Phylogenetic Relationships among Highly Virulent Newcastle Disease Virus Isolates Obtained from Exotic Birds and Poultry from 1989 to 1996

Newcastle disease virus {NDV (avian paramyxovirus type 1 [APMV1])} isolates were recovered from imported exotic birds confiscated following importation into the United States, from waterbirds in the United States, and from poultry. The exotic birds probably originated from Central and South America, Asia, and Africa. The NDV isolates were initially characterized as highly virulent because of a short mean death time in embryonated chicken eggs. The isolates were typed as neurotropic or viscerotropic velogenic by intracloacal inoculation of adult chickens. Intracerebral pathogenicity index values for the virulent NDV isolates ranged from 1.54 to 1.90, compared to a possible maximum value of 2.0. These isolates had a dibasic amino acid motif in the fusion protein cleavage site sequence required for host systemic replication. Sequence differences were detected surrounding the fusion protein cleavage site and the matrix protein nuclear localization signal, indicating evolution of highly virulent NDV. Phylogenetically, these isolates were categorized with other highly virulent NDV strains that caused outbreaks in southern California poultry during 1972 and in cormorants in the north central United States and southern Canada during 1990 and 1992. These isolates are related to NDV that may have the APMV1 strain chicken/Australia/AV/32 or a related virus as a possible progenitor. Recent virulent NDV isolates and those recovered during disease outbreaks since the 1970s are phylogenetically distinct from current vaccine viruses and standard challenge strains.     

Phylogenetic comparison of the S10 genes of recent isolates of bluetongue virus from the United States and French Martinique Island

The sequences of the S10 genes of 28 recent isolates (1994-2004) of bluetongue virus (BTV) from the United States (US) and French Martinique Island (2006) in the Caribbean Basin were compared in phylogenetic analyses to those of viruses previously isolated in the same regions. Although the analyses segregated the recent virus isolates from the two regions into distinct topotype clusters, the analyses also confirm that viruses from the US and the Caribbean Basin/Central America can share similar S10 genes despite the fact that distinct constellations of BTV serotypes occur in the two regions.     

Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.     

Newcastle disease outbreaks in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005 were caused by viruses of the genotypes VIIb and VIId

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world, and infections with virulent NDV strains continue to cause disease outbreaks in poultry and wild birds. To assess the evolutionary characteristics of 28 NDV strains isolated from chickens in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005, we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47–421) was performed using sequences of Kazakhstanian and Kyrgyzstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis demonstrated that the 14 newly characterized strains from years 1998 to 2001 belong to the NDV genotype VIIb, whereas the 14 strains isolated during 2003–2005 were of genotype VIId. All strains possessed a virulent fusion protein cleavage site (R-R-Q-R/K-R-F) and had intracerebral pathogenicity indexes in day-old chickens that ranged from 1.05 to 1.87, both properties typical of NDV strains classified in the mesogenic or velogenic pathotype.     

Emergence and re-emergence of vesicular stomatitis in the United States

Vesicular stomatitis (VS) is an important disease of cattle, horses and pigs. The causal agent is an arbovirus; vesicular stomatitis virus (VSV) of which two distinct serotypes New Jersey (NJ) and Indiana (IN) have been described. The clinical signs in cattle and pigs are undistinguishable from foot-and-mouth disease (FMD), one of the most devastating viral infections of livestock. VSV is the most important cause of vesicular disease in FMD-free countries in the Americas, causing thousands of outbreaks every year from southern Mexico to northern South America. In the United States VS has two different patterns of occurrence; in the southeastern states (Georgia, Alabama, North Carolina and South Carolina) a pattern of yearly occurrence of clinical cases in livestock was reported from early 1900s until the mid 1970s. Since then, viral activity in the region has been focal and limited to isolated wildlife populations. In contrast in the southwestern states (New Mexico, Arizona, Utah and Colorado) VS outbreaks have occurred sporadically at approximately 10-year intervals, with the last cycle of activity occurring from 1995 to 1998. Phylogenetic analyses of VSV have shown that distinct viral lineages occur in the southwestern and southeastern US. Furthermore, in the last 70 years each sporadic outbreak in the Southwest was associated to viral lineages distant from those causing previous outbreaks in the US but closely related to viruses maintained in endemic areas of Mexico. This pattern of viral occurrence contrasts with that observed in endemic areas in Central and South America where viral genetic lineages are maintained in specific ecological areas over long periods of time. The phylogenetic data together with the geographical and temporal distribution of outbreaks indicate that VSV does not have a stable endemic cycle in the western United States.     

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.     

Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000.

Gene sequence analysis of fusion (F) gene cleavage motifs and haemagglutinin-neuraminidase (HN) carboxyl-terminal extension sequences was used to analyse Newcastle disease viruses (NDV) associated with virulent outbreaks of the disease which occurred in New South Wales, Australia in 1998-2000. PCR fragments were amplified directly from diseased tissue or allantoic fluids and sequence analyses used for phylogenetic comparisons between these viruses and Australian reference NDV. F and HN gene sequence comparison showed a strong relationship to sequences derived from endemic Australian NDV rather than those of overseas viruses or wild bird isolates. Prior to notification of the 1998 outbreak, an NDV was isolated from chickens suffering respiratory disease that appeared to be the progenitor virus from which the virulent virus originated. In turn, these viruses are closely related to two previously isolated 'ancestor' viruses that have the same unique HN extension sequence.     

Diversity and phylogenetic relationships among the North American Tacaribe serocomplex viruses (family Arenaviridae)

The purpose of this study was to extend our knowledge of the genetic diversity and phylogenetic relationships among the North American Tacaribe serocomplex viruses. Analyses of glycoprotein precursor gene sequence data separated the North American arenaviruses into 7 major phylogenetic groups. The results of analyses of Z gene and nucleocapsid protein gene sequence data were not remarkably different from the glycoprotein precursor gene tree. In contrast, the tree generated from RNA-dependent RNA polymerase gene sequences differed from the glycoprotein precursor gene tree with regard to phylogenetic relationships among the viruses associated with woodrats captured in the western United States, Texas, or northern Mexico. Further analyses of the polymerase gene sequence data set suggested that the difference in topology was a consequence of incongruence among the gene tree data sets or chance rather than genetic reassortment or recombination between arenaviruses.     

Anaplasma marginale msp1alpha genotypes evolved under positive selection pressure but are not markers for geographic isolates

Anaplasma marginale (order Rickettsiales, family Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur in the United States and have been identified by major surface protein 1a (MSP1a), which varies in sequence and molecular weight due to different numbers of tandem 28- to 29-amino-acid repeats. The present study was undertaken to examine the genetic variations among isolates of A. marginale obtained during 2001 from infected cattle from east-central Oklahoma, where A. marginale is endemic. The gene and protein sequences of MSP1a and msp4 nucleotide sequences were used to infer the phylogenetic relationships among Oklahoma and New World isolates from Argentina, Brazil, Mexico, and the United States. All 11 A. marginale isolates collected from Oklahoma had different MSP1a sequences but identical MSP4 sequences. The phylogenies of the msp4 sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American isolates and 12 U.S. isolates by maximum-parsimony (MP) and maximum-likelihood (ML) analyses, with A. centrale and A. ovis sequences used as outgroups, provided strong bootstrap analysis support for a Latin American clade. Isolates of A. marginale from the southern United States (Florida, Mississippi, and Virginia) and the west-central United States (California, Idaho, Illinois, Oregon, Missouri, and Texas) also grouped into two clades. Both clades contained isolates from Oklahoma, suggesting extensive cattle movement. ML analysis of the msp4 sequences of isolates from Oklahoma provided bootstrap analysis support for east-central and north-central clades in Oklahoma, and both clades included isolates from Stillwater, Okla. Analysis of the codon and amino acid changes among the msp4 sequences of isolates with different phylogenies provided evidence that msp4 is not under positive selection pressure. In contrast, the phylogenies of the MSP1a DNA and protein sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American and 13 isolates from the United States by MP and ML analyses demonstrated no geographic clustering and provided evidence that this gene is under positive selection pressure. The results indicate that msp1alpha is not a marker for the characterization of A. marginale geographic isolates and suggest that the genetic heterogeneity observed among isolates of A. marginale within Oklahoma could be explained by cattle movement and the maintenance of different genotypes by independent transmission events.     

Molecular Characterization of Virulent Newcastle Disease Virus Isolates from Chickens during the 1998 NDV Outbreak in Kazakhstan

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world and has the possibility to cause outbreaks in chicken flocks in future. To assess the evolutionary characteristics of 10 NDV strains isolated from chickens in Kazakhstan during 1998 we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47-421) was performed using sequences of Kazakhstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to the genetic group designated as VIIb. All strains possessed a virulent fusion cleavage site (RRQRR/F) belonging to velogenic or mesogenic pathotypes with intracerebral pathogenicity indexes (ICPI) varying from 1.05 to 1.87.     

Phylogenetic analysis of the envelope gene of St. Louis encephalitis virus

Summary.  The complete nucleotide sequences of the envelope gene of 62 geographic isolates of St. Louis encephalitis (SLE) virus were determined. Phylogenetic analyses of the sequences, conducted using both maximum parsimony and neighbor-joining methods, included four other members of the Japanese encephalitis serogroup. The results indicated that the SLE isolates formed a monophyletic group in which isolates generally clustered according to geographic origin. Isolates from Panama and South America predominantly formed two large groupings, while isolates from the U.S. formed two other major groups. Several South and Central American strains were more closely related to strains isolated in the U.S., e.g., one isolate from Mexico and Panama, each, were closely related to two Tampa Bay, Florida, isolates, and an isolate from Brazil was closely related to three isolates from Texas. The U.S. isolates also were not strictly grouped according to geographic source, e.g., some California isolates were closely related to Texas or midwestern isolates, and a Florida isolate was closely related to three isolates from Maryland. The results of the phylogenetic analyses indicated that SLE virus is predominantly maintained locally, but has been transported occasionally between areas, both within and outside the U.S. Received March 26, 2001 Accepted July 9, 2001     

Analysis of matrix protein gene nucleotide sequence diversity among Newcastle disease virus isolates demonstrates that recent disease outbreaks are caused by viruses of psittacine origin

Nucleotide sequence analysis was completed for isolates of Newcastle disease virus (NDV; avian paramyxovirus 1) from 1992 outbreaks in cormorants and turkeys. These isolates were of the neurotropic velogenic type. The cormorant and turkey NDV isolates had the fusion protein cleavage sequence¹⁰⁹SRGRRQKR/FVG¹¹⁹, as opposed to the consensus sequence¹⁰⁹SGGRRQKR/FIG¹¹⁹ of most known velogenic NDV isolates. The R for G substitution at position 110 may be unique for the cormorant and turkey isolates. For comparative purposes, nucleotide sequencing and analysis of the conserved matrix protein gene coding region were completed for isolates representing all pathotypes. Phylogenetic relationships demonstrated that there are two major groups of NDV isolates. One group includes viruses found in North America and worldwide, such as B1, LaSota, Texas/GB, and Beaudette/C. The second group contains isolates, such as Ulster/2C, Australia/Victoria, and Herts/33, considered exotic to North America. Within this second group are viruses of psittacine origin. The viruses from 1992 outbreaks of Newcastle disease in North America, and an isolate thought to have caused the major outbreak in southern California during the 1970s, are most closely related to an NDV isolate of psittacine origin.     

The origin and global emergence of adamantane resistant A/H3N2 influenza viruses

Resistance to the adamantane class of antiviral drugs by human A/H3N2 influenza viruses currently exceeds 90% in the United States and multiple Asian countries. Adamantane resistance is associated with a single amino acid change (S31N) in the M2 protein, which was shown to rapidly disseminate globally in 2005 in association with a genome reassortment event. However, the exact origin of influenza A/H3N2 viruses carrying the S31N mutation has not been characterized, particularly in South-East Asia. We therefore conducted a phylogenetic analysis of the HA, NA, and M1/2 segments of viral isolates collected between 1997 and 2007 from temperate localities in the Northern hemisphere (New York State, United States, 492 isolates) and Southern hemisphere (New Zealand and Australia, 629 isolates) and a subtropical locality in South-East Asia (Hong Kong, 281 isolates). We find that although the S31N mutation was independently introduced at least 11 times, the vast majority of resistant viruses now circulating globally descend from a single introduction that was first detected in the summer of 2003 in Hong Kong. These resistant viruses were continually detected in Hong Kong throughout 2003-2005, acquired a novel HA through reassortment during the first part of 2005, and thereafter spread globally. The emergence and persistence of adamantane resistant viruses in Hong Kong further supports a source-sink model of global influenza virus ecology, in which South-East Asia experiences continuous viral activity and repeatedly seeds epidemics in temperate areas.     

Phylogenetic and pathogenic analyses of two virulent Newcastle disease viruses isolated from Crested Ibis (Nipponia nippon) in China

The crested ibis is one of the most endangered birds in the world, found only in Shaanxi Province in Central China, and it has been reintroduced in Sadogashima in Japan. Two Newcastle disease virus (NDV) isolates were collected from sick crested ibises, and their pathogenic and phylogenetic characteristics were investigated. The results showed that they are virulent, with intracerebral pathogenicity indices of 1.46–1.83 and a mean time of death of 54.4–84.4 h. They shared the same virulent motif ¹¹²-R-R-Q-K-R-F-¹¹⁷ at the F protein cleavage site. The phylogenetic analysis revealed that both isolates were clustered with class II NDVs, with one in genotype VIId and another in a novel genotype (provisionally designated as VIi). The two isolates shared high homology with the strains isolated from poultry flocks in the same region from 2006 to 2010. We first isolated and characterised the NDV isolates from crested ibises, one of which showed new genetic characteristics and formed a new subgenotype with isolates from pigeons and ostriches in the same area. These data are useful for further epidemiological studies on NDV and the protection of crested ibises.     

Rapid <Emphasis Type="Italic">in vitro</Emphasis> assessment of the virulence of <Emphasis Type="Italic">Newcastle disease virus</Emphasis> isolates using the ligase chain reaction

Summary. The ligase chain reaction was used to assess the virulence of isolates of Newcastle disease virus. In the main study, 18/18 virulent isolates whose nucleotide sequences that code for the cleavage site and fusor peptide regions were known, successfully ligated oligonucleotides in a primer mix for virulent viruses termed VPM. Five of these isolates yielded a more intense ligated product with a second primer mix for virulent viruses called VPM1. No ligation was evident with eight avirulent isolates in tests with VPM or VPM1, however, each of these viruses did yield a strong ligated product with the primer mix for avirulent viruses (AVPM) as did one virulent isolate considered to be a mixture. Two virulent Australian isolates, 1238/1998 and 1248/1998, showed low but seemingly specific ligation with AVPM. In a blind study, 8/9 virulent isolates whose sequences were unknown ligated primers in VPM. Three avirulent and one virulent isolate, the latter again probably a mixture, ligated primers in AVPM. Ligation of oligonucleotides in VPM and AVPM was detectable in mixtures where virulent and avirulent isolates represented 0.1% and 0.01% by volume respectively of the viral population. The results indicate that LCR offers a potential in vitro alternative to current in vivo tests for virulence determination of Newcastle disease virus isolates.Received August 15, 2002; accepted April 9, 2003 Published online June 24, 2003     

Transmission of drug-resistant tuberculosis in Texas and Mexico

To examine the transmission of drug-resistant (DR) tuberculosis between Texas and Mexico, Mycobacterium tuberculosis isolates resistant to one or more of the first-line antimycobacterial drugs were obtained from 606 patients who resided in Texas and 313 patients who resided in Mexico, primarily within the state of Tamaulipas. The isolates were genotyped by IS6110-based restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Of the 919 isolates genotyped, 413 (45%) grouped into 105 clusters containing 2 or more isolates with identical genotypes. In addition to having identical genotypes, identical drug resistance patterns were identified in 250 isolates in 78 clusters (DR clusters). Twenty DR clusters, containing isolates from 32% of the total number of patients infected with DR strains, were geographically distributed across Mexico and Texas. Within this population of 919 patients infected with DR isolates, the probability of being in a DR cluster was the same for residents of Mexico and Texas. In Texas, the significant independent predictors of clustering within DR clusters as opposed to genotype clusters were found to be race, age, country of birth, human immunodeficiency virus (HIV) infection status, and resistance to more than one drug. Specifically, isolates from African Americans, individuals under age 65, individuals born in the United States, and HIV-positive individuals were each more likely to be associated with a DR cluster. By contrast, no significant independent predictors of clustering in a DR cluster were identified in Mexico. Although some DR M. tuberculosis strains are geographically restricted, this study suggests that a number of strains are transmitted between Mexico and the United States.     

Genetic heterogeneity among Saint Louis encephalitis virus isolates of different geographic origin

A series of 57 Saint Louis encephalitis (SLE) virus isolates from humans, birds, rodents, and mosquitoes showed extensive variability in their RNase T₁ oligonucleotide fingerprints. The fingerprints of virion RNA did not contain an obvious poly(A) tract and were identical when the virus was grown in either mosquito or mammalian cells. Analysis and comparison of long oligonucleotides, representing approximately 10% of the genome of SLE isolates from the Central and Atlantic states, indicated the viruses share at least 80% of their long oligonucleotides. Analysis of the large T₁-resistant oligonucleotides by RNase A digestion revealed chemical similarities in the composition of common oligonucleotides derived from the genomes of four isolates. Analysis of 57 SLE strains from North America indicated that on the basis of the similarity of the oligonucleotide fingerprints, SLE isolates could be divided into genotypic sets representing different geographic regions in North America. These geographic varieties, designated “topotypes,” represent isolates from: (1) the Central/Atlantic states, (2) Florida, (3) and the Western United States. Variants of each “topotype” have been characterized whose oligonucleotide fingerprints are similar to that of the “topotype” but are sufficiently distinct to permit separation of the highly and less virulent SLE strains.     

Characterization of recent H5 subtype avian influenza viruses from US poultry.

In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.