Inactivation of the infectivity of two highly pathogenic avian influenza viruses and a virulent Newcastle disease virus by ultraviolet radiation.

Exposure of a virulent isolate of Newcastle disease virus (NDV) and two highly pathogenic avian influenza (HPAI) viruses, one of H7N1 subtype and the other H5N1 subtype, to a continuous ultraviolet B flux of approximately 90µW/cm(2), which models solar ultraviolet radiation, resulted in an exponential decline in infectivity with time. The time taken for a reduction in titre of 1 log10 median tissue culture infectious dose for each virus was: NDV, 69 min; H7N1 HPAI virus, 158 min; and H5N1 HPAI, virus 167 min.

The long view: a selective review of 40 years of Newcastle disease research.

This review is written for the series celebrating the 40th year since the first issue of Avian Pathology. The aim of the authors was to cover the developments in Newcastle disease (ND) research over the last 40 years that they considered significant. During those 40 years there have been several panzootics of this serious disease in poultry and for the last 30 years there has been a continuing panzootic in domestic pigeons, which has spread to wild birds and poultry. The 40-year period began with worldwide outbreaks of severe ND, which served as an important impetus for ND research work. Although early work was concerned with controlling the disease, specifically by improving and developing new vaccines and vaccine regimens, even prior to the 1970s ND virus was seen as a useful laboratory virus for replication and virulence studies. This review covers the historical developments in the following areas: understanding the molecular basis of virulence; epidemiology and relatedness of different ND strains, both antigenically and genetically; the emergence of virulent strains and their relationship with viruses of low virulence; sequencing and understanding the viral genome and genes; the development of rapid molecular-based diagnostic tests; and the phylogeny and molecular taxonomy of ND virus. The authors suggest areas in which future research could or should be undertaken.

Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands.

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.

Newcastle disease in the European Union 2000 to 2009.

Newcastle disease (ND) is a devastating disease of poultry that has to some extent been neglected by those working in the field in the past 10 to 15 years while attention has been focused on the emergence and spread of highly pathogenic avian influenza caused by a H5N1 subtype virus. During 2000 to 2009 in the European Union (EU) member states, ND viruses virulent for chickens have been detected in wild birds, domesticated pigeons and poultry. Based on these isolations it appears that the epizootic in racing pigeons caused by the variant viruses termed pigeon avian paramyxovirus type 1, which form the genetic group 4b(VIb) first seen in Europe in 1981, continued during 2000 to 2009, and the virus is probably enzootic in racing pigeons in some EU countries. This virus appears to have spread regularly to wild birds, especially those of the Columbidae family, and has been the cause of significant outbreaks in poultry. Other avian paramyxovirus type 1 viruses responsible for ND outbreaks in the EU during 2000 to 2009 have been those from genetic groups 5b(VIIb) and 5d(VIId). There is evidence that the former may well represent spread from a wild bird source and these viruses have also been isolated from wild birds, while the latter represents continuing spread from the East. Future legislation or recommendations aimed at the control and eradication of ND will need to encompass these three sources of virulent ND viruses.

Development of an L gene real-time reverse-transcription PCR assay for the detection of avian paramyxovirus type 1 RNA in clinical samples.

A real-time reverse-transcription PCR (rRT-PCR) that targets a region of the polymerase (L) gene was developed to detect all known lineages of avian paramyxovirus type 1 (APMV-1), also known as Newcastle disease virus (NDV). A panel of 23 viruses representing the current known phylogenetic diversity of the APMV-1 population with a bias towards the more recent European strains, which had been grown in embryonated fowls' eggs, were tested. A range of positive and negative clinical samples (n = 350) provided by the National Reference Laboratory and International Reference Laboratory at VLA Weybridge were also tested. Positive clinical material included samples considered representative of lineages 3, 4 and 5 obtained from chickens, ducks, pigeons and partridges. The negative sample population was obtained from chickens, turkeys and ducks. The APMV-1 L gene rRT-PCR gave high relative sensitivity (96.05%) and specificity (98.18%) when compared with virus isolation in embryonated fowls' eggs. It is proposed that this assay could provide a first-line screening tool for the detection of APMV-1 in clinical samples.

Phylogenetic analysis of the nucleotide sequences for the HN gene of 22 avian paramyxovirus type 2 viruses reveals marked heterogeneity.

The nucleotide sequence of the HN gene was determined for 21 isolates of avian paramyxovirus type 2 virus and compared with the published HN gene of APMV-2/chicken/California/Yucaipa/56. The HN gene of the 22 viruses had five different lengths in the range of 1737 to 1755 nucleotides coding for 579 to 585 amino acids. Phylogenetic analysis of a corresponding 1734-nucleotide sequence from the HN gene of each virus established five genetic groups (I to V), two of which (II and IV) could be divided into two sub-groups (IIa and IIb; and IVa and IVb). Although there were some exceptions, generally isolates placed in the same genetic group had >80% similarity in nucleotide sequence and <80% with the other isolates; while those in the same sub-group had >90% nucleotide sequence similarity.

Overview of incursions of Asian H5N1 subtype highly pathogenic avian influenza virus into Great Britain, 2005-2008.

Since 2005 there have been five incursions into Great Britain of highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 related to the ongoing global epizootic. The first incursion occurred in October 2005 in birds held in quarantine after importation from Taiwan. Two incursions related to wild birds: one involved a single dead whooper swan found in March 2006 in the sea off the east coast of Scotland, and the other involved 10 mute swans and a Canada goose found dead over the period extending from late December 2007 to late February 2008 on or close to a swannery on the south coast of England. The other two outbreaks occurred in commercial poultry in January 2007 and November 2007, both in the county of Suffolk. The first of these poultry outbreaks occurred on a large turkey farm, and there was no further spread. The second outbreak occurred on a free-range farm rearing turkeys, ducks, and geese and spread to birds on a second turkey farm that was culled as a dangerous contact. Viruses isolated from these five outbreaks were confirmed to be Asian H5N1 HPAI viruses; the quarantine outbreak was attributed to a clade 2.3 virus and the other four to clade 2.2 viruses. This article describes the outbreaks, their control, and the possible origins of the responsible viruses.

Avian influenza viruses in poultry products: a review.

The extensive circulation of the H5N1 highly pathogenic avian influenza virus, and the human health threat that it poses, has raised concerns over the food safety implications of this virus infecting poultry. In addition, among the most important risk factors for the possible emergence of avian influenza in the European Union and the United States, the European and Food Safety Agency and the US Department of Agriculture's Animal and Plant Health Inspection Service, respectively, have identified legal and illegal importations of infected poultry commodities. The present paper reviews existing knowledge on the presence of viable avian influenza viruses in poultry commodities.

Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally.

Asian H5N1 (hereafter referred to as panzootic H5N1) highly pathogenic avian influenza (HPAI) virus has caused large numbers of deaths in both poultry and wild-bird populations. Recent isolates of this virus have been reported to cause disease and death in commercial ducks, which has not been seen with other HPAI viruses. However, little is known about either the dissemination of this H5N1 within the organs or the cause of death in infected ducks. Nineteen 4-week-old Pekin ducks were infected with 10(6.7) median egg infectious doses of HPAI A/turkey/Turkey/1/05 (H5N1, clade 2.2) in 0.1ml via the intranasal and intraocular routes. Cloacal and oropharyngeal swabs were taken daily before three animals were selected randomly and killed humanely for postmortem examination, when samples of tissues were taken for real-time reverse transcriptase-polymerase chain reaction, histopathological examination and immunohistochemistry. Clinical signs were first observed 4 days post infection (d.p.i.) and included depression, reluctance to feed, in-coordination and torticollis resulting in the death of all the birds remaining on 5d.p.i. Higher levels of virus shedding were detected from oropharyngeal swabs than from cloacal swabs. Real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry identified peak levels of virus at 2d.p.i. in several organs. In the spleen, lung, kidney, caecal tonsils, breast muscle and thigh muscle the levels were greatly reduced at 3d.p.i. However, the highest viral loads were detected in the heart and brain from 3d.p.i. and coincided with the appearance of clinical signs and death. Our experimental results demonstrate the systemic spread of this HPAI H5N1 virus in Pekin ducks, and the localization of virus in the brain and heart tissue preceding death.

Animal and human health implications of avian influenza infections.

Avian influenza (AI) is a listed disease of the World Organisation for Animal Health (OIE) that has become a disease of great importance both for animal and human health. Until recent times, AI was considered a disease of birds with zoonotic implications of limited significance. The emergence and spread of the Asian lineage highly pathogenic AI (HPAI) H5N1 virus has dramatically changed this perspective; not only has it been responsible of the death or culling of millions of birds, but this virus has also been able to infect a variety of non-avian hosts including human beings. The implications of such a panzootic reflect themselves in animal health issues, notably in the reduction of a protein source for developing countries and in the management of the pandemic potential. Retrospective studies have shown that avian progenitors play an important role in the generation of pandemic viruses for humans, and therefore these infections in the avian reservoir should be subjected to control measures aiming at eradication of the Asian H5N1 virus from all sectors rather than just eliminating or reducing the impact of the disease in poultry.

Summary of avian influenza activity in Europe, Asia, Africa, and Australasia, 2002-2006.

Between December 2003 and January 2004 highly pathogenic avian influenza (HPAI) H5N1 infections of poultry were declared in China, Japan, South Korea, Laos, Thailand, Cambodia, Vietnam, and Indonesia. In 2004 an outbreak was reported in Malaysia. In 2005 H5N1 outbreaks were recorded in poultry in Russia, Kazakhstan, Mongolia, Romania, Turkey, and Ukraine, and virus was isolated from swans in Croatia. In 2004 HPAI H5N1 virus was isolated from smuggled eagles detected at the Brussels Airport and in 2005 imported caged birds held in quarantine in England. In 2006 HPAI was reported in poultry in Iraq, India, Azerbaijan, Pakistan, Myanmar, Afghanistan, and Israel in Asia; Albania, France, and Sweden in Europe; and Nigeria, Cameroon, and Niger in Africa; as well as in wild birds in some 24 countries across Asia and Europe. In 2003, over 25,000,000 birds were slaughtered because of 241 outbreaks of HPAI caused by virus of H7N7 subtype in the Netherlands. The virus spread into Belgium (eight outbreaks) and Germany (one outbreak). HPAI H5N2 virus was responsible for outbreaks in ostriches in South Africa during 2005. HPAI H7N3 virus was isolated in Pakistan in 2004. Low-pathogenicity avian influenza (LPAI) H5 or H7 viruses were isolated from poultry in Italy (H7N3 2002-2003; H5N2 2005), The Netherlands (H7N3 2002), France (H5N2 2003), Denmark (H5N7 2003), Taiwan (H5N2 2004), and Japan (H5N2 2005). Many isolations of LPAI viruses of other subtypes were reported from domestic and wild birds. Infections with H9N2 subtype viruses have been widespread across Asia during 2002-06.

Epidemiological Risk Factors for Animal Influenza A Viruses Overcoming Species Barriers.

Drivers and risk factors for Influenza A virus transmission across species barriers are poorly understood, despite the ever present threat to human and animal health potentially on a pandemic scale. Here we review the published evidence for epidemiological risk factors associated with influenza viruses transmitting between animal species and from animals to humans. A total of 39 papers were found with evidence of epidemiological risk factors for influenza virus transmission from animals to humans; 18 of which had some statistical measure associated with the transmission of a virus. Circumstantial or observational evidence of risk factors for transmission between animal species was found in 21 papers, including proximity to infected animals, ingestion of infected material and potential association with a species known to carry influenza virus. Only three publications were found which presented a statistical measure of an epidemiological risk factor for the transmission of influenza between animal species. This review has identified a significant gap in knowledge regarding epidemiological risk factors for the transmission of influenza viruses between animal species.

Avian influenza and human health.

Natural infections with influenza A viruses have been reported in a variety of animal species including humans, pigs, horses, sea mammals, mustelids and birds. Occasionally devastating pandemics occur in humans. Although viruses of relatively few HA and NA subtype combinations have been isolated from mammalian species, all 15 HA subtypes and all 9 NA subtypes, in most combinations, have been isolated from birds. In the 20th century the sudden emergence of antigenically different strains transmissible in humans, termed antigenic shift, has occurred on four occasions, 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each time resulting in a pandemic. Genetic analysis of the isolates demonstrated that 'new' strains most certainly emerged after reassortment of genes of viruses of avian and human origin in a permissive host. The leading theory is that the pig represents the 'mixing vessel' where this genetic reassortment may occur. In 1996, an H7N7 influenza virus of avian origin was isolated from a woman with a self-limiting conjunctivitis. During 1997 in Hong Kong, an H5N1 avian influenza virus was recognised as the cause of death of 6 of 18 infected patients. Genetic analysis revealed these human isolates of H5N1 subtype to be indistinguishable from a highly pathogenic avian influenza virus that was endemic in the local poultry population. More recently, in March 1999, two independent isolations of influenza virus subtype H9N2 were made from girls aged one to four who recovered from flu-like illnesses in Hong Kong. Subsequently, five isolations of H9N2 virus from humans on mainland China in August 1998 were reported. H9N2 viruses were known to be widespread in poultry in China and other Asian countries. In all these cases there was no evidence of human to human spread except with the H5N1 infections where there was evidence of very limited spread. This is in keeping with the finding that all these viruses possessed all eight genes of avian origin. It may well be that infection of humans with avian influenza viruses occurs much more frequently than originally assumed, but due to their limited effect go unrecognised. For the human population as a whole the main danger of direct infection with avian influenza viruses appears to be if people infected with an 'avian' virus are infected simultaneously with a 'human' influenza virus. In such circumstances reassortment could occur with the potential emergence of a virus fully capable of spread in the human population, but with antigenic characteristics for which the human population was immunologically naive. Presumably this represents a very rare coincidence, but one which could result in a true influenza pandemic.

Antigenic and genotypical characterization of Newcastle disease viruses isolated in Taiwan between 1969 and 1996.

Three major epidemics of Newcastle disease (ND) occurred in Taiwan over the past three decades (in 1969, 1984, and 1995). In order to gain a better understanding of the relationships between past ND epizootics in Taiwan, 36 ND viruses (NDVs) isolated between 1969 and 1996 were characterized antigenically and genotypically. The antigenicity of these viruses was analysed by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Using a panel of 22 mAbs to divide NDVs into subgroups, a total of 18 binding patterns were revealed. The sequences covering the cleavage site of the fusion protein gene of these isolates were also determined. The results of the phylogenetic analysis placed 36 NDVs into I, II, VIb, VIIa, VIII and two novel genotypes (provisionally termed X and VIh). The 1969 velogenic isolates were of genotypes X and VIh; the 1984-1985 velogenic isolates were genotyped VIb, VIh, VIIa, and X; while the 1995-1996 velogenic isolates were genotyped VIIa or VIII. Some 1969 and 1984 velogenic isolates were of the same mAbs binding pattern and genotype, and the mAbs binding patterns of the 1995-1996 isolates have not been seen before. It is concluded that velogenic NDVs of different genotype and antigenic type have co-circulated in Taiwan at least since 1969. Also there were epizootiological links between strains isolated in 1969 and 1984, whereas the 1995-1996 epidemic was caused by new antigenic variants.

Evaluation of two different swab transport systems in the detection of avian influenza virus excretion from infected Pekin ducks (Anas platyrhynchos).

The role of wild birds in the epidemiology and ecology of influenza A viruses has long been recognised (Alexander, 2007a). As a result of the emergence of a H5N1 highly pathogenic avian influenza (HPAI) virus and the apparent role of wild birds in its spread across Asia, Europe and Africa, avian influenza (AI) wild bird surveillance has been implemented in many countries including, since February 2006, a mandatory programme in the European Union (CEC, 2006a). In the present study the detection of virus excreted from Pekin ducks (Anas platyrhynchos) infected experimentally with A/mallard/England/2126/07 (H3N6) was investigated over a fourteen day period post-infection using cloacal and oropharyngeal swabs, with (wet) and without (dry) viral transport medium which were collected from each duck in alternating order. For influenza A virus matrix gene RNA detection, wet oropharyngeal swabs were significantly more sensitive than dry oropharyngeal on days 4-5 after infection. For cloacal samples, dry swabs were equivalent or superior to wet swabs throughout the study. Although differences in detection between dry and wet swabs were observed, the qualitative bird-level results were unaffected, meaning that the infection status of individual birds was correctly determined.

The effect of age on the pathogenesis of a highly pathogenic avian influenza (HPAI) H5N1 virus in Pekin ducks (Anas platyrhynchos) infected experimentally.

BACKGROUND: Highly pathogenic avian influenza (HPAI) H5N1 viruses have recently displayed increased virulence for wild waterfowl. OBJECTIVES: To study the effect of host age on the shedding and tissue dissemination of a HPAI H5N1 virus in infected Pekin ducks. METHODS: Pekin ducks in two age-matched groups (n = 18), 8 and 12 weeks old (wo) were each infected with 10(6) EID(50)/0.1 ml of HPAI A/turkey/Turkey/1/05 (H5N1, clade 2.2). Each day for 5 days, birds were monitored clinically, and cloacal and oropharyngeal swabs collected, before three birds from each group were selected randomly for post-mortem examination. Tissue samples were collected for examination by real-time RT-PCR, histopathology and immunohistochemistry (IHC). RESULTS: Severe clinical signs, including incoordination and torticollis were observed in the 8 wo group resulting in 100% mortality by 4 dpi. Mild clinical signs were observed in the 12 wo group with no mortality. Real-time RT-PCR and IHC results demonstrated the systemic spread of H5N1 virus in birds of both age groups. Higher levels of virus shedding were detected in oropharyngeal swabs than in cloacal swabs, with similar levels of shedding detected in both age groups. Variations in level and temporal dissemination of virus within tissues of older ducks, and the presence of the virus in brain and heart were observed, which coincided with the appearance of clinical signs preceding death in younger birds. CONCLUSIONS: These results are consistent with reports of natural infections of wild waterfowl and poultry possibly indicating an age-related association with dissemination and clinical outcome in ducks following infection with H5N1 HPAI virus.

A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health.

Real-time, reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods in the field of molecular diagnostics and research. The potential of this format to provide sensitive, specific and swift detection and quantification of viral RNAs has made it an indispensable tool for state-of-the-art diagnostics of important human and animal viral pathogens. Integration of these assays into automated liquid handling platforms for nucleic acid extraction increases the rate and standardisation of sample throughput and decreases the potential for cross-contamination. The reliability of these assays can be further enhanced by using internal controls to validate test results. Based on these advantageous characteristics, numerous robust rRT-PCRs systems have been developed and validated for important epizootic diseases of livestock. Here, we review the rRT-PCR assays that have been developed for the detection of five RNA viruses that cause diseases that are notifiable to the World Organisation for Animal Health (OIE), namely: foot-and-mouth disease, classical swine fever, bluetongue disease, avian influenza and Newcastle disease. The performance of these tests for viral diagnostics and disease control and prospects for improved strategies in the future are discussed.

Avian influenza vaccines and vaccination in birds.

Although the use of vaccines against avian influenza viruses in birds has been discouraged over the years, the unprecedented occurrence of outbreaks caused by avian influenza (AI) viruses in recent times has required review of this policy. A variety of products are now available on the market, ranging from inactivated conventional to live recombinant products. The general consensus on the use of vaccination is that if complying to GMP standards and properly administered, birds will be more resistant to field challenge and will exhibit reduced shedding levels in case of infection. However, viral circulation may still occur in a clinically healthy vaccinated population. This may result in an endemic situation and in the emergence of antigenic variants. In order to limit these risks, monitoring programmes enabling the detection of field exposure in vaccinated populations are recommended by international organisations and are essential to allow the continuation of international trade. Adequate management of a vaccination campaign, including monitoring, improved biosecurity and restriction is essential for the success of any control program for AI.

An overview of the epidemiology of avian influenza.

Only viruses of the Influenzavirus A genus have been isolated from birds and termed avian influenza [AI] viruses, but viruses with all 16 haemagglutinin [H1-H16] and all 9 neuraminidase [N1-N9] influenza A subtypes in the majority of possible combinations have been isolated from avian species. Influenza A viruses infecting poultry can be divided into two groups. The very virulent viruses causing highly pathogenic avian influenza [HPAI], with flock mortality as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. All other viruses cause a milder, primarily respiratory, disease [LPAI], unless exacerbated. Until recently HPAI viruses were rarely isolated from wild birds, but for LPAI viruses extremely high isolation rates have been recorded in surveillance studies, with overall figures of about 11% for ducks and geese and around 2% for all other species. Influenza viruses may infect all types of domestic or captive birds in all areas of the world, the frequency with which primary infections occur in any type of bird usually depending on the degree of contact there is with feral birds. Secondary spread is usually associated with human involvement, either by bird or bird product movement or by transferring infective faeces from infected to susceptible birds, but potentially wild birds could be involved. In recent years there have been costly outbreaks of HPAI in poultry in Italy, The Netherlands and Canada and in each millions of birds were slaughtered to bring the outbreaks under control. Since the 1990s AI infections due to two subtypes have been widespread in poultry across a large area of the World. LPAI H9N2 appears to have spread across the whole of Asia in that time and has become endemic in poultry in many of the affected countries. However, these outbreaks have tended to have been overshadowed by the H5N1 HPAI virus, initially isolated in China, that has now spread in poultry and/or wild birds throughout Asia and into Europe and Africa, resulting in the death or culling of hundreds of millions of poultry and posing a significant zoonosis threat.

Avian influenza infections in birds--a moving target.

Avian influenza (AI) is a complex infection of birds, of which the ecology and epidemiology have undergone substantial changes over the last decade. Avian influenza viruses infecting poultry can be divided into two groups. The very virulent viruses cause highly pathogenic avian influenza (HPAI), with flock mortality as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. All other viruses cause a milder, primarily respiratory, disease (low pathogenic avian influenza, LPAI), unless exacerbated by other infections or environmental conditions. Until recently, HPAI viruses were rarely isolated from wild birds, but for LPAI viruses extremely high isolation rates have been recorded in surveillance studies, particularly in feral waterfowl. In recent years, there have been costly outbreaks of HPAI in poultry in Italy, the Netherlands and Canada and in each of these countries millions of birds were slaughtered to bring the outbreaks under control. However, these outbreaks tend to have been overshadowed by the H5N1 HPAI virus, initially isolated in China, that has now spread in poultry and/or wild birds throughout Asia and into Europe and Africa, resulting in the death or culling of hundreds of millions of poultry and posing a significant zoonosis threat. Since the 1990s, AI infections due to two subtypes, LPAI H9N2 and HPAI H5N1,have been widespread in poultry across large areas of the world, resulting in a modified eco-epidemiology and a zoonotic potential. An extraordinary effort is required to manage these epidemics from both the human and animal health perspectives.