WGS) is now available to study molecular epidemiology and identify MeV transmission pathways. In the present study, WGS of 23 MeV strains of genotype H1, collected in Mainland China between 2006 and 2018, were generated and compared to 31 WGSs from the public domain to analyze genomic characteristics, evolutionary rates and date of emergence of H1 genotype. The noncoding region between M and F protein genes (M/F NCR) was the most variable region throughout the genome. Although the nucleotide substitution rate of H1 WGS was around 0.75 × 10-3 substitution per site per year, the M/F NCR had an evolutionary rate three times higher, with 2.44 × 10-3 substitution per site per year. Phylogenetic analysis identified three distinct genetic groups. The Time of the Most Recent Common Ancestor (TMRCA) of H1 genotype was estimated at approximately 1988, while the first genetic group appeared around 1995 followed by two other genetic groups in 1999-2002. Bayesian skyline plot showed that the genetic diversity of the H1 genotype remained stable even though the number of MeV cases decreased 50 times between 2014 (52 628) and 2020 (993). The current coronavirus disease 2019 (COVID-19) pandemic might have some effect on the measles epidemic and further studies will be necessary to assess the genetic diversity of the H1 genotype in a post-COVID area.

Comparison of the Whole-Genome Sequence of an Oka Varicella Vaccine from China with Other Oka Vaccine Strains Reveals Sites Putatively Critical for Vaccine Efficacy.

Varicella-zoster virus (VZV) infection results in varicella mostly in children. Reactivation of the virus causes herpes zoster (HZ), mostly in adults. A live attenuated vaccine (vOka-Biken) was originally derived from the parental strain pOka. Several live attenuated vaccines based on the Oka strain are currently available worldwide. In China, varicella vaccines have been licensed by four manufacturers. In this study, we analyze the whole-genome sequence (WGS) of vOka-BK produced by Changchun BCHT Biotechnology also known as Baike. vOka-BK WGS was compared against the genomic sequences of four other Oka strains: pOka, vOka-Biken, vOka-Varilrix from GlaxoSmithKline, and vOka-Varivax from Merck & Co. A previous study identified 137 single nucleotide polymorphisms (SNPs) shared by all vOkas. The current analysis used these data as a reference to compare with vOka-BK WGS and focused on 54 SNPs located in the unique regions of the genome. Twenty-eight nonsynonymous substitutions were identified, ORF62 and ORF55 featuring the most amino acid changes with 9 and 3, respectively. Among the 54 SNPs, 10 had a different mutation profile in vOka-BK compared to the other three vaccines. A comparison with the clade 3 strain Ellen, known to be attenuated, identified three shared amino acid changes: *130R in ORF0 and R958G and S628G in ORF62. This analysis provides the first comparison of a Chinese varicella vaccine to the other vaccines available worldwide and identifies sites potentially critical for VZV vaccine efficacy.IMPORTANCE Varicella, also known as chickenpox, is a highly contagious disease, caused by varicella-zoster virus (VZV). Varicella is a common childhood disease that can be prevented by a live attenuated vaccine. The first available vaccine was derived from the parental Oka strain in Japan in 1974. Several live attenuated vaccines based on the Oka strain are currently available worldwide. Among the four vaccines produced in China, the vaccine manufactured by Changchun BCHT Biotechnology, also known as Baike, has been reported to be very efficacious. Comparative genomic analysis of the Baike vaccine with other Oka vaccine strains identified sites that might be involved in vaccine efficacy, as well as important for the biology of the virus.

Revisiting the genotyping scheme for varicella-zoster viruses based on whole-genome comparisons.

We report whole-genome sequences (WGSs) for four varicella-zoster virus (VZV) samples from a shingles study conducted by Kaiser Permanente of Southern California. Comparative genomics and phylogenetic analysis of all published VZV WGSs revealed that strain KY037798 is in clade IX, which shall henceforth be designated clade 9. Previously published single nucleotide polymorphisms (SNP)-based genotyping schemes fail to discriminate between clades 6 and VIII and employ positions that are not clade-specific. We provide an updated list of clade-specific positions that supersedes the list determined at the 2008 VZV nomenclature meeting. Finally, we propose a new targeted genotyping scheme that will discriminate the circulating VZV clades with at least a twofold redundancy. Genotyping strategies using a limited set of targeted SNPs will continue to provide an efficient 'first pass' method for VZV strain surveillance as vaccination programmes for varicella and zoster influence the dynamics of VZV transmission.

Genomic analysis of the causative agents of coccidiosis in domestic chickens.

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

A distinct lineage of influenza A virus from bats.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

Herpes zoster caused by vaccine-strain varicella zoster virus in an immunocompetent recipient of zoster vaccine.

We report the first laboratory-documented case of herpes zoster caused by the attenuated varicella zoster virus (VZV) contained in Zostavax in a 68-year-old immunocompetent adult with strong evidence of prior wild-type VZV infection. The complete genome sequence of the isolate revealed that the strain carried 15 of 42 (36%) recognized varicella vaccine-associated single-nucleotide polymorphisms, including all 5 of the fixed vaccine markers present in nearly all of the strains in the vaccine. The case of herpes zoster was relatively mild and resolved without complications.

Evolutionary genetics of genotype H1 measles viruses in China from 1993 to 2012.

Virologic surveillance is a critical component of measles management. One of the criteria for verification of elimination of endemic measles is genetic analysis of wild-type viruses to demonstrate lack of an indigenous genotype. Measles is yet to be eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very active in China, providing a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a timespan of nearly two decades. Phylogenetic analysis performed on the 450 nt coding sequence for the C-terminal 150 amino acids of the nucleoprotein (N-450), fusion (F) gene and haemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence of selective pressure for the H protein was found. The substitution rates ranged from 0.75×10(-3) substitutions site(-1) year(-1) for H to 1.65×10(-3) substitutions site(-1) year(-1) for N-450. The time of most recent common ancestor (TMRCA) for genotype H1 was estimated as approximately 1985 (95 % highest probability density, 1979-1989). Finally, the overall diversity of measles sequences from China decreased from 2005 to 2012, coincident with a substantial decrease in measles cases. The results suggest that detailed evolutionary analyses should facilitate the documentation of eventual measles elimination in China. Moreover, the molecular approaches used in this study can be applied in other countries approaching measles elimination.

Phylogenetic analysis of rubella viruses identified in Uganda, 2003-2012.

Molecular data on rubella viruses are limited in Uganda despite the importance of congenital rubella syndrome (CRS). Routine rubella vaccination, while not administered currently in Uganda, is expected to begin by 2015. The World Health Organization recommends that countries without rubella vaccination programs assess the burden of rubella and CRS before starting a routine vaccination program. Uganda is already involved in integrated case-based surveillance, including laboratory testing to confirm measles and rubella, but molecular epidemiologic aspects of rubella circulation have so far not been documented in Uganda. Twenty throat swab or oral fluid samples collected from 12 districts during routine rash and fever surveillance between 2003 and 2012 were identified as rubella virus RNA positive and PCR products encompassing the region used for genotyping were sequenced. Phylogenetic analysis of the 20 sequences identified 19 genotype 1G viruses and 1 genotype 1E virus. Genotype-specific trees showed that the Uganda viruses belonged to specific clusters for both genotypes 1G and 1E and grouped with similar sequences from neighboring countries. Genotype 1G was predominant in Uganda. More epidemiological and molecular epidemiological data are required to determine if genotype 1E is also endemic in Uganda. The information obtained in this study will assist the immunization program in monitoring changes in circulating genotypes.

Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007-2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

Adaptation of a duck influenza A virus in quail.

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)--and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.

Multiple hosts and Influenza A viruses genetic mixing.

Influenza A viruses have a segmented, negative-stranded RNA genome. These viruses are classified according to the antigenic properties of the two glycoproteins, expressed on the surface of the virus particles, the hemagglutinin (HA or H) and the neuraminidase (NA or N). To date, 17 H and 10 N have been described and 116 HxNy combinations or subtypes reported. Except for the H17N10 subtype recently identified in bats, all identified subtypes have been identified in wild aquatic birds. These birds are considered to be the natural reservoir of influenza A viruses, from which some subtypes can be transmitted to other bird and mammal species, including humans. Interspecies transmissions seem to occur regularly, and can occasionally lead to the adaptation and stable establishment of a new viral lineage in a given species. This review recalls the genetic diversity of avian, swine and human influenza viruses and focuses on lesser-known influenza A viruses, identified in horses, dogs and very recently in bats. It discusses the genetic mixing that may result from interspecies transmission, and the associated risks of epizootics, zoonosis and pandemics.

Metagenomic analysis of a throat swab sample collected in China on a patient infected with Varicella Zoster Virus.

Varicella Zoster Virus (VZV) is endemic worldwide, causing varicella in children and zoster upon reactivation in adults. This study concerned a metagenomic analysis of a throat swab sample collected in China, on a young patient suffering from Systemic Lupus Erythematosus (SLE) and diagnosed with varicella. The complete genome sequence of a VZV strain of clade 2 has been generated. Clade 2 strains are the most prevalent in Asian countries. A comparison of 223 VZV genomes identified 77 clade specific markers, 20 of them specific to clade 2. The metagenomic analysis also identified sequences covering most of the genome of the bacteria Schaalia odontolytica also known as Actinomyces odontolyticus. VZV infection and bacterial infection in the context of SLE is further discussed. Even though the patient presented only mild symptoms, this study is a reminder that vaccination against VZV is critical to avoid severe complications like bacterial superinfection or even death in the case of immunodeficiency.

Detection of multiple viruses potentially infecting humans in sewage water from Xinjiang Uygur Autonomous Region, China.

The progress of sequencing technologies has facilitated metagenomics projects on environmental samples like sewage water. The present study concerned the analysis of sewage samples collected from 3 locations in Xinjiang Uygur Autonomous Region in China. The analysis focused on RNA viruses known to infect humans and identified viruses from 10 families. The proportion of human virus species in the sewage samples was relatively stable with an average of 17%. Thirty virus species known to infect humans were identified and they belonged to 6 families: Picornaviridae (12), Astroviridae (11), Reoviridae (3), Caliciviridae (2), Papillomaviridae (1) and Picobirnaviridae (1). A total of 16 full-length genomes were generated from Astroviridae, Picornaviridae (Salivirus and Kobuvirus) and Picobirnaviridae. Astroviruses appeared to be the most present viruses and were detected in all sewage samples. Analyzing the virome of sewage samples should help to monitor any potential risks to public health.

Synonymous and nonsynonymous polymorphisms versus divergences in bacterial genomes.

Comparison of the ratio of nonsynonymous to synonymous polymorphisms within species with the ratio of nonsynonymous to synonymous substitutions between species has been widely used as a supposed indicator of positive Darwinian selection, with the ratio of these 2 ratios being designated as a neutrality index (NI). Comparison of genome-wide polymorphism within 12 species of bacteria with divergence from an outgroup species showed substantial differences in NI among taxa. A low level of nonsynonymous polymorphism at a locus was the best predictor of NI < 1, rather than a high level of nonsynonymous substitution between species. Moreover, genes with NI < 1 showed a strong tendency toward the occurrence of rare nonsynonymous polymorphisms, as expected under the action of ongoing purifying selection. Thus, our results are more consistent with the hypothesis that a high relative rate of between-species nonsynonymous substitution reflects mainly the action of purifying selection within species to eliminate slightly deleterious mutations rather than positive selection between species. This conclusion is consistent with previous results highlighting an important role of slightly deleterious variants in bacterial evolution and suggests caution in the use of the McDonald-Kreitman test and related statistics as tests of positive selection.

Recombination analysis of Human mastadenovirus C whole genomes.

This study aims at analyzing all publicly available HAdV-C whole genome sequences (WGSs) and describes the genetic relationships between these genomes as well as identifies potential hotspots for recombination throughout the viral genome. In addition to the 4 prototypical genomic sequences, this analysis identified 20 HAdV-C WGSs which should be relevant for future recombination analysis of HAdV-C. This report confirmed the recombinogenic property of HAdV-C genomes and identified two main regions for breakpoints, within the hexon gene and around the fiber genomic region. No obvious recombination was detected between HAdV-Cs and non-human mastadenoviruses or non-C HAdVs. Finally, it highlighted the need for a surveillance of HAdVs in order to detect novel recombinant types that might represent health risks and develop possible prevention measures. Genetic analyses of recombination between recently collected HAdV-Cs and the assessment of their potential virulence are necessary steps towards the establishment of a surveillance of HAdVs in the future.

Multiple divergent Human mastadenovirus C co-circulating in mainland of China.

The human mastadenovirus C (HAdV-C) cause respiratory infections in children. Homologous recombination was clearly involved in the molecular evolution of HAdV-A, B, and D, but little is known about the molecular evolution of HAdV-C. From 2000 to 2016, 201 HAdV-C strains were collected from nine provinces covering six administrative regions of mainland of China via 3 existing surveillance programs, namely the febrile respiratory syndrome surveillance, the acute flaccid paralysis surveillance, and the hand, foot, and mouth disease surveillance system. The genes coding for the capsid protein (penton base, hexon, and fiber) of 201 HAdV-C strains were sequenced and compared with representative sequences publicly available. In addition, the whole genome sequence of 24 representative strains of HAdV-C was generated for further recombination analysis. Phylogenetic analysis of the penton base sequences of HAdV-C revealed six genetic groups (labelled as Px1-6), which showed that the penton base had more variation than previously thought. Based on the penton base, hexon, and fiber gene sequences, 16 new genetic patterns of HAdV-C circulating in mainland of China were identified in this study. Whole genome sequence analysis revealed frequent recombination events among HAdV-C genomes. This study is highly beneficial for case classification, tracking the transmission chain, and further epidemiological exploration of HAdV-C-related severe clinical diseases in the near future. Our data demonstrated that multiple newly divergent HAdV-C co-circulated across mainland China during the research period.

Phylogeny and recombination history of gallid herpesvirus 2 (Marek's disease virus) genomes.

Phylogenetic analyses based on concatenated amino acid sequences from orthologous loci from eight genomes of alpha herpesviruses infecting birds provided strong support for the following hypotheses: (1) gallid HV3 is a sister taxon to gallid HV2 but gallid HV1 is not closely related to the other two chicken herpesviruses; (2) meleagrid HV1 is closer to both gallid HV2 and gallid HV3 than is gallid HV1; (3) within gallid HV2, the virulent GA genome forms an outgroup to both the avirulent CVI988 genome and the highly virulent Md5 and Md11 genomes. Analysis of the pattern of synonymous nucleotide substitution between orthologous genes shared by four complete genomes of gallid HV2 showed strong evidence of past events of homologous recombination that homogenized certain loci between genomes. Eight of these loci represented cases of loci homogenized between the CVI988, on the one hand, and the Md5 and Md11 genomes, on the other hand. Two others represented loci where the GA genome was homogenized with those of Md5 and Md11. The two loci (UL49.5 and RLORF12) that were homogenized among the virulent genomes GA, Md5, and Md11 are candidates for contributing to viral virulence.

Whole genomic analysis of two potential recombinant strains within Human mastadenovirus species C previously found in Beijing, China.

Human mastadenovirus species C (HAdV-C) are the most common etiologic agents of respiratory disease in young children and are frequently detected worldwide including China. Two recombinant HAdV-C strains (BJ04 and BJ09) were isolated from infants with acute respiratory infection (ARI) in Beijing in 2012-2013. The whole genome sequences (WGS) of BJ04 and BJ09 were generated and compared to other 35 HAdV-C WGSs publicly available. Phylogenetic analyses showed that the BJ04 strain might be the result of three homologous recombination events involving the parental strains JX173086 (HAdV-1), NC_001405 (HAdV-2) and LC068718 (HAdV-6), whereas BJ09 viral genome might be made of genetic elements from JX173083 (HAdV-1), KF268199 (HAdV-5), and KR699642 (strain CBJ113). Despite intratypic recombination, amino acid analysis showed that the gene repertoire of BJ04 and BJ09 were similar to type 2 viruses. Finally, this analysis revealed that at least three lineages of HAdV-C have been identified in China, represented by BJ04 related to NC_001405, BJ09 related to CBJ113, and KF951595 (strain DD28) related to virus isolated in Japan. This study showed that the frequent recombination played an important driving force for complexity of the HAdV-C epidemic in Beijing, thereby demonstrating the necessity for epidemiological and virological surveillance for HAdV-C in China.

Evolutionary analysis of mumps viruses of genotype F collected in mainland China in 2001-2015.

Mumps incidence in mainland China remains at a high level. Genotype F has been the predominant genotype of mumps virus (MuV) in the last 20 years in mainland China. To better understand the genetic characteristics of MuV in China, the sequences of the Small Hydrophobic (SH), Hemagglutinin-Neuraminidase (HN) and Fusion (F) genes of MuVs of genotype F collected during 2001-2015 were determined. The evolutionary rates of the HN and F genes were similar (0.5 × 10-3 substitutions/site/year) whereas the SH gene evolutionary rate was three times faster. The most recent common ancestor of genotype F was traced back to 1980. Four lineages were identified within HN and F MuV sequences. A phylogeographic analysis indicated that the genotype F viruses originally spread from the Liaoning and Shandong provinces followed by a spread to the South and East of China. This study provides important genetic baseline data for the development of prevention and control measures of mumps.

Evolutionary analysis of rubella viruses in mainland China during 2010-2012: endemic circulation of genotype 1E and introductions of genotype 2B.

Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010-2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010-2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China.