Sequencing and phylogenetic analysis of the coding region of six common rotavirus strains: evidence for intragenogroup reassortment among co-circulating G1P[8] and G2P[4] strains from the United States.

The segmented genome of rotaviruses provides an opportunity for rotavirus strains to generate a large genetic diversity through reassortment; however, this mechanism is considered to play little role in the generation of mosaic gene constellations between Wa-like and DS-1-like strains in genes other than the neutralization antigens. A pilot study was undertaken to analyze these two epidemiologically important strains at the genomic level in order to (i) identify intergenogroup reassortment and (ii) to make available additional reference genome sequences of G1P[8] and G2P[4] for future genomics analyses. The full or nearly complete coding region of all 11 genes for 3 G1P[8] (LB2719, LB2758, and LB2771) and 3 G2P[4] (LB2744, LB2764, and LB2772) strains isolated from children hospitalized with severe diarrhea in Long Beach, California, where these strains were circulating at comparable rates during 2005-2006 are described in this study. Based on the full-genome classification system, all G1P[8] strains had a conserved genomic constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1 and were mostly identical to the few Wa-like strains whose genome sequences have already been determined. Similarly, the genome sequences of the 3 G2P[4] strains were highly conserved: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-E2-H2 and displayed an overall lesser genetic divergence with reference DS-1-like strains. While intergenogroup reassortment was not seen between the G1P[8] and G2P[4] strains studied here, evidence for intragenogroup reassortment events was identified. Similar studies in the post-rotavirus genomic era will help uncover whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children.

Knockout reactions from p-shell nuclei: tests of ab initio structure models.

Absolute cross sections have been determined following single neutron knockout reactions from 10Be and 10C at intermediate energy. Nucleon density distributions and bound-state wave function overlaps obtained from both variational Monte Carlo (VMC) and no core shell model (NCSM) ab initio calculations have been incorporated into the theoretical description of knockout reactions. Comparison to experimental cross sections demonstrates that the VMC approach, with the inclusion of 3-body forces, provides the best overall agreement while the NCSM and conventional shell-model calculations both overpredict the cross sections by 20% to 30% for 10Be and by 40% to 50% for 10C, respectively. This study gains new insight into the importance of 3-body forces and continuum effects in light nuclei and provides a sensitive technique to assess the accuracy of ab initio calculations for describing these effects.

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples.

AIM: To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples. METHODS AND RESULTS: Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples. CONCLUSION: Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.

Diversity of Rotavirus Strains Circulating in Botswana before and after introduction of the Monovalent Rotavirus Vaccine.

BACKGROUND: Globally, rotavirus is the leading cause of acute gastroenteritis (AGE) in children aged <5 years. Botswana introduced the monovalent rotavirus vaccine (Rotarix) in July 2012. To study the impact of this vaccine on rotavirus genotypes circulating in Botswana, a comparison of the genotypes pre-vaccination (2011-2012) and post-vaccination (2013-2018) periods was conducted. SUBJECTS AND METHODS: Residual samples from 284 children <5 years of age that tested positive for rotavirus by enzyme immunoassay were genotyped. One hundred and five samples were from the pre-vaccination period and 179 were from the post-vaccination period. Genotyping was performed using two multiplexed one-step reverse transcription polymerase chain reaction (RT-PCR) assays for the amplification and genotyping of rotavirus VP7 (G) and VP4 (P) genes. RESULTS: Prior to vaccine introduction, the predominant rotavirus circulating genotypes were G9P[8] (n = 63, 60%) and G1P[8] (n = 22, 21%). During the vaccine period, G2P[4] was the predominant genotype (n = 49, 28%), followed by G9P[8] (n = 40, 22%) and G1P[8] (n = 33, 18.5%). There was a significant decline in the prevalence of G9P[8] (p = 0.001) in the post-vaccination period. There was also a notable decline in G1P[8]. A spike in G2P[4] was observed in 2013, one year post-vaccine introduction. Rotavirus strain G3P[4] (n = 8) was only detected in the post-vaccine introduction period. In 2018 there was a marked increase in genotype G3P[8] (p = 0.0003). CONCLUSIONS: The distribution of circulating rotavirus genotypes in Botswana changed after vaccine implementation. Further studies are needed to examine whether these changes are related to vaccination or simply represent natural secular variation.

Genetic characterization of a human isolate of Puumala hantavirus from France.

PUU90-13 is a strain of Puumala (PUU) virus (family Bunyaviridae: genus Hantavirus) isolated from a human in northeastern France (Rollin et al., 1995). This report describes the full-length sequences of the small (S) and medium (M) genomic RNAs of PUU90-13. The terminal sequences of both the S and M genomic RNAs were found to be conserved and imperfectly complementary. The S RNA of PUU90-13 is 1847 nt in length and contains the nucleocapsid (N) protein gene and a potential overlapping open reading frame (ORF-2) previously described in other hantaviruses. Statistical analysis of the third base substitution frequency in the N ORFs of PUU90-13 and other PUU viruses suggests that the ORF-2 is functional. The M RNA is 3681 nt in length and encodes the glycoprotein precursor. Both genomic segments share the highest degree of nucleotide and amino acid sequence identity with PUUBerkel, a PUU virus from Germany. Phylogenetic analyses of sequences from both segments indicate that PUU90-13 occupies a distinct Western European PUU virus lineage that it shares with PUUBerkel. Both PUU90-13 and PUUBerkel lack a potential N-linked glycosylation site found on the G2 glycoprotein of other PUU viruses.

Vector competence of California mosquitoes for California encephalitis and California encephalitis-like viruses.

Mosquitoes collected from coastal, inland valley, and alpine locations in California were evaluated for their experimental vector competence for two viruses in the California serogroup (Bunyaviridae:Bunyavirus). Aedes squamiger, a coastal salt marsh mosquito, was an efficient vector of a California encephalitis (CE)-like virus isolated from its habitat (89% of the pledget-fed females became infected and 61% transmitted virus). Aedes dorsalis, a coastal mosquito, and Ae. melanimon, an inland valley mosquito, were competent vectors of prototype CE virus (98% and 100% of the pledget-fed females became infected and 56% and 30%, respectively, transmitted virus). Aedes squamiger and Ae. dorsalis transmitted both viruses vertically to one or more of 20 of their progeny. Culiseta inornata was susceptible to infection with both viruses, but 5% or less transmitted virus perorally. Alpine mosquitoes, Ae. cataphylla, Ae. increpitus, and Ae. tahoensis, became infected with both CE and CE-like viruses, but 3% or less transmitted virus. All species of mosquitoes were more efficient vectors of both viruses following intrathoracic inoculation than following pledget feeding, suggesting the presence of mesenteronal barriers.

Short report: isolation and partial characterization of a Puumala virus from a human case of nephropathia epidemica in France.

Puumala (PUU) virus (Bunyaviridae: Hantavirus), the etiologic agent of nephropathia epidemica (NE), the mid form of hemorrhagic fever with renal syndrome, is enzootic in Europe and has been known to occur in France since 1983. We report the first isolation of PUU virus in France and western Europe from a case of NE acquired in France. The virus was isolated from a serum collected in the acute phase of the clinical course by successive blind passages in Vero E6 cells. Serologic typing using monoclonal antibodies confirmed the identity of the virus as PUU. The sequence of an 832-nucleotide fragment of the virus medium RNA segment obtained by the polymerase chain reaction (PCR) also classified it as a PUU virus. The sequence of this isolate from a human case in France is closely related to the sequence of a PUU virus obtained by the PCR from a German patient.

Geographic distribution and serologic and genomic characterization of Morro Bay virus, a newly recognized bunyavirus.

More than 75,000 immature mosquitoes in three genera were collected from coastal California, reared to the adult stage, and tested for virus by plaque assay in Vero cell cultures. Twenty-six strains of Morro Bay (MB) virus, a newly recognized member of the California (CAL) serogroup, were isolated from Aedes squamiger, a pestiferous salt marsh mosquito species restricted to intertidal salt marshes in coastal California and Baja California. The geographic distribution of the isolates was 10 from San Luis Obispo County, one each from Santa Barbara and Orange Counties, and 14 from San Diego County. No virus isolations were made from 23,157 Ae. squamiger collected north of San Luis Obispo County (midpoint in the geographic range of this species in California). Thus, MB virus infection in Ae. squamiger appears to be restricted to the southern range of this species in California. Serum dilution neutralization tests indicated that MB virus represents a novel subtype of the California encephalitis (CE) serotype within the CAL serogroup. Comparative analyses of genomic sequence data from four geographically distinct MB virus isolates indicated that the isolates are genetically similar to each other and distinct from other CE serotype bunyaviruses. Phylogenetic analysis of nucleocapsid protein gene sequence data indicated that MB virus represents a distinct lineage within the CE serotype and thus supports the serologic classification of MB virus as a distinct CAL serogroup virus.

Natural rodent host associations of Guanarito and pirital viruses (Family Arenaviridae) in central Venezuela.

The objective of this study was to elucidate the natural rodent host relationships of Guanarito and Pirital viruses (family Arenaviridae) in the plains of central Venezuela. Ninety-two arenavirus isolates from 607 animals, representing 10 different rodent species, were characterized to the level of serotype. The 92 isolates comprised 19 Guanarito virus strains and 73 Pirital virus strains. The 19 Guanarito virus isolates were from Zygodontomys brevicauda; 72 (98.6%) of the 73 Pirital virus isolates were from Sigmodon alstoni. These results indicate that the natural rodent associations of these 2 sympatric arenaviruses are highly specific and that Z brevicauda and S. alstoni are the principal rodent hosts of Guanarito and Pirital viruses, respectively.

Isolation and characterization of pirital virus, a newly discovered South American arenavirus.

Specific rodent species are principal hosts for each of the well-characterized members of the virus family Arenaviridae. Guanarito virus (Arenaviridae) is the etiologic agent of Venezuelan hemorrhagic fever. A previous study on the epidemiology of Venezuelan hemorrhagic fever revealed extensive arenavirus infection (presumed to be caused by Guanarito virus) in two rodent species. Sigmodon alstoni and Zygodontomys brevicauda, collected from the region of Venezuela in which the disease is endemic. In the present study, four arenavirus isolates recovered from the Municipality of Guanarito (two isolates each from S. alstoni and Z. brevicauda) were characterized to learn more about the natural rodent host relationships of Guanarito virus. Serologic tests and analyses of nucleocapsid protein gene sequence data indicated that the two isolates from Z. brevicauda are strains of Guanarito virus and that the two isolates from S. alstoni are representatives of a novel New World arenavirus (proposed name Pirital) that is antigenically and phylogenetically distinct from all known New World arenaviruses. The results of the present study provide further evidence that the cane mouse Z. brevicauda is a natural host of Guanarito virus and suggest that the cotton rat S. alstoni is the natural reservoir host of Pirital but not Guanarito virus.

Characterization of Oliveros virus, a new member of the Tacaribe complex (Arenaviridae: Arenavirus).

Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years.

Vector competence of selected mosquito species (Diptera: Culicidae) for California strains of Northway virus (Bunyaviridae: Bunyavirus).

Selected mosquito species from Central Valley, coastal, and alpine habitats of California were evaluated for their vector competence for Northway (NOR) virus. Culiseta incidens Thomson, Culiseta inornata (Williston), and Anopheles freeborni Aitken were the only competent vectors when fed virus. Aedes sierrensis (Ludlow), as well as alpine snow pool Aedes (i.e., Ae. cataphylla Dyar, Ae. hexodontus Dyar, Ae. increpitus Dyar and Ae. tahoensis Dyar), Ae. melanimon Dyar, Ae. washinoi Lanzaro & Eldridge, Culex erythrothorax Dyar, and Cx. tarsalis Coquillett were highly refractory to peroral infection. Alpine snow pool species were poor vectors even following parenteral infection, but 21-35% of intrathoracically inoculated Ae. sierrensis from several lower elevation localities and 39-46% of parenterally infected Cx. tarsalis were able to transmit NOR virus per os. Perorally infected Cs. inornata transmitted NOR virus vertically to their F1 adult progeny at a low rate (minimal filial infection rate, 1:248).

Vector competence of alpine, Central Valley, and coastal mosquitoes (Diptera: Culicidae) from California for Jamestown Canyon virus.

Mosquitoes collected from alpine, Central Valley, and coastal habitats in California were evaluated for their vector competence for four strains of Jamestown Canyon (JC) virus. Three of the viral strains examined were isolated from alpine Aedes species collected in California, and one, the prototype JC virus, was isolated from Culiseta inornata (Williston) collected in Colorado. Alpine Aedes tahoensis Dyar, Ae. cataphylla Dyar, Ae. hexodontus Dyar, Ae. increpitus Dyar, Ae. clivis Lanzaro and Eldridge, and coastal Aedes washinoi Lanzaro and Eldridge were variably susceptible to alpine strains of JC virus. Infection rates ranged from 22 to 77%, and peroral transmission rates of the infected females ranged from 0 to 26%. The differences were related to both mosquito species and viral strain. Coastal populations of Cs. inornata, Ae. washinoi, and Ae. sierrensis (Ludlow) were incompetent vectors when fed an alpine strain of JC virus, whereas Ae. squamiger (Coquillet) and Ae. dorsalis (Meigen) were competent vectors. Peroral transmission rates following parenteral infection of females of most species were about twofold higher than those for perorally infected females. A population of Cs. inornata from the Central Valley was highly susceptible when fed an alpine strain of JCV and transmitted virus both horizontally and vertically. Alpine strains of JC virus also were transmitted vertically by Ae. tahoensis, Ae. washinoi, and Ae. squamiger following parenteral infection of females.

Human G9P[8] rotavirus strains circulating in Cameroon, 1999-2000: Genetic relationships with other G9 strains and detection of a new G9 subtype.

Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and genotype G9P[8] is one of the five most common genotypes detected in humans. In order to gain insight into the degree of genetic variability of G9P[8] strains circulating in Cameroon, stool samples were collected during the 1999-2000 rotavirus season in two different geographic regions in Cameroon (Southwest and Western Regions). By RT-PCR, 15 G9P[8] strains (15/89=16.8%) were identified whose genomic configurations was subsequently determined by complete or partial gene sequencing. In general, all Cameroonian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6-100% nucleotide identity amongst themselves and 81.2-99.5% nucleotide identity with global G9 strains. The full genome classification of all Cameroonian strains was G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinct lineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P[8], which shared the genomic constellation of other Cameroonian G9P[8] strains, contained a novel G9 subtype which diverged significantly (18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide and amino acid alignments revealed that the 3' end of this gene is highly divergent from other G9 VP7 genes suggesting that it arose through extensive accumulation of point mutations. The results of this study demonstrate that diverse G9 strains circulated in Cameroon during 1999-2000.

Genome sequence based molecular epidemiology of unusual US Rotavirus A G9 strains isolated from Omaha, USA between 1997 and 2000.

After discovery in the early 1980s, Rotavirus A serotype G9 was detected infrequently for almost a decade. Since the mid-1990s, however, serotype G9 has emerged to become a globally common strain linked to the introduction of a single, new genetic variant of G9 VP7 gene. Studies have demonstrated that genetically divergent G9 strains co-circulated at low frequency with the emerging variants. Examples include unique U.S. G9 strains Om46/Hu/USA/1998 and Om67/Hu/USA/1998, isolated in Omaha during the 1997-1998 rotavirus season, that are more closely related phylogenetically to reference strains from the 1980s than to most emerging G9 strains from the U.S. and globally. Here, we sequenced the VP7 full open reading frame for all available G9 strains (n=12) identified in Omaha during 1996-2000 seasons to investigate their epidemiology and evolution. In addition, the full or partial length open reading frames of the remaining 10 genes for five divergent Om46-like strains and one modern G9 variant were sequenced to evaluate their potential origin. Our findings suggest that Om46-like G9 strains may have been introduced into humans recently, perhaps in 1997-1998 when it was first detected, and the presumed original host of this VP7 gene variant may have been an animal species based on the unexpected detection of porcine rotavirus related NSP2 gene in the genome. The relatively high fitness of Om46-like strains during the 1997-1998 rotavirus season, 1 year after the globally important G9 variant was documented to be already spreading in the study area and other sites of the United States, appears to parallel findings on seasonal replacement of various genetic and antigenic variants of other common human rotavirus antigen specificities.

Genomic characterization of human rotavirus G10 strains from the African Rotavirus Network: relationship to animal rotaviruses.

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. The aim of this study was to elucidate the genetic and evolutionary relationships of short fragments of all 11 gene segments of G10 HRV strains identified in West Africa through the African Rotavirus Network (ARN) system. During 1998-2004 surveillance within the ARN, we identified 5 G10 P[8] HRV strains. Fragments of all 11 gene segments of these G10 strains were sequenced. Phylogenetic and sequence analyses of each gene segment revealed high nucleotide similarities amongst the ARN strains (97-100%) except in the case of the VP1(85-96%) and NSP2 genes (87.8-99.7%) where some strains were divergent. All genes of the ARN strains were classified as Wa-like (genotype 1) with the exception of their VP7 gene of all strains (genotype G10) and the VP6 gene of a single strain, 6755/2002/ARN (DS-1 like, genotype 2). While classified as Wa-like, the NSP2 genes of four of the ARN strains occupied a distinct sub-lineage related to simian strain Tuch, while the NSP2 of strain 6755/2002/ARN and NSP5 genes of all strains were closely related to the cognate genes of both human and animal strains belonging to the Wa-like genogroup. Although these findings help to elucidate the evolution of ARN G10 strains, additional sequence studies of cognate animal rotavirus genes are needed to determine irrefutably the specific origin of those genes relative to both human and animal rotavirus strains.

Spectroscopy of 36Mg: interplay of normal and intruder configurations at the neutron-rich boundary of the "island of inversion".

We report on the first spectroscopy study of the very neutron-rich nucleus (36)(12)Mg24 using the direct two-proton knockout reaction 9Be(38Si,36Mg+gamma)X at 83 MeV/nucleon. The energy of the first excited 2+ state of 36Mg, E(2+(1)=660(6) keV, was measured. The magnitude of the partial cross sections to the ground state and the 2+(1) state is indicative of strong intruder admixtures in the lowest-lying states as suggested by Monte Carlo shell-model calculations.

Measurement of excited states in (40)Si and evidence for weakening of the N=28 shell gap.

Excited states in (40)Si have been established by detecting gamma rays coincident with inelastic scattering and nucleon removal reactions on a liquid hydrogen target. The low excitation energy, 986(5) keV, of the 2(1)(+) state provides evidence of a weakening in the N=28 shell closure in a neutron-rich nucleus devoid of deformation-driving proton collectivity.

Phylogenetic analysis of the Arenaviridae: patterns of virus evolution and evidence for cospeciation between arenaviruses and their rodent hosts.

Viruses of the Arenaviridae cause hemorrhagic fevers and neurologic disease in humans. Historically, the arenaviruses have been divided into two complexes (LASV-LCMV, Tacaribe) through the use of antigenic typing. The phylogeny of the Arenaviridae as a whole has not been estimated previously due to a lack of sequence data for all members of the family. In this study, nucleocapsid protein gene sequence data were obtained for all currently known arenaviruses and used to estimate, for the first time, a phylogeny of the entire virus family. The LCMV-LASV complex arenaviruses are monophyletic and comprise three distinct lineages. The Tacaribe complex viruses also are monophyletic and occupy three distinct lineages. Comparisons of arenavirus phylogeny with rodent host phylogeny and taxonomic relationships provide several examples in which virus-host cospeciation is potentially occurring. The pathogenic arenaviruses do not appear to be monophyletic, suggesting that the pathogenic phenotype has arisen in multiple independent events during virus evolution.

The phylogeny of New World (Tacaribe complex) arenaviruses.

Several New World (Tacaribe complex) arenaviruses (Arenaviridae) are known to cause severe hemorrhagic disease in humans. Phylogenetic reconstruction of the Tacaribe complex arenaviruses previously has been limited by the relative scarcity of sequence data for arenavirus genomes. In the present study, oligonucleotide primers were designed based on conserved regions of the nucleocapsid (N) protein gene and then used to amplify, by reverse transcription--polymerase chain reaction, a 613-to 649-nucleotide region of the N gene of all known Tacaribe complex arenaviruses. This has allowed completion of the first detailed genetic characterization and phylogenetic analysis of all known members of the Tacaribe complex. These viruses formed three lineages. Lineage A contained Flexal, Parana, Pichinde, and Tamiami viruses; lineage B contained Amapari, Guanarito (GUA), Junin (JUN), Machupo (MAC), Sabia (SAB), and Tacaribe viruses. Latino and Oliveros viruses occupied lineage C. The highly pathogenic Tacaribe complex arenaviruses (GUA, JUN, MAC, SAB) were all members of lineage B, suggesting the possibility that the highly pathogenic phenotype is the result of evolutionary radiation from a common ancestor. The approach described here provides a rapid method for characterization of novel Tacaribe complex arenaviruses and may provide clues as to their potential public health importance.