Psittacine beak and feather disease in a free-living ring-necked parakeet (Psittacula krameri) in Great Britain

The ring-necked parakeet (RNP), Psittacula krameri, is an invasive species in Great Britain (GB) which is undergoing rapid population expansion in the wild. Although it has been suggested that RNPs could be a potential source of infectious disease, little research has been done on the pathogens infecting this species in GB. Psittacine beak and feather disease (PBFD), caused by beak and feather disease virus (BFDV), is an important infectious disease of psittacines, including captive RNPs, in GB and elsewhere. A wild RNP with marked feather abnormalities observed in an urban garden in London was euthanased and examined post mortem. Plucked contour feathers and pooled liver and spleen were PCR-positive for BFDV DNA. Histopathological examination of affected skin demonstrated BFDV-compatible lesions. A feather from another RNP from a different location also was PCR-positive for BFDV. This is the first report of PBFD in a wild free-living bird in GB. BFDV only affects psittacines; therefore, there is no known risk to native British birds. The presence of BFDV in free-living RNPs, however, could pose a disease threat to captive psittacines. Further work is required to determine the distribution and impact of BFDV infection in free-living RNPs in GB. Whether this case represents sporadic disease associated with established endemic infection or the index case of an emergent disease is currently unknown.     

Identification and Characterization of a Distinct Strain of Beak and Feather Disease Virus in Southeast China

Beak and feather disease virus(BFDV) is an infectious agent responsible for feather degeneration and beak deformation in birds. In March 2017, an epidemic of psittacine beak and feather disease(PBFD) struck a farm in Fuzhou in the Fujian Province of southeast China, resulting in the death of 51 parrots. In this study, the disease was diagnosed and the pathogen was identified by PCR and whole genome sequencing. A distinct BFDV strain was identified and named as the FZ strain.This BFDV strain caused severe disease symptoms and pathological changes characteristic of typical PBFD in parrots, for example, loss of feathers and deformities of the beak and claws, and severe pathological changes in multiple organs of the infected birds. Phylogenetic analysis showed that the FZ strain was more closely related to the strain circulating in New Caledonia than the strains previously reported in China. Nucleotide homology between the FZ strain and other 43 strains of BFDV ranged from 80.0% to 92.0%. Blind passage experiment showed that this strain had limited replication capability in SPF Chicken Embryos and DF-1 Cells. Furthermore, the capsid(Cap) gene of this FZ strain was cloned into the p GEX-4 T-1 expression vector to prepare the polyclonal anti-Cap antibody. Western blotting analysis using the anti-Cap antibody further confirmed that the diseased parrots were infected with BFDV. In this study, a PBFD and its pathogen was identified for the first time in Fujian Province of China, suggesting that future surveillance of BFDV should be performed.     

Plasmodium falciparum mitochondrial genetic diversity exhibits isolation-by-distance patterns supporting a sub-Saharan African origin

The geographical distribution of single nucleotide polymorphism (SNP) in the mitochondrial genome of the human malaria parasite Plasmodium falciparum was investigated. We identified 88 SNPs in 516 isolates from seven parasite populations in Africa, Southeast Asia and Oceania. Analysis of the SNPs postulated a sub-Saharan African origin and recovered a strong negative correlation between within-population SNP diversity and geographic distance from the putative African origin over Southeast Asia and Oceania. These results are consistent with those previously obtained for nuclear genome-encoded housekeeping genes, indicating that the pattern of inheritance does not substantially affect the geographical distribution of SNPs.     

Mitochondrial DNA variation of domestic sheep (Ovis aries) in Kenya

The history of domestic sheep (Ovis aries) in Africa remains largely unknown. After being first introduced from the Near East, sheep gradually spread through the African continent with pastoral societies. The eastern part of Africa was important either for the first diffusion of sheep southward or for putative secondary introductions from the Arabian Peninsula or southern Asia. We analysed mitochondrial DNA control region sequences of 91 domestic sheep from Kenya and found a high diversity of matrilines from the widespread haplogroup B, whereas only a single individual from haplogroup A was detected. Our phylogeography analyses of more than 500 available mitochondrial DNA sequences also identified ancestral haplotypes that were probably first introduced in Africa and are now widely distributed. Moreover, we found no evidence of an admixture between East and West African sheep. The presence of shared haplotypes in eastern and ancient southern African sheep suggests the possible southward movement of sheep along the eastern part of Africa. Finally, we found no evidence of an extensive introduction of sheep from southern Asia into Africa via the Indian Ocean trade. The overall findings on the phylogeography of East African domestic sheep set the grounds for understanding the origin and subsequent movements of sheep in Africa. The richness of maternal lineages in Kenyan breeds is of prime importance for future conservation and breeding programmes.     

Further Evidence for Limited Genetic Diversity among East African Isolates of Sweet potato chlorotic stunt virus

In Africa, the crinivirus Sweet potato chlorotic stunt virus (SPCSV) exists in two serologically and genetically distinct strains, geographically distinguished as a West African (SPCSV_WA) and an East African (SPCSV_EA) strain. To obtain a better understanding of the genetic diversity among SPCSV_EA isolates, the major coat protein (CP) and heat shock protein 70 homologue (Hsp70h) gene sequences of 24 further isolates of SPCSV_EA were determined and compared. SPCSV_EA diversity was also examined using available monoclonal antibodies (mAbs) to SPCSV_EA but there was no apparent coincidence between CP and partial Hsp70h gene nucleotide sequences and the subdivision of SPCSV_EA isolates by the mAbs into two serotypes, suggesting this latter may not be of great biological significance. The nucleotide (nt) sequences of isolates of SPCSV_EA displayed a high degree of conservation and the only variation observed consisted of a few base exchanges. Pairwise alignments of CP nucleotide sequences revealed differences of <4% between SPCSV_EA isolates. Comparisons with published SPCSV sequences confirmed a more distant relationship (up to 34.6% nt; 12% amino acid divergence) between the Hsp70h sequences of isolates of SPCSV_EA and SPCSV_WA and indicated that SPCSV_EA in East and Southern Africa is the more homogeneous than SPCSV_WA isolates from West Africa, North and South America, which were up to 12.4% nt divergent among themselves.     

我国一株鹦鹉幼雏病病毒全序列测定与初步分析

采用鸡胚成纤维细胞(CEF)培养增殖首次从湖北省云梦县分离的鹦鹉幼雏病病毒(Budgerigar fledgling dis ease virus,BFDV)分离株(BFDV HBYM02),经 PCR分段扩增法获得全基因组并完成序列测定。HBYM02 株全序列测定结果与GenBank中仅有的六株BFDV全序列进行同源性与进化分析。经BLAST分析,HBYM02株与其他六株BFDV同源性为98%~99%,为同一个基因型。运用Phylip3.5软件构建进化树,分析显示,来源于不同宿主的BFDV与宿主关系紧密,与地理分布没有明显的相关性。     

The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus.     

Analysis of Coat Protein of Peanut stunt virus Subgroup II Isolates from Alfalfa Fields in West Iran

Alfalfa fields in three western provinces of Iran were surveyed for Peanut stunt virus (PSV) during 2011 and 2012. Forty‐seven of 115 samples tested (41%) were infected with PSV. Phylogenetic analysis using coat protein (CP) gene sequences showed that the Iranian isolates belong to the subgroup II of PSV. Pairwise identity analysis revealed four groups representing four phylogenetic subgroups. PSV strains in subgroups III and IV are closely related to each other, as supported by the lowest nucleotide diversity, high pairwise nucleotide identity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck. Using the maximum likelihood method, amino acid 86S in the CP gene of the Iranian PSV isolates was found to be under positive selection, although the likelihood ratio test statistics is not significant. This is the first report of the occurrence and phylogenetic relationships of Iranian PSV isolates in west Iran.     

Book Review

To study the variability and to identify the species of Begomovirus associated with yellow mosaic disease of blackgram in Andhra Pradesh, India, infected blackgram samples were collected from six districts belonging to three regions of Andhra Pradesh. The total DNA was isolated by modified CTAB method and amplified with coat protein gene-specific primers (RHA-F and AC abut) resulting in 900?bp gene product. The PCR products were cloned, sequenced and deposited in GenBank. The sequence analysis of six clones showed that the size of amplified CP gene of YMV was 920?bp. Based on nucleotide sequence identity of six isolates representing three regions of Andhra Pradesh, the isolates from Rayalaseema and Telangana region are the same variant of YMV (>99.5% identity) and isolate from coastal Andhra is another variant of YMV (>95.4%) when compared with other region isolates. Comparison of CP gene sequence of YMV-TPT isolate with 27 other isolates in database revealed more than 93.2 and 86.2% identity with MYMIV isolates and less than 80 and 64% identity with MYMY isolates that originate from Indian sub-continent and South-East Asia at nucleotide and amino acid level, respectively. Phylogenetic tree based on CP gene sequences of six isolates with other isolates from GenBank formed unique cluster with MYMIV. Hence the YMV infecting blackgram in Andhra Pradesh is caused by MYMIV rather than MYMY as reported in Tamil Nadu which is adjoining state in southern India.     

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.     

On the Dispersal of Bottle Gourd [<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> (Mol.) Standl.] Out of Africa: a Contribution from the Analysis of Nuclear Ribosomal DNA Haplotypes,Divergent Paralogs and Variants of 5.8S Protein Sequences

Bottle gourd (Lagenaria siceraria), a multipurpose crop, is among the first domesticates of humans. This study analyses nuclear ribosomal DNA (nrDNA) of the two cultivated subspecies to improve our understanding on the African origin and the dispersal to Asia. A total of 146 nrDNA sequences representing 79 individuals from African cultivars and 67 individuals from Asian cultivars were compared; the resulting nrDNA sequences were composed of 35 and 16 haplotypes specific to Africa and Asia, respectively, and two additional haplotypes shared by both continents. When all the rDNA haplotypes were bulked, the genetic differentiation (F _ST ) was significant between the subspecies (P?<?0.001), within Africa (P?<?0.001) and within Asia (P?<?0.05), and the nucleotide diversity was 2.5-fold higher in Africa. Sorting the haplotypes by classes of paralogs revealed more classes in Africa, and in classes where African and Asian cultivars were represented, the diversity was higher in Africa, in general. The 5.8S-coding regions showed two to four amino acid differences resulting to nine protein sequence variants, one of which encompassed all the Asian cultivars. The nucleotide diversity at that shared variant was 1.43-fold higher in Africa than in Asia. Analyses of phylogenetic networks revealed major shared haplotypes containing 23.91 % of the cultivars and having founder locations. We suggest that African cultivars reached Asia. The study tags for the first time nrDNA haplotypes capable of discriminating between and within the subspecies. Thirty single nucleotide polymorphisms (SNPs) and five insertion-deletions (Indels) derived from the haplotypes and registered in GenBank are provided.     

Phylogenetic placement and population structure of Indo‐Pacific bottlenose dolphins (Tursiops aduncus) off Zanzibar,Tanzania, based on mtDNA sequences

Phylogenetic placement of bottlenose dolphins from Zanzibar, East Africa and putative population differentiation between animals found off southern and northern Zanzibar were examined using variation in mtDNA control region sequences. Samples (n= 45) from animals bycaught in fishing gear and skin biopsies collected during boat surveys were compared to published sequences (n= 173) of Indo‐Pacific bottlenose dolphin, Tursiops aduncus, from southeast Australian waters, Chinese/Indonesian waters, and South African waters (which recently was proposed as a new species) and to published sequences of common bottlenose dolphin, Tursiops truncatus. Bayesian and maximum parsimony analyses indicated a close relationship between Zanzibar and South African haplotypes, which are differentiated from both Chinese/Indonesian and Australian T. aduncus haplotypes. Our results suggest that the dolphins found off Zanzibar should be classified as T. aduncus alongside the South African animals. Further, analyses of genetic differentiation showed significant separation between the T. aduncus found off northern and southern Zanzibar despite the relatively short distance (approximately 80 km) between these areas. Much less differentiation was found between southern Zanzibar and South Africa, suggesting a more recent common evolutionary history for these populations than for the northern and southern Zanzibar populations.     

Molecular characterization of simian lentiviruses from east African green monkeys

Asymptomatic infection with simian lentiviruses (also called simian immunodeficiency viruses, or SIV) is common among feral African green monkeys. To characterize the range of SIV genetic diversity among infected African green monkeys, we have determined nucleotide sequences from complete or partial molecular clones of four distinct SIVagm isolates from Kenya and Ethiopia. The nucleotide and amino acid variability we observed among the SIVagm isolates was greater than the variability within any other group of primate lentiviruses. These data suggest that: a) African green monkeys have been infected with simian lentiviruses for many years; and b) novel and uncharacterized primate lentiviruses may exist in the feral African green monkey population in other parts of Africa.     

Comparison of acetylcholinesterase genes from cattle ticks

We describe the isolation and characterisation of two putatively new acetylcholinesterase genes from the African cattle ticks Boophilus decoloratus and Rhipicephalus appendiculatus. The nucleotide sequences of these genes had 93% homology to each other and 95% and 91% identity, respectively, to the acetylcholinesterase gene from an Australian strain of another cattle tick, Boophilus microplus. Translation of the nucleotide sequences revealed putative amino acids that are essential for acetylcholinesterase activity: the active site serine, and the histidine and glutamate residues that associate with this serine to form the catalytic triad. All known acetylcholinesterases have three sets of cysteines that form disulfide bonds; however, the acetylcholinesterase genes of these three species of ticks encode only two sets of cysteines. Acetylcholinesterases of B. microplus from South Africa, Zimbabwe, Kenya and Mexico had 98-99% identity with acetylcholinesterase from B. microplus from Australia, whereas acetylcholinesterase from B. microplus from Indonesia was identical to that from Australia. Preliminary phylogenetic analyses surprisingly indicate that the acetylcholinesterases of ticks are closer phylogenetically to acetylcholinesterases of vertebrates than they are to those of other arthropods.     

Hakuna Nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae

Caenorhabditis elegans and C. briggsae have many parallels in terms of morphology, life history and breeding system. Both species also share similar low levels of molecular diversity, although the global sampling of natural populations has been limited and geographically biased. In this study, we describe the first cultured isolates of C. elegans and C. briggsae from sub-Saharan Africa. We characterize these samples for patterns of nucleotide polymorphism and vulva precursor cell lineage, and conduct a series of hybrid crosses in C. briggsae to test for genetic incompatibilities. The distribution of genetic diversity confirms a lack of geographic structure to C. elegans sequences but shows genetic differentiation of C. briggsae into three distinct clades that may correspond to three latitudinal ranges. Despite low levels of molecular diversity, we find considerable variation in cell division frequency in African C. elegans for the P3.p vulva precursor cell, and in African C. briggsae for P4.p, a variation that was not previously observed in this species. Hybrid crosses did not reveal major incompatibilities between C. briggsae strains from Africa and elsewhere, and there was some evidence of inbreeding depression. These new African isolates suggest that important ecological factors may be shaping the patterns of diversity in C. briggsae, and that despite many similarities between C. elegans and C. briggsae, there may be more subtle differences in their natural histories than previously appreciated.     

Patterns of species diversity and endemism in comparable temperate brown algal floras

Brown algal species diversity is compared in 100 km sections of the coastlines of four warm temperate regions: southern Australia, California, southwestern Africa, north-central Chile. The highest diversity (over 140 species per section) is found in southern Australia. California has a reasonable diversity (around 70 species per section), and both southern Australia and California have high regional endemism. Sections of north-central Chile and southwestern Africa have similar patterns, with low diversity (< 30 species per 100 km section), low endemism, few or no fucoids, and up to 25% of the brown algal flora are environmentally tolerant species of Scytosiphonales. Species turnover between contiguous sections of coast is generally related to relative change in temperature regime. Thus the high diversity of southern Australia is due to high species diversity within the 100 km sections, with little turnover, except for a rapid reduction in eastern Victoria likely to be related to lack of rocky substatum. It is hypothesized that low diversity and endemism in Chile and southwestern Africa can be explained by the occurrence of major environmental perturbations (upwelling and El Nino effects) in these regions, producing variable inter-annual temperature conditions that select out tolerant species from the local floras.     

Population structure of the African savannah elephant inferred from mitochondrial control region sequences and nuclear microsatellite loci

Two hundred and thirty-six mitochondrial DNA nucleotide sequences were used in combination with polymorphism at four nuclear microsatellite loci to assess the amount and distribution of genetic variation within and between African savannah elephants. They were sampled from 11 localities in eastern, western and southern Africa. In the total sample, 43 haplotypes were identified and an overall nucleotide diversity of 2.0% was observed. High levels of polymorphism were also observed at the microsatellite loci both at the level of number of alleles and gene diversity. Nine to 14 alleles per locus across populations and 44 alleles in the total sample were found. The gene diversity ranged from 0.51 to 0.72 in the localities studied. An analysis of molecular variance showed significant genetic differentiation between populations within regions and also between regions. The extent of subdivision between populations at the mtDNA control region was approximately twice as high as shown by the microsatellite loci (mtDNA F(ST) = 0.59; microsatellite R(ST) = 0.31). We discuss our results in the light of Pleistocene refugia and attribute the observed pattern to population divergence in allopatry accompanied by a recent population admixture following a recent population expansion.     

Duplex shuttle PCR for differential diagnosis of budgerigar fledgling disease and psittacine beak and feather disease

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298-bp fragment of the t/T antigen region of APV genome and a 495-bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV-positive, 15 PBFDV-positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV-positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.     

Ultrastructural, protein composition, and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds

Psittacine beak and feather disease (PBFD) virus, was purified from diseased tissues of a lesser sulphur-crested cockatoo (Cacatua sulphurea), a black palm cockatoo (Probosiger aterrimus), a red-lored Amazon parrot (Amazona autumnalis), and a peach-faced lovebird (Agapornis roseicollis). The histopathology of diseased feathers and follicular epithelium from the different species was compared; basophilic intranuclear inclusion bodies were identified in the follicular epithelium and intracytoplasmic globular inclusions were observed within macrophages located in the feather pulp from the four species. Psittacine beak and feather disease virus antigen was specifically detected by colloidal gold immunoelectron microscopy. The different preparations of purified virions displayed an icosahedral symmetry, were non-enveloped, and had a mean diameter that varied from 12 to 15 nm when negatively stained. Two major viral-associated proteins with approximate molecular weights of 26 and 23 kilodaltons (kd) were consistently demonstrated from the four viral preparations. Purified virions from the four genera were antigenically related. These findings suggest that the PBFD virus purified from numerous genera of diseased birds is similar based on ultrastructural characteristics, protein composition and antigenic reactivity.