Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa

Two isolates of a virus of the genus Orthobunyavirus (family Bunyaviridae) were obtained from hemorrhagic fever cases during a large disease outbreak in East Africa in 1997 and 1998. Sequence analysis of regions of the three genomic RNA segments of the virus (provisionally referred to as Garissa virus) suggested that it was a genetic reassortant virus with S and L segments derived from Bunyamwera virus but an M segment from an unidentified virus of the genus Orthobunyavirus. While high genetic diversity (52%) was revealed by analysis of virus M segment nucleotide sequences obtained from 21 members of the genus Orthobunyavirus, the Garissa and Ngari virus M segments were almost identical. Surprisingly, the Ngari virus L and S segments showed high sequence identity with those of Bunyamwera virus, showing that Garissa virus is an isolate of Ngari virus, which in turn is a Bunyamwera virus reassortant. Ngari virus should be considered when investigating hemorrhagic fever outbreaks throughout sub-Saharan Africa.     

Iquitos virus: a novel reassortant Orthobunyavirus associated with human illness in Peru

Oropouche (ORO) virus, a member of the Simbu serogroup, is one of the few human pathogens in the Orthobunyavirus genus in the family Bunyaviridae. Genetic analyses of ORO-like strains from Iquitos, Peru, identified a novel reassortant containing the S and L segments of ORO virus and the M segment of a novel Simbu serogroup virus. This new pathogen, which we named Iquitos (IQT) virus, was first isolated during 1999 from a febrile patient in Iquitos, an Amazonian city in Peru. Subsequently, the virus was identified as the cause of outbreaks of "Oropouche fever" during 2005 and 2006 in Iquitos. In addition to the identification of 17 isolates of IQT virus between 1999 and 2006, surveys for neutralizing antibody among Iquitos residents revealed prevalence rates of 14.9% for ORO virus and 15.4% for IQT virus. Limited studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. Identification of a new Orthobunyavirus human pathogen in the Amazon region of Peru highlights the need for strengthening surveillance activities and laboratory capabilities, and investigating the emergence of new pathogens in tropical regions of South America.     

Species-specific variants of GB virus A in captive monkeys .

Sequences from the putative 5' nontranslated region of GB virus A were isolated from mystax, owl monkeys, and tamarins. Though sequences of isolates from each animal species are virtually identical at the nucleotide level (95%), isolates from different species are dramatically different (52 to 79% identical) and genetically cluster on this basis.     

Unusual Antigenic and Genetic Characteristics of Human Respiratory Syncytial Viruses Isolated in Cuba

The G protein of 23 strains of human respiratory syncytial virus isolated in Havana, Cuba, between October 1994 and January 1995 was analyzed at the antigenic and genetic level. All viruses reacted with 10 of 11 antibodies specific for the Long strain. Moreover, the G protein gene of the Cuban isolates had only five nucleotide differences from the sequence of the Long gene. The homogeneity of the Cuban isolates and their resemblance to an ancient strain, such as Long, are at odds with previous findings for viruses isolated in countries with a temperate climate and different socioeconomic status. The G proteins of three of four other viruses isolated in Havana 2 years later (1996) were also identical to those of the 1994-to-1995 isolates, and the fourth virus had a single extra nucleotide difference. This, again, is unusual, since no identical viruses had been isolated in different epidemics previously. The singular characteristics of the Cuban isolates reported here are discussed in terms of the epidemiological, climatic, and socioeconomic characteristics of Cuba.     

The cellular slime mold guild and its bacterial prey: growth rate variation at the inter- and intraspecific levels

Summary A guild of cellular slime molds (CSM) consisting of two isolates from each of five species, representing two genera, and obtained from the same square meter of forest soil exhibited extensive growth rate variation when tested on a suite of 18 bacteria isolated from the same soil. Significant growth rate differences were found at each taxonomic level examined: among species of different genera, between genera, among species within genera, and between isolates (=clones) within species. The type of bacteria used as prey determined the relative rank of the growth rates in different CSM isolates, as well as the taxonomic level at which significant differences were found. We suggest a possible reconciliation between a previous hypothesis, based on competition, and contradictory experimental work on resource partitioning in this guild of bacterial predators. Our results raise a question about the efficacy of using single genotypes to represent a species when ecological ideas are developed through laboratory investigations.     

Coat colour in dogs: identification of the Merle locus in the Australian shepherd breed

Background

A panel of 37 rabies virus isolates were collected and studied, originating mainly from the northern and central regions of Namibia, between 1980 and 2003.

Results

These virus isolates demonstrated a high degree of genetic similarity with respect to a 400 bp region of the nucleoprotein gene, with the virus isolates originating from kudu antelope (n = 10) sharing 97.2–100% similarity with jackal isolates, and 97–100% similarity with those isolated from domestic dogs. Phylogenetic analysis suggested that these viruses were all of the canid rabies biotype of southern Africa. The viruses from kudu were closely associated with jackal isolates (n = 6), bat-eared fox isolates (n = 2) and domestic dog isolates (n = 2) at the genetic level and identical at the amino acid level, irrespective of the year of isolation.

Conclusion

These data suggest that jackal and kudu may form part of the same epidemiological cycle of rabies in Namibian wildlife, and might demonstrate the close-relationship between rabies virus strains that circulate within Namibia and those that circulate between Namibia and its neighbouring countries such as Botswana and South Africa.     

DNase SISPA-Next Generation Sequencing Confirms Schmallenberg Virus in Belgian Field Samples and Identifies Genetic Variation in Europe

In 2011, a novel Orthobunyavirus was identified in cattle and sheep in Germany and the Netherlands. This virus was named Schmallenberg virus (SBV). Later, presence of the virus was confirmed using real time RT-PCR in cases of congenital malformations of bovines and ovines in several European countries, including Belgium. In the absence of specific sequencing protocols for this novel virus we confirmed its presence in RT-qPCR positive field samples using DNase SISPA-next generation sequencing (NGS), a virus discovery method based on random amplification and next generation sequencing. An in vitro transcribed RNA was used to construct a standard curve allowing the quantification of viral RNA in the field samples. Two field samples of aborted lambs containing 7.66 and 7.64 log(10) RNA copies per μL total RNA allowed unambiguous identification of SBV. One sample yielded 192 SBV reads covering about 81% of the L segment, 56% of the M segment and 13% of the S segment. The other sample resulted in 8 reads distributed over the L and M segments. Three weak positive field samples (one from an aborted calf, two from aborted lambs) containing virus quantities equivalent to 4.27-4.89 log(10) RNA copies per μL did not allow identification using DNase SISPA-NGS. This partial sequence information was compared to the whole genome sequence of SBV isolated from bovines in Germany, identifying several sequence differences. The applied viral discovery method allowed the confirmation of SBV in RT-qPCR positive brain samples. However, the failure to confirm SBV in weak PCR-positive samples illustrates the importance of the selection of properly targeted and fresh field samples in any virus discovery method. The partial sequences derived from the field samples showed several differences compared to the sequences from bovines in Germany, indicating sequence divergence within the epidemic.     

Genetic heterogeneity among isolates of Ross River virus from different geographical regions.

The RNase T1 maps of 80 isolates of Ross River virus from different regions of mainland Australia and the Pacific Islands were compared. Four different clusters of isolates with greater than an estimated 5 to 6% diversity at the nucleotide level were found. There was a pattern of differences between eastern and western Australian strains; however, the pattern was disturbed by overlaps and incursants. Pacific Islands isolates belonged to the eastern Australian topotype. Our findings suggest that certain genetic types of Ross River virus predominate in different geographical regions. In contrast, populations of other important Australian arboviruses (Murray Valley encephalitis, Kunjin, and Sindbis viruses) are distributed across the Australian continent as minor variants of one strain. Our data also show that in one region, strains of Ross River virus with identical RNase T1 maps circulate during both years when epidemics occur and years when they do not. This finding suggests that Ross River virus epidemics are not dependent on the introduction or evolution of new strains of the virus. Two strains, belonging to the eastern Australian topotype, were isolated in Western Australia. It is likely that viremic humans or possibly domestic livestock travelling by aircraft were responsible for this movement.     

Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses.

Respiratory syncytial (RS) virus causes repeated infections throughout life. Between the two main antigenic subgroups of RS virus, there is antigenic variation in the attachment protein G. The antigenic differences between the subgroups appear to play a role in allowing repeated infections to occur. Antigenic differences also occur within subgroups; however, neither the extent of these differences nor their contributions to repeat infections are known. We report a molecular analysis of the extent of diversity within the subgroup B RS virus attachment protein genes of viruses isolated from children over a 30-year period. Amino acid sequence differences as high as 12% were observed in the ectodomains of the G proteins among the isolates, whereas the cytoplasmic and transmembrane domains were highly conserved. The changes in the G-protein ectodomain were localized to two areas on either side of a highly conserved region surrounding four cysteine residues. Strikingly, single-amino-acid coding changes generated by substitution mutations were not the only means by which change occurred. Changes also occurred by (i) substitutions that changed the available termination codons, resulting in proteins of various lengths, and (ii) a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame of the protein in comparison to the other G genes analyzed. Fifty-one percent of the G-gene nucleotide changes observed among the isolates resulted in amino acid coding changes in the G protein, indicating a selective pressure for change. Maximum-parsimony analysis demonstrated that distinct evolutionary lineages existed. These data show that sequence diversity exists among the G proteins within the subgroup B RS viruses, and this diversity may be important in the immunobiology of the RS viruses.     

Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals.

In the course of human immunodeficiency virus type 1 (HIV-1) infection, patients develop a strong and persistent immune response characterized by the production of HIV-specific antibodies. The aim of our study was to analyze the appearance of autologous and heterologous neutralizing antibodies in the sera of HIV-infected individuals. For this purpose, primary strains have been isolated from 18 HIV-1-infected subjects prior to seroconversion (in one case) or within 1 to 8 months after seroconversion. Sera, collected at the same time as the virus was isolated and at various times after isolation, have been analyzed for their ability to neutralize the autologous primary strains isolated early after infection, heterologous primary isolates, and cell-line adapted strains. Our neutralization assay, which combines serial dilutions of virus and serial dilutions of sera, is based on the determination of the serum dilution at which a fixed reduction in virus titer (90%) occurs. We have shown that (i) we could not detect autologous neutralizing antibodies in sera collected at the same time as we isolated viruses; (ii) we detected neutralizing antibodies against the autologous strains about 1 year after seroconversion, occasionally after 8 months, but sera were not always available to exclude the presence of neutralizing antibodies at earlier times; (iii) after 1 year, the neutralization response was highly specific to virus present during the early phase of HIV infection; and (iv) heterologous neutralization of primary isolates was detected later (after about 2 years). These results reveal the enormous diversity of neutralization determinants on primary isolates as well as a temporal evolution of the humoral response generating cross-reactive neutralizing antibodies.     

Molecular characterisation of Australasian isolates of aquatic birnaviruses

An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 1998, has continued to be re-isolated on an infrequent but regular basis. Due to its low pathogenicity, there has been little urgency to undertake a comprehensive characterisation of this aquatic birnavirus. However, faced with possible incursions of any new aquatic birnaviruses, specific identification and differentiation of this virus from other, pathogenic, aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV) are becoming increasingly important. The present study determined the nucleic acid sequence of the aquatic birnavirus originally isolated in 1998, as well as a subsequent isolate from 2002. The sequences of the VP2 and VP5 genes were compared to that of other aquatic birnaviruses, including non-pathogenic aquatic birnavirus isolates from New Zealand and pathogenic infectious pancreatic necrosis virus isolates from North America and Europe. The deduced amino acid (aa) sequences indicate that the Australian and New Zealand isolates fall within Genogroup 5 together with IPNV strains Sp, DPL, Fr10 and N1. Thus, Genogroup 5 appears to contain aquatic birnavirus isolates from quite diverse host and geographical ranges. Using the sequence information derived from this study, a simple diagnostic test has been developed that differentiates the current Australian isolates from all other aquatic birnaviruses, including the closely related isolates from New Zealand.     

Molecular characterization of lactic acid bacteria from sourdough breads produced in Sardinia (Italy) and multivariate statistical analyses of results

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in sourdough used for the production of traditional breads (Carasau, Moddizzosu, Spianata, Zichi) in Sardinia. 16S rDNA sequencing and Randomly Amplified Polymorphic DNA (RAPD-PCR) was applied for the identification and typing of the LAB isolated from 25 samples of sourdoughs. Multivariate statistical techniques were applied to RAPD-PCR pattern to study the biological diversity of sourdough samples. Twelve different species of LAB were identified, and most isolates were classified as facultative heterofermentative lactobacilli. Lactobacillus pentosus dominated the lactic microflora of many samples while Lactobacillus sanfranciscensis was isolated only from a limited number of samples. Although heterofermentative species represented between between 30% and 60% of the isolates in Carasau, Spianata and Zichi sourdoughs, only 2% of the isolates from Moddizzosu sourdoughs were identified as heterofermentative LAB. RAPD-PCR with a single primer followed by cluster analysis did not allow the identification of the isolates at the species level. However, a multidimensional scaling/bootstrapping approach on the RAPD-PCR patterns uncovered the diversity of the LAB communities of LAB showing differences both within and between bread types.     

Characterization of genetic changes occurring in attenuated poliovirus 2 during persistent infection in mouse central nervous systems.

Genomic changes occurring in the attenuated W-2 strain of poliovirus 2 during persistent infection of the central nervous system of immunosuppressed mice were analyzed. The RNase T1 oligonucleotide fingerprints of 34 different viruses, isolated from the brains and spinal cords of paralyzed and nonparalyzed mice during a 105-day period, were used to quantitate and compare the mutations occurring in each isolate. Although mice were inoculated with plaque-purified virus, genetically distinct viruses were recovered from the central nervous system. The number of oligonucleotide changes occurring in isolates from paralyzed mice generally was greater than that observed in isolates from nonparalyzed mice. However, differences in the extent of mutation in isolates from the two groups of mice did not appear to be related to the level of virus replication. In paralyzed mice, the number of oligonucleotide changes on average was greater in viruses isolated during the first 60 days of the infection than in the last 45 days. The number of oligonucleotide changes was essentially constant throughout the infection, however, in viruses isolated from the brains of nonparalyzed mice. In addition, several specific oligonucleotide changes were found only in viruses isolated from paralyzed animals.     

Naturally occurring human parainfluenza type 3 viruses exhibit divergence in amino acid sequence of their fusion protein neutralization epitopes and cleavage sites.

Many human parainfluenza type 3 virus (PIV3) strains isolated from children with respiratory illness are resistant to neutralization by monoclonal antibodies (MAbs) which recognize epitopes in antigenic site A or B of the fusion (F) protein of the prototype 1957 PIV3 strain. The F protein genes of seven PIV3 clinical isolates were sequenced to determine whether their neutralization-resistant phenotypes were associated with specific differences in amino acids which are recognized by neutralizing MAbs. Several clinical strains which were resistant to neutralization by site A or B MAbs had amino acid differences at residues 398 or 73, respectively. These specific changes undoubtedly account for the neutralization-resistant phenotype of these isolates, since identical substitutions at residues 398 or 73 in MAb-selected escape mutants confer resistance to neutralization by site A or B MAbs. The existence of identical changes in naturally occurring and MAb-selected neutralization-resistant PIV3 strains raises the possibility that antigenically different strains may arise by immune selection during replication in partially immune children. Three of the seven clinical strains examined had differences in their F protein cleavage site sequence. Whereas the prototype PIV3 strain has the cleavage site sequence Arg-Thr-Lys-Arg, one clinical isolate had the sequence Arg-Thr-Arg-Arg and two isolates had the sequence Arg-Thr-Glu-Arg. The different cleavage site sequences of these viruses did not affect their level of replication in either continuous simian or bovine kidney cell monolayers (in the presence or absence of exogenous trypsin or plasmin) or in the upper or lower respiratory tract of rhesus monkeys. We conclude that two nonconsecutive basic residues within the F protein cleavage site are sufficient for efficient replication of human PIV3 in primates.     

Genetic diversity of enzootic isolates of vesicular stomatitis virus New Jersey.

The RNA genomes of 43 vesicular stomatitis virus (VSV) isolates of the New Jersey (NJ) serotype were T1-ribonuclease fingerprinted to compare the extent of genetic diversity of virus from regions of epizootic and enzootic disease activity. Forty of these viruses were obtained from Central America during 1982 to 1985. The other three were older isolates, including a 1970 isolate from Culex nigripalpus mosquitos in Guatemala, a 1960 bovine isolate from Panama, and a 1976 isolate from mosquitos (Mansonia indubitans) in Ecuador. The data indicate that extensive genetic diversity exists among virus isolates from this predominantly enzootic disease zone. Six distinct T1 fingerprint groups were identified for the Central American VSV NJ isolates from 1982 to 1985. The 1960 VSV NJ isolate from Panama and the 1976 isolate from Ecuador formed two additional distinct fingerprint groups. This finding is in sharp contrast to the relatively close genetic relationship existing among VSV NJ isolates obtained from predominantly epizootic disease areas of the United States and Mexico during the same period (S. T. Nichol, J. Virol. 61:1029-1036, 1987). In this previous study, RNA genome T1 fingerprint differences were observed among isolates from different epizootics; however, the isolates were all clearly members of one large T1 fingerprint group. The eight T1 fingerprint groups described here for Central American and Ecuadorian viruses are distinct from those characterized earlier for virus isolates from the United States and Mexico and for the common laboratory virus strains Ogden and Hazelhurst. Despite being isolated 14 years earlier, the 1970 insect isolate from Guatemala is clearly a member of one of the 1982 to 1985 Central American virus fingerprint groups. This indicates that although virus genetic diversity in the region is extensive, under certain natural conditions particular virus genotypes can be relatively stably maintained for an extended period. The implications of these findings for the evolution of VSV NJ and epizootiology of the disease are discussed.     

Genomic analysis of pandemic (H1N1) 2009 reveals association of increasing disease severity with emergence of novel hemagglutinin mutations

Experimental studies and epidemiological observations during the first wave of the pandemic (H1N1) 2009 suggest that a novel influenza A (H1N1) virus has significant pandemic potential based on high transmissibility of the virus. Substantial uncertainty remains regarding evolution of the clinical severity of this pandemic during the transition to the second wave which is currently underway in the Northern Hemisphere. We carried-out analysis of large volume of clinical, epidemiological and genomics data for assessment of evolution of the current pandemic in United States, Canada, United Kingdom, Australia and Japan based on official reports of public health agencies of corresponding countries. Analysis of reported sequences of virus strains isolated from postmortem samples indicates that 42.9% of individuals who died from laboratory-confirmed cases of the pandemic (H1N1) were infected with the hemagglutinin (HA) Q310H mutant virus. Overall, six of seven (86%) of virus isolates recovered from the necropsy samples have at least one mutation within the HA 301–316 or HA 219–240 regions. During the second wave of the pandemic (H1N1) 2009, there is an increased number of reported double mutant virus isolates with mutations within both of these HA regions. Mutations within HA 219-240 region at the position D239 (D239E/G/N) are reported with higher frequency. In addition, D239G mutants were detected more frequently in viruses isolated from patients with fatal outcomes and in isolates from lungs. Multiple viral isolates with the novel HA 301-316 mutations (I312V and P314S) have been documented. Statistically significant increase of detection of mutant viruses and H1N1-related death rates is documented in July-September reporting time periods. Our analysis seems to indicate that evolution of current pandemic is associated with notable changes in mortality rate among hospitalized patients and increasing number of reported cases of novel mutations of HA gene. Recently emerged HA mutants are: (1) detected in large proportion of virus isolates recovered from the postmortem samples; (2) documented in multiple independent reports around the world; (3) expanding within global viral population; (4) manifesting spatial and temporal patterns of association with increased mortality rate of hospitalized patients. Identification of candidate virus mutants with potential association to increasing disease severity should facilitate clinical and experimental testing of the validity of both “antigenic drift” and increase virulence hypotheses. The results of these follow-up experiments may have a significant impact on ultimate outcomes of current pandemic. Our analysis indicates the urgent need for international surveillance systems that track disease severity and individual patient influenza sequence data in a representative fashion. Information gained from this type of surveillance will direct experimental work that assesses influenza strain-specific features of virulence and transmissibility through carefully designed and timely executed laboratory studies. Practical implementation of these surveillance systems would facilitate the timely evidence-based resolution of critically important relationships between the antigenic drift of mutant strains and immunogenicity of existing vaccines which should be assessed in the laboratory setting during the course of the ongoing pandemic.     

Virus recovery rates for wild-type and live-attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species

Previously reported virus recovery rates from Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel (Diptera, Ceratopogonidae) orally infected with vaccine strain of African horse sickness virus serotype 7 (AHSV-7) were compared with results obtained from concurrently conducted oral infections with five recent AHSV-7 isolates from naturally infected horses from various localities in South Africa. Culicoides were fed sheep bloods spiked with 10(7.6) TCID(50)/mL of a live-attenuated vaccine strain AHSV-7, and with five field isolates in which virus titre in the bloodmeals ranged from 10(7.1) to 10(8.2) TCID(50)/mL). After an extrinsic incubation of 10 days at 23.5 degrees C, virus recovery rates were significantly higher in C. imicola (13.3%) and C. bolitinos (4.2%) infected with the live-attenuated virus than in midges infected with any of the field isolates. The virus recovery rates for the latter groups ranged from 0% to 9.5% for C. imicola and from 0% to 1.5% for C. bolitinos. The C. imicola population at Onderstepoort was significantly more susceptible to infection with AHSV-7 isolated at Onderstepoort than to the virus strains isolated from other localities. Results of this study suggest that tissue culture attenuation of AHSV-7 does not reduce its ability to orally infect competent Culicoides species and may even lead to enhanced replication in the vector. Furthermore, oral susceptibility in a midge population appears to vary for geographically distinct isolates of AHSV-7.     

Alfalfa mosaic virus isolates from lucerne in South Australia: biological variability and antigenic similarity

Alfalfa mosaic virus (AMV) was isolated from lucerne (Medicago sativa) plants with a variety of disease symptoms in eight of 13 sites in South Australia indicating that the virus is widespread in the state. The host ranges and symptomatology of the virus isolates varied considerably. Twelve selected local lesion isolates were shown to be distinct when mechanically inoculated to a range of plant species and cultivars. However, agar-gel diffusion and enzyme-linked immunoassay tests with polyclonal antisera prepared against glutaraldehyde-fixed virus preparations of the five most readily distinguishable AMV isolates, failed to reveal significant antigenic differences between the 12 virus isolates. This indicates that serological tests with polyclonal antisera can detect a wide range of AMV variants but would be unlikely to differentiate between strains. The wide host range and variability of AMV precluded the grouping of isolates into strains of the virus.     

Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks

Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.