MIT: Genetic Changes In A 2014 Indian H1N1pdm09 Virus

Flu Virus binding to Receptor Cells – Credit CDC

 

# 9810

 

A little over a week ago, in EID Journal: Emergence of D225G Variant A/H1N1, 2013–14 Flu Season, Florida, we re-visited one of the mutations linked to greater virulence in the (formerly pandemic, now seasonal) A/H1N1pdm09 virus.

 

This relatively rare amino acid substitution at position 225 (222 using H1 Numbering) from aspartic acid (D) to glycine (G) allows the virus to bind to receptors found deeper in the lungs, and is linked to the development of more severe pneumonia.

.

A 2013 study in Influenza Other Respir Viruses called A(H1N1)pdm09 hemagglutinin D222G and D222N variants are frequently harbored by patients requiring extracorporeal membrane oxygenation and advanced respiratory assistance for severe A(H1N1)pdm09 infection linked this, and the D225N mutation to more severe respiratory symptoms.

While probably the best known, and most studied, of the virulence enhancing mutations in the pH1N1 virus, D225G/N is certainly not the only one.  And in truth, there are likely other  mutations – or combinations of amino acid substitutions – that would increase the virus’s transmissibility, replication, antiviral resistance, or pathogenicity that we don’t even know about.

 

With the D225G/N mutation, it is believed that while it increases virulence, it reduces transmissibility.   But that may simply mean that the right combination of concurrent changes hasn’t emerged to increase both . . . yet.

 

Last November, in When Influenza Goes Rogue, we looked at the long history of extreme variability between flu seasons, and the emergence of mutated, or `drifted’ viruses.  And by now everyone knows that this year we are experiencing exactly that scenario with an antigenically drifted H3N2 virus.

 

The point being that flu viruses – even seasonal flu viruses that have been around for a long time – can occasionally throw us a curve. 

 

This winter we’ve seen India reporting a large number of pH1N1 cases – which they call `swine flu’ – supposedly with an unusually high mortality rate.    We’ve seen these sorts of reports from India in the past, and so it has been difficult to determine if anything is really different about this year’s flu strain (see India swine flu toll inches towards 1500).

 

Although the numbers seem high (and likely represent only the smallest tip of much larger iceberg), in a nation of over a billion people –  millions would be expected to contract the flu in an average year - and of those, thousands would likely die. 

A couple of weeks ago, in India’s H1N1 Outbreak, we looked at some of these reports along the Indian Government’s denials that anything untoward was occurring . India’s National Institute of Virology (NIV) and their National Centre for Disease Control (NCDC) both reported No mutation of H1N1.

 

Today, however, we’ve a report that suggests (based on very limited data) that perhaps something has changed with the H1N1 virus, and that it may be affecting its transmissibility, and severity, in India.

 

First, this press release, and then a link to the study.

 

Analysis suggests a more virulent swine flu virus in the Indian subcontinent

Cell Press

A flu outbreak in India that has claimed over 1200 lives may not be identical to the 2009 North American strain, as recently reported in India. A comparative analysis conducted by scientists at the Massachusetts Institute of Technology (MIT) shows that the flu virus in India seems to have acquired mutations that could spread more readily and therefore requires deeper studies. As flu season in India winds down, the researchers call on officials to increase surveillance of this and future flu outbreaks and rethink vaccination strategies to account for potential new viruses.

The MIT analysis, which compared viral proteins important for virulence and transmissibility in the 2009 and 2014 flu epidemics, was conducted by professor Ram Sasisekharan, PhD, at the Koch Institute for Integrative Cancer Research, and his research scientist colleague Kannan Tharakaraman, PhD. It appears in the March 11 issue of the journal Cell Host & Microbe.

"It has been extensively reported in India that a virus similar to A/California/07/2009 is responsible for the current outbreak," Sasisekharan says. "Examination of the Indian H1N1 flu viruses that circulated in 2014 shows amino acid mutations that make them distinct (in terms of receptor binding, virulence, and antigenic drift) from the A/California/07/2009 virus."

"It is widely believed that the current H1N1 flu vaccine is still effective for the most part," he adds.  "Effectiveness of the current H1N1 flu vaccine is debatable, and there have been calls for updating the vaccine. The Indian H1N1 viruses that circulated in 2014 are different compared to the 2009 vaccine strain A/California/07/2009."

(Continue . . .. )

 

This commentary (see below), which calls for greater testing and genetic sequencing on the Indian Subcontinent, notes that - despite the vastness of the Indian subcontinent, only two sequences have been deposited during 2014–2015 from India, suggesting poor surveillance and potentially limiting the response to a deadly outbreak.

Based on an analysis of Indian-origin strain A/India/6427/2014, they reported finding:

 

Although there are limited Indian-origin influenza sequences available in the public database to make any causal inference on the perceived increased fatalities in India, examination of the 2014 Indian H1N1 HA sequences shows traits with potential cause for concern. Amino acid changes in specific positions in the receptor binding site (RBS) of 2009pdmH1N1 have been shown to impact glycan RBS specificity and have been linked to increased virulence and disease severity.

Among these changes, the Indian-origin strain A/India/6427/2014 contains amino acid changes T200A and D225N compared to the 2009pdmH1N1 pandemic strain. The T200A amino acid change has been shown to improve human glycan receptor-binding of 2009pdmH1N1 HA (Xu et al., 2012b). The D225N mutation has been linked to increased virulence and disease severity in patients infected by the 2009 pdm virus (Ruggiero et al., 2013).

 

A third mutation,  K166Q, was also detected and has been linked to increased severity of pH1N1 in middle-aged adults during the 2013-14 flu season (see CIDRAP Study: Middle-aged adults susceptible to recent flu virus mutation).

The lack of any recent sequencing of H1N1 viruses from India is particularly frustrating in light of recent news reports. The authors write:  It is unknown if the strain A/India/6427/2014 is still in circulation; however, the apparent severity of the current outbreak seems to suggest that it could be.

 

The entire report/commentary – which emphasizes the need for more robust and timely influenza surveillanece and sequencing data -  may be accessed at:

 

Influenza Surveillance: 2014–2015 H1N1 “Swine”-Derived Influenza Viruses from India

Kannan Tharakaraman , Ram Sasisekharan

DOI: http://dx.doi.org/10.1016/j.chom.2015.02.019

Summary

The 2014-15 H1N1 outbreak in India has reportedly led to 800 fatalities. The reported influenza hemagglutinin sequences from India indicate that these viruses contain amino acid changes linked to enhanced virulence and are potentially antigenically distinct from the current vaccine containing 2009 (Cal0709) H1N1 viral hemagglutinin.

NARO: Miyazaki H5N8 Outbreak A Different Sub Clade

Credit Wikipedia

 

# 9488

 

While not a huge surprise – given the ever-growing diversity of H5 avian viruses in general and previous reports of multiple reassortants of H5N8 in Korea – this morning Japan’s National Agriculture and Food Research Organization (NARO) has announced that an analysis of the recent H5N8 outbreak in Miyazaki Prefecture shows it differs enough from previous Japanese outbreaks to be considered a different sub clade.

 

First some excerpts from the press release (machine translated), then I’ll return with a bit more.

 

December 26, 2014 
Highly pathogenic avian influenza occurred in Miyazaki Prefecture in 2014 - analysis of genome sequences for verification of the origin of the virus -

Analysis of genomic sequences for the verification from the virus -

Point

The whole genome sequence of the causative virus of highly pathogenic avian influenza that occurred in the poultry farm in Miyazaki Prefecture in December 2014 (HPAI) I was deciphered.

This occurs with the cases of wild birds in Chiba Prefecture of poultry farms and in November of Kumamoto Prefecture of this year in April, has been shown that there is no direct association.

Summary

• January 2014 or later, Korean house Kinya has continued to prevalent in wild bird H5N8 subtype highly pathogenic avian influenza virus ¹⁾ (Highly Pathogenic Avian Influenza Virus: HPAIV), the poultry of Kumamoto Prefecture in April in Japan cause the occurrence of HPAI in place since November, feces of Chiba Prefecture of wild birds (ducks), and is separated from Kagoshima Prefecture of vine, etc.. In addition, has been reported the occurrence of HPAI is caused by viruses of the same subtype in November and later Europe and North America of poultry, etc..

• The NARO National Institute of Animal Health, HPAI causative virus of that occurred in the poultry farm in Miyazaki Prefecture in December 16, 2014 is H5N8 subtype ²⁾ as well as to identify that it is a HPAIV, of all of the genes of this virus The nucleotide sequence I was quickly determined.

• And are separated by the South Korea and Europe on the Chiba strains and public gene information database that has been separated from the feces of ducks in Kumamoto stocks and November 2014 that has been separated from dead chickens the determined nucleotide sequence in April 2014 I made a comparative analysis of the H5N8 subtype HPAIV gene.

• This time, isolated virus (Miyazaki Ltd.) is, Kumamoto stock, Chiba stock, and South Korea since January, is derived from a common ancestor with the H5N8 subtype HPAIV that have occurred in the European poultry since November It is now clear.

• On the other hand, Miyazaki strain, from the difference of gene sequences, Kumamoto stocks and Chiba stock, and Europe generated stock has been clearly distinguished.

• HPAI that occurred in this Miyazaki Prefecture, Kumamoto Prefecture of poultry farms in April this year, the cases of wild birds in Chiba Prefecture in November found that direct relationship does not. This will be different from the one that occurred in April 2014, after the fall, we have suggested that at least two types of H5N8 subtype HPAIV are newly entering the country.

• From the fact that mutation of the amino acid sequence deduced to be involved in the infection to humans was observed, Miyazaki strains I are considered less likely to infect humans directly.

  (Continue . . . )

Until now, all of the Japanese H5N8 samples (and most Korean and European samples) have all fallen from the same branch of the H5 2.3.4.6 phylogenic tree (highlighted in yellow below), a clade that includes the H5N1 and H5N6 viruses as well. 

Adapted from FAO-EMPRES Report On The Emergence And Threat Of H5N6.

It will be interesting to see exactly where this Miyazaki virus falls into the H5 phylogenic tree.

 

Influenza viruses are constantly throwing the genetic dice, and while most of these variants will be evolutionary failures and fade away quickly, sometimes a `biologically fit’ and competitive strain appears and thrives.  

 

Influenza viruses normally evolve through a process called antigenic drift – a relatively slow process where minor amino acid substitutions accrue during (flawed) reproduction –  but sometimes a new `hybrid’ virus will emerge from a process called antigenic shift, or reassortment.

Reassortment of two Avian Viruses Producing a Hybrid (Reassortant) Virus

Shift occurs when one virus swap out chunks of their genetic code with gene segments from another virus.  Like viral tinker toys, these interchangeable parts allow for the creation of many different hybrids.  While far less common than drift, shift can produce abrupt and sometimes dramatic changes to the virus (see NIAID Video: How Influenza Pandemics Occur).

 

When an avian influenza virus remains in its reservoir host (predominantly waterfowl) – to which it is already well adapted – it usually changes very slowly. When it enters a new host population – like commercial poultry  – it can begin to evolve rapidly, producing numerous clades and subclades.

 

After a decade of pretty much only having to worry about H5N1 (2003-2013), over the past couple of years we’ve seen the sudden emergence and/or spread of multiple clades of HPAI avian H5N8, H5N6, H5N3, and H5N2 along side H7N9, H10N8 and a handful of `minor players’ like canine & equine H3N8, canine H3N2, and even H10N7 in marine mammals.

 

The concern is - while one can’t predict where any of these viruses will end up - the greater the diversity of novel viruses in circulation, the greater the chances of someday seeing one jump to, and adapt to, humans.

 

For more on this rapidly expanding array of novel flu viruses you may wish to revisit:

The Expanding Array Of Novel Flu Strains

EID Journal: Predicting Hotspots for Influenza Virus Reassortment

Viral Reassortants: Rocking The Cradle Of Influenza

Study: Ebola Virus Is Rapidly Evolving

Credit CDC PHIL

 

# 9015

 

One of the concerns we have when any zoonotic virus spills over into the human population is that over time, as it passes from one person to the next, it could pick up host adaptations – mutations – that could make the virus a greater threat over time.

 

In the laboratory, researchers will often conduct serial passage experiments (see Serial Passage Of H5N2 In Mice) to observe these evolutionary changes, and try to figure out what they mean.

 

Often, these genetic changes are of little or no effect, and can sometimes even be detrimental to the `biological fitness’ of the virus. Those that favor replication in the new found host, however, tend to carry on to produce more progeny, advancing their new lineage forward,  drowning out the earlier `wild type’ virus in the host.

 

A recent concern has been that Ebola - which up until now has never really spread in kind of long chains of human cases that we are seeing now – could better adapt to human physiology over time.

 

Today we’ve a study appearing in the Journal Science where scientists sequenced 99 Ebola viruses taken from 78 people from Sierra Leone during the month of June, and found that the virus is showing a marked propensity to accumulate `interhost and intrahost genetic variation’ as it passages through the population.

 

First a bit from the study, then I’ll be back with more.

Published Online August 28 2014

Science DOI: 10.1126/science.1259657

Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak

Stephen K. Gire¹,²,*, Augustine Goba³,*,†, Kristian G. Andersen¹,²,*,†, Rachel S. G. Sealfon²,⁴,*, Daniel J. Park²,*, Lansana Kanneh³, Simbirie Jalloh³, Mambu Momoh³,⁵, Mohamed Fullah³,⁵,‡, Gytis Dudas⁶, Shirlee Wohl¹,²,⁷, Lina M. Moses⁸, Nathan L. Yozwiak¹,², Sarah Winnicki¹,², Christian B. Matranga², Christine M. Malboeuf², James Qu², Adrianne D. Gladden², Stephen F. Schaffner¹,², Xiao Yang², Pan-Pan Jiang¹,², Mahan Nekoui¹,², Andres Colubri¹, Moinya Ruth Coomber³, Mbalu Fonnie³,‡, Alex Moigboi³,‡, Michael Gbakie³, Fatima K. Kamara³, Veronica Tucker³, Edwin Konuwa³, Sidiki Saffa³, Josephine Sellu³, Abdul Azziz Jalloh³, Alice Kovoma³,‡, James Koninga³, Ibrahim Mustapha³, Kandeh Kargbo³, Momoh Foday³, Mohamed Yillah³, Franklyn Kanneh³, Willie Robert³, James L. B. Massally³, Sinéad B. Chapman², James Bochicchio², Cheryl Murphy², Chad Nusbaum², Sarah Young², Bruce W. Birren², Donald S. Grant³, John S. Scheiffelin⁸, Eric S. Lander²,⁷,⁹, Christian Happi¹⁰, Sahr M. Gevao¹¹, Andreas Gnirke²,§, Andrew Rambaut⁶,¹²,¹³,§, Robert F. Garry⁸,§, S. Humarr Khan³,‡§, Pardis C. Sabeti¹,²,†§

 

In its largest outbreak, Ebola virus disease is spreading through Guinea, Liberia, Sierra Leone, and Nigeria. We sequenced 99 Ebola virus genomes from 78 patients in Sierra Leone to ~2,000x coverage. We observed a rapid accumulation of interhost and intrahost genetic variation, allowing us to characterize patterns of viral transmission over the initial weeks of the epidemic. This West African variant likely diverged from Middle African lineages ~2004, crossed from Guinea to Sierra Leone in May 2014, and has exhibited sustained human-to-human transmission subsequently, with no evidence of additional zoonotic sources. Since many of the mutations alter protein sequences and other biologically meaningful targets, they should be monitored for impact on diagnostics, vaccines, and therapies critical to outbreak response.

• In Depth Infectious Disease Genomes reveal start of Ebola outbreak

  • Gretchen Vogel
  Science 29 August 2014: 989-990.
  • Summary
  • Full Text
  • Full Text (PDF)

These researchers found that the virus had evolved into three distinct lineages in Sierra Leone during the month of June (one of which appears to have died out), along with accumulating scores of amino acid changes to its genome.

 

It should be noted that while scientists have the ability to sequence and compare these variant viruses, they don’t necessarily know what these individual mutations (or their aggregate) means to the virus, or how it might change its behavior. 

 

Based on the location of some these changes, there are concerns that the PCR primers currently used to detect it patients may need adjusting, and that some of the antiviral drugs being developed could be impacted as well. 

 

And while it is theoretically possible that changes to the genome could affect the transmissibility of the virus, we haven’t seen any evidence of that happening.

 

Unknown at this time are what genetic changes might be occurring in the virus in Liberia and Guinea, or even Nigeria. The bottom line, however, is that the longer this virus circulates in humans, the better chance it has of producing a mutation we really don’t want to see.

 

For some more coverage on this report, NPR’s Goats & Soda Blog has:

 

Ebola Is Rapidly Mutating As It Spreads Across West Africa

by Michaeleen Doucleff

 

 

This from Scientific American:

 

Patient Zero Believed to be Sole Source of Ebola Outbreak

By pinpointing the virus’s source, a new report validates steps health care workers are taking to battle the disease

Aug 28, 2014 |By Dina Fine Maron

And this from Nature News. 

 

Ebola virus mutating rapidly as it spreads

Outbreak likely originated with a single animal-to-human transmission.

Referral: VDU Blog On MERS-CoV Partial Spike Sequence Results

MERS detections by month  - Credit VDU Blog

# 8526

 

This morning Dr. Ian Mackay – Virologist, researcher, and editor of the VDU blog – takes a look at a report posted yesterday on Science Insider by Kai Kupferschmidt called Soaring MERS Cases Cause Pandemic Jitters, but Causes Are Unclear, that announces the results of some rapid genetic sequencing of the MERS virus  from the Jeddah cluster.

 

Last week virologist Christian Drosten  of the University of Bonn in Germany received 31 samples of the virus, and since then has sequenced (n=30)  a subset of the genome (a section from  the `spike protein’) – that, at least with the SARS virus – was considered probative for finding genetic changes of significance.

Early results found `nothing special’ to suggest an evolutionary change in the virus, but as we learn from Ian’s article, this isn’t the end of the story.  As Ian underscores, there are a lot of different factors at play, and much more that needs to be learned.

 

Although some of this discussion is - by its very nature - a bit technical and the issues involved complex, Ian does a great job sorting things out.  Follow the link to read:

 

MERS-CoV partial spike gene sequences do not implicate viral change in April's Jeddah human case cluster

Friday, 25 April 2014

With a new article at ScienceInsider written by Kai Kupferschmidt (@kakape on Twitter)[1], it seems that the idea of a Spring start to human detections of MERS-CoV in Saudi Arabia is gaining some support from other scientists.

(Continue . . . )

 

mBio: Spread, Circulation, and Evolution of MERS-CoV

Distribution of MERS-CoV clades in time and space. - mBio

 

# 8313

 

Although MERS has receded from the headlines over the past couple of months, it continues to circulate in the Middle East, and occasionally jump to humans.   Exactly how it circulates, and how it jumps to humans, isn’t known – although recent research has pointed a finger at both camels and bats as being possible reservoir hosts for the virus.

 

Today, the open access journal mBio carries a review of what is known about the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), with an emphasis on the evolutionary changes observed in the virus over time. 

 

As the number of genetic sequences on deposit has increased, so have the number of MERS variants. None, however, have shown the kind of persistence one would associate with efficient human-to-human transmission.

 

The primary assumption is that the virus is maintained in an (as yet, unidentified) animal reservoir, and that intermittent spillovers from animal hosts to humans has resulted in localized clusters. But thus far, none of these outbreaks has demonstrated a basic reproductive number (R0) high enough (>1.0) to sustain an outbreak (see The Lancet: Transmissibility Of MERS-CoV for additional background).

R0 (pronounced R-nought) or Basic Reproductive Number.

Essentially, the number of new cases in a susceptible population likely to arise from a single infection. With an R0 below 1.0, a virus (as an outbreak) begins to sputter and dies out. Above 1.0, and an outbreak can have `legs’.

 

The authors do offer an alternative explanation, however, writing:

 

An alternative hypothesis is that the virus has now infected a sufficient number of humans to account for the observed distribution and diversity of the virus but the infection is asymptomatic in many individuals. A recent serosurvey of 363 individuals in the Saudi Arabia failed, however, to find MERS-CoV-seropositive individuals (13).

 

First a link to the study, and  some excerpts, after which I’ll have a bit more.

 

Spread, Circulation, and Evolution of the Middle East Respiratory Syndrome Coronavirus

Matthew Cotten^a, Simon J. Watson^a, Alimuddin I. Zumla^b,c,d, Hatem Q. Makhdoom^e, Anne L. Palser^a, Swee Hoe Ong^a, Abdullah A. Al Rabeeah^b, Rafat F. Alhakeem^b, Abdullah Assiri^b, Jaffar A. Al-Tawfiq^f, Ali Albarrak^g, Mazin Barry^h, Atef Shibl^h, Fahad A. Alrabiahⁱ, Sami Hajjarⁱ, Hanan H. Balkhy^j, Hesham Flemban^k, Andrew Rambaut^l,m, Paul Kellam^a,c,d, Ziad A. Memish^b,n

ABSTRACT

The Middle East respiratory syndrome coronavirus (MERS-CoV) was first documented in the Kingdom of Saudi Arabia (KSA) in 2012 and, to date, has been identified in 180 cases with 43% mortality. In this study, we have determined the MERS-CoV evolutionary rate, documented genetic variants of the virus and their distribution throughout the Arabian peninsula, and identified the genome positions under positive selection, important features for monitoring adaptation of MERS-CoV to human transmission and for identifying the source of infections.

Respiratory samples from confirmed KSA MERS cases from May to September 2013 were subjected to whole-genome deep sequencing, and 32 complete or partial sequences (20 were ≥99% complete, 7 were 50 to 94% complete, and 5 were 27 to 50% complete) were obtained, bringing the total available MERS-CoV genomic sequences to 65. An evolutionary rate of 1.12 × 10⁻³ substitutions per site per year (95% credible interval [95% CI], 8.76 × 10⁻⁴; 1.37 × 10⁻³) was estimated, bringing the time to most recent common ancestor to March 2012 (95% CI, December 2011; June 2012).

Only one MERS-CoV codon, spike 1020, located in a domain required for cell entry, is under strong positive selection.

Four KSA MERS-CoV phylogenetic clades were found, with 3 clades apparently no longer contributing to current cases. The size of the population infected with MERS-CoV showed a gradual increase to June 2013, followed by a decline, possibly due to increased surveillance and infection control measures combined with a basic reproduction number (R₀) for the virus that is less than 1

<SNIP>

IMPORTANCE

MERS-CoV adaptation toward higher rates of sustained human-to-human transmission appears not to have occurred yet. While MERS-CoV transmission currently appears weak, careful monitoring of changes in MERS-CoV genomes and of the MERS epidemic should be maintained. The observation of phylogenetically related MERS-CoV in geographically diverse locations must be taken into account in efforts to identify the animal source and transmission of the virus.

<SNIP>

DISCUSSION

In conclusion, the rapid identification and isolation of cases, combined with an R₀ of less than 1, may control the human-to-human transmission as long as the virus transmission properties remain the same. Full control of the MERS epidemic requires identification of the source of infections to prevent the initiation of the observed human-to-human transmission chains.

(Continue . . . )

 

The conclusion is simply an academic’s way of saying, so far, we’ve been lucky with this virus.

It didn’t come fully transmissible `out of the box’, and it still requires some evolutionary tweaking before it can spark a greater epidemic threat. 

 

But each human infection is another opportunity for MERS-CoV to `figure us out’, and the big challenge right now is to find the reservoir host of the virus (camels, bats, baboons, rodents, etc. . . ) in order to stop these spillovers before the virus learns to adapt to us.

 

I’ll leave it to Dr. Vincent Racaniello or Dr. Ian Mackay to parse and interpret the more technical aspects of this study, as a lot of it is truly above my pay grade. 

 

In the meantime, for some additional background on MERS, check out some of Ian’s recent  blogs on the topic:

 

Middle East respiratory syndrome coronavirus (MERS-CoV): summing up 100 weeks

Monkey magic: Vero cells make more MERS-CoV RNA than any other animal's...

MERS-CoV antibodies in dromedary camels from Dubai, UAE, as far back as 2005...

A date with Middle East respiratory syndrome coronavirus (MERS-CoV)..

Referral: Dr. Mackay On Newly Released MERS Sequences

 

Photo Credit WHO

# 7784

 

 

Dr. Ian Mackay takes an early look at the recently deposited (at GenBank) MERS coronavirus sequences in advance of an upcoming Lancet article, and the good news is these sequences precisely match the existing primers used in the current PCR assays. 

 

Ian has two posts on this topic (which is well above my pay grade, but luckily, not above his . . . ) and so I’m relieved to be able to pass these links onto my readers.

 

 

MERS-CoV genomes on GenBank...[UPDATE]

Click to enlarge. A scale schematic of the first
MERS-CoV genome, EMC/2012.

45 subgenomic (the smallest is 361 nucleotides [nt]) to full length genome (only 13; >30,000nt) sequences of the MERS-CoV have been released onto GenBank ahead of a Lancet Infectious Diseases paper arriving in days. The GenBank accession numbers range from KF600612 - KF600656 and repsenst human cases form 2012 & 2013.

(Continue . . . )

 

17 new MERS-CoV sequences bind perfectly to frontline screening PCR assay for MERS...

Only 17 of the 45 sequences seem to include the region covered by the upE laboratory assay I just posted about in the WHO laboratory testing update but of those, the forward and reverse oligonucleotide primers and the probe all bind without any mismatch.

While that may sound like an obvious statement considering that these viruses were probably detected using that assay it isn't.

(Continue . . .)

Referral: Dr. MacKay On The Saudi MERS-CoV Sequences

Distribution of MERS-CoV cases - Credit VDU

 

 

# 7389

 

Ian Mackay  has taken a first look at the new Saudi MERS-CoV sequences deposited at GenBank, and he has some good news when it comes to the real-time PCR assays being used to identify this virus.

These four new sequences – all isolated recently in Saudi Arabia – still match the targets established using isolates taken last year, and no decrease in detection efficiency is expected.

 

To read more, follow the link to Ian’s Virology Down Under website:

 

New MERS-CoV genome sequences from Al-Ahsa.

Lancet: Origin and Diversity of Novel Avian Influenza A H7N9

 

The H7N9 Reassortment – Credit Eurosurveillance

 

# 7212

 

 

Two weeks ago, in Eurosurveillance: Sequence Analysis Of H7N9 Suggests `Widespread Circulation’, we looked at research that examined 7 early H7N9 virus samples collected in China’s outbreak and calculated the genetic distance between them.

 

Simply put, the genetic distance is the accumulated genetic difference between species or between different samples taken within a species.

 

We know that viruses circulating in the wild pick up mutations at a roughly determinable rate. Once you figure out that rate (it varies among viruses), you can compare two similar viruses, and count the number differences between them.

 

With that information you can get an idea of how long it has been since their  tMRCA  (Time To Most Recent Common Ancestor).

 

The Eurosurveillance study (above) – which utilized mutation rates from two previous H7 outbreaks (in 1999 and 2003) – calculated that H7N9 may have been widely circulating for months before it was finally detected.

But, in exactly what host, remains a subject of debate.

 

Today the Lancet has the most complete phylogenic analysis of the H7N9 virus to date, that not only suggests this virus evolved through multiple reassortments (likely involving wild birds -> wild ducks -> domesticated ducks -> poultry), it pushes back its estimated tMRCA to roughly January 2012.

 

doi:10.1016/S0140-6736(13)60938-1

Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses

Di Liu PhD , Weifeng Shi PhD , Yi Shi PhD, Dayan Wang PhD , Haixia Xiao , Wei Li MSc , Yuhai Bi PhD , Ying Wu PhD , Xianbin Li BS, Prof Jinghua Yan PhD , Prof Wenjun Liu PhD, Prof Guoping Zhao PhD , Prof Weizhong Yang MD , Prof Yu Wang MD , Prof Juncai Ma PhD , Prof Yuelong Shu PhD , Prof Fumin Lei PhD , Prof George F Gao DPhil

Summary

Background

On March 30, 2013, a novel avian influenza A H7N9 virus that infects human beings was identified. This virus had been detected in six provinces and municipal cities in China as of April 18, 2013. We correlated genomic sequences from avian influenza viruses with ecological information and did phylogenetic and coalescent analyses to extrapolate the potential origins of the virus and possible routes of reassortment events.

<SNIP>

 

Findings

The novel avian influenza A H7N9 virus originated from multiple reassortment events. The HA gene might have originated from avian influenza viruses of duck origin, and the NA gene might have transferred from migratory birds infected with avian influenza viruses along the east Asian flyway. The six internal genes of this virus probably originated from two different groups of H9N2 avian influenza viruses, which were isolated from chickens. Detailed analyses also showed that ducks and chickens probably acted as the intermediate hosts leading to the emergence of this virulent H7N9 virus. Genotypic and potential phenotypic differences imply that the isolates causing this outbreak form two separate subclades.

Interpretation

The novel avian influenza A H7N9 virus might have evolved from at least four origins. Diversity among isolates implies that the H7N9 virus has evolved into at least two different lineages. Unknown intermediate hosts involved might be implicated, extensive global surveillance is needed, and domestic-poultry-to-person transmission should be closely watched in the future.

 

There is a lot here to digest (and I’ve only hit the highlights), so follow the link to read this paper in its entirety (free registration req.).

 

There also remains a lot we don’t know about this virus, including where it spends its time when its not infecting people. While linked to poultry exposure, questions remain over exactly what host (or hosts) are silently carrying this virus and giving it to humans.

 

The authors conclude by writing:

 

. . .  on the basis of available evidence, we believe that the novel avian influenza A H7N9 virus was a multiple reassortant. The HA and NA genes might originate from duck avian influenza viruses, which might have obtained the viral genes from migratory birds a year previously, whereas the internal genes might come from chicken avian influenza viruses. We believe that the estimated times to most recent common ancestor for the eight genomic fragments and the frequent poultry transportation in China account for the increased number of confirmed sporadic cases of human infection. In particular, this novel H7N9 virus has diversified into different lineages since its emergence several months ago.

 

Nature: The Row Over H7N9 Sequencing Data

Photo Credit – CDC 

 

 

# 7210

 

There is an old adage of uncertain parentage – but most often attributed to political scientist Wallace Sayre – that (paraphrased) states, `Academic infighting is so bitter, because the stakes are so small’.

 

Of course, that isn’t nearly as true as it once was.

 

The stakes in academia now extend far beyond the publish or perish incentive in order to achieve tenure.  In addition to prestige, the fruits of research today can be worth millions in terms of government and private grants, patents, or licensing agreements to individuals, universities, organizations, and even governments.

 

These incentives have often led to research being closely held until papers can be published and proper credit bestowed to the originators.

 

In the fast moving world of virology, where – as we’ve recently witnessed – new, and potentially dangerous viruses can pop up and begin to spread quickly, delays in releasing data can have serious consequences for public health.

 

To overcome this hoarding of data - which can impede other researcher's progress for months or even years - GISAID was set up in 2008 to allow researchers to deposit, and get credit for, genetic sequences they have worked out.

 

But as the following  Nature article from Declan Butler & David Cyranoski explains, once sequences are deposited, the process gets complicated and things don’t always go as well as planned. 

 

Follow the link to read:

 

 

Nature | News

Flu papers spark row over credit for data

Rush to publish on H7N9 avian flu upsets Chinese scientists.

• Declan Butler
• & David Cyranoski

01 May 2013

On 31 March, China reported the first human cases of infection with a new H7N9 avian flu virus. The same day, a team at the Chinese National Influenza Center (CNIC) in Beijing uploaded to a research database the genetic sequences of viruses isolated from the first three human cases. But Nature has learned that in the days that followed, Chinese scientists and officials grew increasingly concerned that China might lose credit for its work in isolating and sequencing the virus.

 

(Continue . . . )

Study: Source Identification Of H7N9

 

# 7188

 

 

Although this study was actually published several days ago – and bits and pieces have surfaced in recent news stories - the steady barrage of breaking news has prevented me from posting it until now. From the Chinese Science Bulletin we get a genetic analysis of H7N9 samples pulled from live poultry markets in Shanghai.

 

Teams led by Professor Chen Hualan (National Avian Influenza Reference Laboratory in Harbin) collected 970 test samples from drinking water, feces, contaminated soil, and cloacal and tracheal swabs from birds in live markets in Shanghai and Anhui province.

 

Of these 970 samples, 20 were positive for the H7N9 avian flu virus; 10 from chickens, 3 from pigeons, and 7 were from environmental samples.  Furthermore, all of the positive samples were collected in Shanghai.

 

Analysis showed these H7N9 viruses to be a reassortant, with six internal genes lifted from the H9N2 avian virus, while the source of the H7 (HA) and N9 (NA) were not completely clear.

 

The found the HA genes were most closely related to a duck H7N3, while the NA shared the highest homology with H4N9 or H11N9 influenzas from ducks.

 

The H7N9 Reassortment – Credit Eurosurveillance

 

Some of their other findings match what we’ve seen in other studies (see Eurosurveillance: Genetic Analysis Of Novel H7N9 Virus), such as finding the amino acid leucine at position 226 of the HA, a change that is typically found in human flu viruses, and is viewed as a sign of partial adaptation from avian to mammalian hosts.

 

All of the poultry samples tested contained the 627E residue in their PB2 protein – while the first three human samples sequenced carried the E627K substitution; the swapping out of the amino acid Glutamic acid (E) at position 627 for Lysine (K).

 

Glutamic acid (E) at this position is a hallmark of avian influenza viruses, and is believed to make the virus better adapted to replicate at the higher temperatures commonly found in birds (41C).

 

Human flu viruses normally have Lysine (K) at position 627. That mutation supposedly makes the virus better adapted to replicate at the lower temperatures (roughly 33C) normally found in the upper human respiratory tract.

The authors write that this change `may have significantly contributed to their pathogenicity and lethality in humans.’

 

Follow the link below to read the study in its entirety, and for a quick synopsis, you’ll find a press release here.

 

 

Isolation and characterization of H7N9 viruses from live poultry markets—Implication of the source of current H7N9 infection in humans

JianZhong Shi, GuoHua Deng, PeiHong Liu, JinPing Zhou, LiZheng Guan, WenHui Li, XuYong Li, Jing Guo, GuoJun Wang, Jun Fan, JinLiang Wang, YuanYuan Li, YongPing Jiang, LiLing Liu, GuoBin Tian, ChengJun Li, HuaLan Chen

Download PDF (626 KB)

Abstract

On March 31, 2013, the National Health and Family Planning Commission announced that human infections with a previously undescribed influenza A (H7N9) virus had occurred in Shanghai and Anhui Province, China.

 

To investigate the possible origins of the H7N9 viruses causing these human infections, we collected 970 samples, including drinking water, soil, and cloacal and tracheal swabs of poultry from live poultry markets and poultry farms in Shanghai and Anhui Province.

 

Twenty samples were positive for the H7N9 influenza virus. Notably, all 20 viruses were isolated from samples collected from live poultry markets in Shanghai.

 

Phylogenetic analyses showed that the six internal genes of these novel human H7N9 viruses were derived from avian H9N2 viruses, but the ancestor of their HA and NA genes is uncertain. When we examined the phylogenetic relationship between the H7N9 isolates from live poultry markets and the viruses that caused the human infections, we found that they shared high homology across all eight gene segments. We thus identified the direct avian origin of the H7N9 influenza viruses that caused the human infections.

 

Importantly, we observed that the H7N9 viruses isolated from humans had acquired critical mutations that made them more “human-like”. It is therefore imperative to take strong measures to control the spread of H7N9 viruses in birds and humans to prevent further threats to human health.

(Continue . . .)

 

The authors conclude by writing:

 

The novel features that the new H7N9 viruses possess, including: previously unidentified HA and NA composition; wide prevalence in avian hosts and the environment; an exceptional adaptive ability in humans; and the potential to acquire multiple basic amino acids at the HA cleavage site and evolve into a highly pathogenic form, are a major cause for concern with respect to public health worldwide.

 

Forceful measures, such as continued surveillance in avian and human hosts, control of animal movement, shutdown of live  poultry markets and culling of poultry in affected areas must be taken during this initial stage of virus prevalence to
prevent a possible pandemic.

It is also imperative to evaluate the pathogenicity and transmissibility of these H7N9 viruses, and to develop effective vaccines and antiviral drugs to combat them and reduce, if not eliminate, their threat to human health.

Eurosurveillance: Sequence Analysis Of H7N9 Suggests `Widespread Circulation’

The H7N9 Reassortment – Credit Eurosurveillance

 

# 7161

 

 

Like crime scene investigators looking to identify a killer, scientists are using modern laboratory tests on collected evidence to profile, identify, and hopefully halt the outbreak of H7N9 that has suddenly erupted in Eastern China.

 

One of the most sophisticated tools in their arsenal is genetic sequencing, which can often tell scientists where a virus has been, and just importantly, where it might be going.

 

Virologists know that viruses tend to produce single nucleotide substitutions as they replicate, and the longer that a virus circulates, the more of these point mutations it will accrue.

 

Some types of point mutations are more likely to occur when a virus replicates in an avian host, while others are more apt to show up when infecting a human or other mammalian host.

By looking at these specific genetic markers, they can often make intelligent guesses as to where the virus has been.

By the same token, some amino acid substitutions are known to increase a virus’s affinity for certain types of hosts, which can give us a clue as to where a virus might be heading.

Which brings us to a report, published today in the journal Eurosurveillance, that examines 7 H7N9 virus samples collected early in China’s outbreak and calculates the genetic distance between them.

 

The researchers then compared that genetic distance to samples collected from two other H7 outbreaks of the past (Italy H7N1 and Netherlands H7N7), in order to estimate how long this H7N9 virus may have been circulating.

 

First the link, abstract, and some excerpts (slightly reparagraphed for readability). 

 

 

Rapid communications

Guiding outbreak management by the use of influenza A(H7Nx) virus sequence analysis

M Jonges , A Meijer, R A Fouchier, G Koch, J Li, J C Pan, H Chen, Y L Shu, M P Koopmans

The recently identified human infections with avian influenza A(H7N9) viruses in China raise important questions regarding possible source and risk to humans.

 

Sequence comparison with an influenza A(H7N7) outbreak in the Netherlands in 2003 and an A(H7N1) epidemic in Italy in 1999–2000 suggests  that widespread circulation of A(H7N9) viruses must have occurred in China.

 

The emergence of human adaptation marker PB2 E627K in human A(H7N9) cases parallels that of the fatal A(H7N7) human case in the Netherlands.  

<SNIP>

In the current study, we compared the sequence diversity observed during the Dutch A(H7N7) outbreak and Italian A(H7N1) epidemic with the initial A(H7N9) virus sequences from the current outbreak in China.

 

The maximum genetic distance generated during the three months of the Dutch HPAI A(H7N7) outbreak in concatenated HA, NA and PB2 segments of A(H7N7) viruses was 25 nucleotide substitutions.

 

For the Italian LPAI A(H7N1) epidemic, the distance generated during a nine-month period was 66 nucleotide substitutions.

 

For the A(H7N9) outbreak strains, this genetic distance is 35 substitutions, or 21 substitutions when the outlier strain A/Shanghai/1/2013 is ignored (Figure).

<SNIP>

Conclusion

Comparative analysis of the first virological findings from the current outbreak of influenza A(H7N9) virus infection in China with those from other influenza A(H7Nx) outbreaks suggests that widespread circulation must have occurred, resulting in major genetic diversification.

 

Such diversification is of concern, given that several markers associated with increased risk for public health are already present. Enhanced monitoring of avian and mammalian animal reservoirs is of utmost importance as the public health risk of these A(H7N9) viruses may change following limited additional modification.

---

 

  

The genetic distance between samples collected in China suggest that this virus may have been circulating for months before it was finally detected, but in what host remains a subject of debate.

 

Also of concern to these researchers was the finding of the PB2 E627K mutation in the human isolates, but not in those collected from birds. 

 

This substitution - the swapping out of the amino acid Glutamic acid (E) at position 627 for Lysine (K) – has been linked to increased influenza virulence in the past.

 

The authors write:

 

Remarkably, the PB2 segments of the four available human virus genome sequences from China all carry this E627K substitution, which is absent in the virus isolates obtained from birds and the environment [2]. In addition, three of the four infections with the virus with PB2 E627K were fatal. There are two plausible explanations for this observation:

1. the mammalian adaptation markers are selected during replication in humans following exposure to viruses that do not have this mutation, which are circulating in animals; 
2. the mammalian adaptation markers result from virus replication in animals from which humans become infected.

The relatively protracted disease course in the current outbreak of A(H7N9) virus infection, with relatively mild symptoms at first, followed by exacerbation in the course of a week or longer, is suggestive of the first hypothesis, similar to the outbreak in the Netherlands.

 

In this scenario, an important difference in the A(H7N7) observations from the Netherlands is the frequency of finding the PB2 E627K  mutation in humans (4/4 A(H7N9) sequenced patient strains compared with 1/61 sequenced A(H7N7) patient strains).

 

Therefore, an outstanding question is whether the A(H7N9) viruses are more readily mutating in humans or milder cases are being missed.

 

Contact investigations have found no mild cases and only one asymptomatic case), but in order to address this issue, more enhanced testing of persons exposed to a similar source is needed, using the most sensitive tests available on the optimal clinical specimen type obtained at the right time.

 

 

While this study adds substantially to our knowledge of this virus, we are still a long way from understanding where it came from, how it is circulating, and exactly how much of a public health threat it poses.

 

Still, it is absolutely remarkable that just a little over 2 weeks into this outbreak, we are seeing this type of in-depth research published online.

Eurosurveillance: Genetic Analysis Of Novel H7N9 Virus

The H7N9 Reassortment – Credit Eurosurveillance

 

# 7118

 

Less than two weeks after the announcement from China that they had isolated a novel H7N9 virus from a handful of patients, we are seeing a flood of scientific analyses describing the virus and its impact.

 

A few examples from the past two days include:

 

NEJM On The H7N9 Virus

OIE: H7N9 Represents An `Exceptional Situation’

Eurosurveillance: The Implications Of H7N9 For Europe

 

Adding to this growing knowledge base is an exploration of the genetic makeup of this viral hybrid, produced  by researchers primarily based in Japan. 

 

Attached to this research you’ll find such familiar names as Yoshihiro Kawaoka and Masato Tashiro. 

 

Eurosurveillance, Volume 18, Issue 15, 11 April 2013

Rapid communications

Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013

T Kageyama, S Fujisaki, E Takashita, H Xu, S Yamada, Y Uchida, G Neumann, T Saito, Y Kawaoka, M Tashiro

 

Novel influenza viruses of the H7N9 subtype have infected 33 and killed nine people in China as of 10 April 2013. Their haemagglutinin (HA) and neuraminidase genes probably originated from Eurasian avian influenza viruses; the remaining genes are closely related to avian H9N2 influenza viruses. Several characteristic amino acid changes in HA and the PB2 RNA polymerase subunit probably facilitate binding to human-type receptors and efficient replication in mammals, respectively, highlighting the pandemic potential of the novel viruses.

 

The abstract above is both brief and simplified, but be assured you’ll find ample details in the main body of the paper, which is well worth reading in its entirety.

 

Briefly, researchers found several worrying genetic changes, including the discovery of mammalian-adapting mutations in the RBS (receptor-binding site) of the surface HA protein. 

 

The authors describe their findings:

 

The amino acid sequence of the receptor-binding site (RBS) of HA determines preference for human- or avian-type receptors.

 

At this site, A/Shanghai1/2013 encodes an S138A mutation (H3 numbering; Figure 4, Table 3), whereas A/Shanghai/2/2013, A/Anhui/1/2013, the two avian isolates, and the virus from the environmental sample encode G186V and Q226L mutations; any of these three mutations could increase the binding of avian H5 and H7 viruses to human-type receptors [12-14].

 

The finding of mammalian-adapting mutations in the RBS of these novel viruses is cause for concern. The A/Hangzhou/1/2013 isolate encodes isoleucine at position 226, which is found in seasonal influenza A(H3N2) viruses.

 

In addition, all seven influenza A(H7N9) viruses possess a T160A substitution (H3 numbering; Table 3) in HA, which is found in recently circulating H7 viruses; this mutation leads to the loss of an N-glycosylation site at position 158 (H3 numbering; position 149 in H7 numbering), which results in increased virus binding to human-type receptors [15].

 

We’ve discussed receptor binding often in the past (see Study: Dual Receptor Binding H5N1 Viruses In China & PLoS: Human-Type H5N1 Receptor Binding In Egypt) but to review:

 

Flu Virus binding to Receptor Cells – Credit CDC

 

Human adapted influenza viruses have an RBS - Receptor Binding Site (the area of its genetic sequence that allows it to attach to, and infect, host cells) that – like a key slipping into a padlock -`fit’ the receptor cells commonly found in the human upper respiratory tract; the alpha 2,6 receptor cell.

 

Avian adapted flu viruses, like the H5N1 virus, bind preferentially to the alpha 2,3 receptor cells found in the gastrointestinal tract of birds.

 

While there are some alpha 2,3 cells deep in the lungs of humans, for an influenza to be successful in a human host, most researchers believe it needs to a able to bind to the a 2,6 receptor cell.

 

Another item generating concern is finding the (E627K) substitution in the (PB2) protein; The swapping out of the amino acid Glutamic acid (E) at position 627 for Lysine (K).

 

Glutamic acid (E) at this position is a hallmark of avian influenza viruses, and is believed to make the virus better adapted to replicate at the higher temperatures commonly found in birds (41C).

 

Human flu viruses normally have Lysine (K) at position 627.  That mutation supposedly makes the virus better adapted to replicate at the lower temperatures (roughly 33C) normally found in the upper human respiratory tract.

 

Again, the authors write:

 

Lysine at position 627 of the polymerase PB2 protein is essential for the efficient replication of avian influenza viruses in mammals [16] and has been detected in highly pathogenic avian influenza A(H5N1) viruses and in the influenza A(H7N7) virus isolated from the fatal case in the Netherlands in 2003 [17]. PB2-627K is rare among avian H9N2 PB2 proteins (i.e. it has been found in only five of 827 isolates). In keeping with this finding, the avian and environmental influenza A(H7N9) viruses analysed here encode PB2-627E. By contrast, all four human H7N9 viruses analysed here encode PB2-627K.

 

Antiviral susceptibility can often be inferred from various genetic changes, although this is an imprecise science. All 7 viruses showed signs of resistance to the older Amantadine-type ion channel inhibitors, while one had changes often associated with oseltamivir (Tamiflu ®) resistance. 

 

The authors write:

 

Based on the sequences of their NA proteins, all H7N9 viruses analysed here, with the exception of A/Shanghai/1/2013, should be sensitive to neuraminidase inhibitors (Table 3).

 

However, the R294K mutation in the NA protein of A/Shanghai/1/2013 is known to confer resistance to NA inhibitors in N2 and N9 subtype viruses [20], and is therefore of great concern.

 

 

There is a good deal more to be gleaned from this paper, but in conclusion the authors write:

 

In conclusion, we here present a biological evaluation of the sequences of the avian influenza A(H7N9) viruses that caused fatal human infections in China.

 

These viruses possess several characteristic features of mammalian influenza viruses, which are likely to contribute to their ability to infect humans and raise concerns regarding their pandemic potential.