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 x 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.

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.

Recent studies have shown that the currently circulating rubella viruses are mostly members of two genotypes, 1E and 2B. Also, genetically distinct viruses of genotype 1G have been found in East and West Africa. This study used a Mantel test to objectively include both genetic diversity and geographic location in the definition of lineages, and identified statistically justified lineages (n=13) and sub-lineages (n=9) of viruses within genotypes 1G, 1E and 2B. Genotype 2B viruses were widely distributed, while viruses of genotype 1E as well as 1G and 1J were much more geographically restricted. This analysis showed that more precise groupings for rubella viruses are possible, which should improve the ability to track rubella viruses worldwide. A year by year analysis revealed gaps in surveillance that need to be resolved in order to support the surveillance needed for enhanced control and elimination goals for rubella.

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.

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.

Virologic surveillance is a critical component of measles surveillance because 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 has not yet been eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very strong in China and this provided a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a time span of nearly 20 years. Phylogenetic analysis performed on the 450 nucleotides coding for the COOH terminal 150 amino acids of the nucleoprotein (N-450), the fusion (F) gene and the hemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence was found for selective pressure on the H protein. The substitution rates ranged from 0.75 x 10-3 substitutions/site/year for H to 1.65 x10-3 substitutions/site/year for N-450. An estimate of the time of the most recent common ancestor for genotype H1 was approximately 1985 (95% Highest Probability Density = 1979-1989). Finally, the overall diversity of the measles sequences from China decreased from 2005 to 2012 which is coincident with a substantial decrease in measles cases. Overall, the results suggest that detailed evolutionary analyses will help to document the eventual elimination of measles in China, and the molecular approaches used in this study can be applied in other countries that are approaching measles elimination.

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3' untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5' UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007-2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.

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.

The evolutionary classification of influenza genes into lineages is a first step in understanding their molecular epidemiology and can inform the subsequent implementation of control measures. We introduce a novel approach called Lineage Assignment By Extended Learning (LABEL) to rapidly determine cladistic information for any number of genes without the need for time-consuming sequence alignment, phylogenetic tree construction, or manual annotation. Instead, LABEL relies on hidden Markov model profiles and support vector machine training to hierarchically classify gene sequences by their similarity to pre-defined lineages. We assessed LABEL by analyzing the annotated hemagglutinin genes of highly pathogenic (H5N1) and low pathogenicity (H9N2) avian influenza A viruses. Using the WHO/FAO/OIE H5N1 evolution working group nomenclature, the LABEL pipeline quickly and accurately identified the H5 lineages of uncharacterized sequences. Moreover, we developed an updated clade nomenclature for the H9 hemagglutinin gene and show a similarly fast and reliable phylogenetic assessment with LABEL. While this study was focused on hemagglutinin sequences, LABEL could be applied to the analysis of any gene and shows great potential to guide molecular epidemiology activities, accelerate database annotation, and provide a data sorting tool for other large-scale bioinformatic studies.

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.

We report on the genetic analysis of 213 highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry in Vietnam between 2007 and 2010. Phylogenetic analyses of the viral genomes revealed 38 distinct viral genotypes, 29 were novel and 9 were reported in Vietnam or neighboring countries in recent years. Viruses from only six genotypes persisted beyond one season or year. Thus, most reassortant viruses were transient, suggesting that such genotypes lacked significant fitness advantages. Viruses with clade 2.3.2.1 HA were re-introduced into Vietnam in 2009 and their prevalence rose steeply towards the end of 2010. Clade 2.3.4-like viruses (genotype V) were predominant in northern Vietnam and caused the majority of zoonotic infections, whereas clade 1.1 (genotype Z) viruses were only detected in the Mekong delta region, in southern Vietnam. Antigenic analysis of representative viruses from the four clades indicated substantial drift.

BACKGROUND: Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking. METHODOLOGY/PRINCIPAL FINDINGS: Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The d(N)/d(S) ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding. CONCLUSIONS/SIGNIFICANCE: The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance.

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-alpha2-3-galactose [Siaalpha2-3Gal] linkages on sialyloligosaccharides)--and human (Siaalpha2-6Gal)-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.

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.

The evolution of classical swine influenza viruses receptor specificity preceding the emergence of the 2009 H1N1 pandemic virus was analyzed in glycan microarrays. Classical swine influenza viruses from the alpha, beta, and gamma antigenic clusters isolated between 1945 and 2009 revealed a binding profile very similar to that of 2009 pandemic H1N1 viruses, with selectivity for alpha2-6-linked sialosides and very limited binding to alpha2-3 sialosides. Despite considerable genetic divergence, the 'human-like' H1N1 viruses circulating in swine retained strong binding preference for alpha2-6 sialylated glycans. Interspecies transmission of H1N1 influenza viruses from swine to humans or from humans to swine has not driven selection of viruses with distinct novel receptor binding specificities. Classical swine and human seasonal H1N1 influenza viruses have conserved specificity for similar alpha2-6-sialoside receptors in spite of long term circulation in separate hosts, suggesting that humans and swine impose analogous selection pressures on the evolution of receptor binding function.

Influenza virus, subtype H3N8, was transmitted from horses to greyhound dogs in 2004 and subsequently spread to pet dog populations. The co-circulation of H3N8 viruses in dogs and horses makes bi-directional virus transmission between these animal species possible. To understand the dynamics of viral transmission, we performed virologic surveillance in dogs and horses between 2005 and 2008 in the United States. The genomes of influenza A H3N8 viruses isolated from 36 dogs and horses were sequenced to determine their origin and evolution. Phylogenetic analyses revealed that H3N8 influenza viruses from horses and dogs were monophyletic and distinct. There was no evidence of canine influenza virus infection in horses with respiratory disease or new introductions of equine influenza viruses into dogs in the United States. Analysis of a limited number of equine influenza viruses suggested substantial separation in the transmission of viruses causing clinically apparent influenza in dogs and horses.

A type A avian influenza (AI) virus was isolated from dead or severely ill red-winged tinamous (Rhynchotus rufescens) found in a hunting ground in April 2008 in Argentina. The subtype of A/red-winged tinamou/Argentina/MP1/2008 was determined as H1N1 by sequence analysis. The cleavage site of the viral hemagglutinin corresponded to a low pathogenic influenza virus, although the clinical presentation and pathological studies suggest that the virus was pathogenic for red-winged tinamous. Phylogenetic analysis of the viral genome suggested that while the hemagglutinin and neuraminidase genes were related to AIV from North America, the internal genes were most closely related to other South American isolates. These findings support the postulated South American phylogenetic lineage for AIV PB2, PB1, PA, M and NS genes, and suggest that the evolutionary pathways of HA and NA genes involve exchanges between the Northern and Southern hemispheres.

Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).